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Some aspects of the metabolism of Endamoeba histolytica

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Some aspects of the metabolism of Endamoeba histolytica

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Content SOME ASPECTS OP THE METABOLISM OP ENDAMOEBA HISTOLYTICA Herbert Blumenthal A Dissertation Presented to the FACULTY OP THE GRADUATE SCHOOL UNIVERSITY OP SOUTHERN CALIFORNIA In partial Fulfillment of the Requirements for the Degree D OCT OR OP PHILOSOPHY (Biochemistry and Nutrition) August 1955 UMI Number: DP21568 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI DP21568 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6 Pf\ - D B This dissertation, written by Herbert Blumenthal under the direction of Y&r&Guidance Committee, and approved by all its members, has been pre sented to and accepted by the Faculty of the Graduate School, in partial fulfillment of re quirements for the degree of D O C T O R O F P H IL O S O P H Y Guidance Committee . i d L J Z ACKNCWIEDGEMENTS I I i It is with sincere gratitude that I acknowledge the I inspiration and guidance of Doctors James N. DeLamater and i John W. Mehl. I wish to express my appreciation to Prances A. Hallman, Joseph B. Michaelson, and William C. T. Yang for many stimulating discussions, I would like to express my appreciation to the , Faculty of the Department of Biochemistry and Nutrition for their guidance during my graduate career. Appreciation is !also extended to the Commission on Enteric Infections of i the United States Armed Forces Epidemiological Board for its financial support of this research program, and to the Allen Hancock Foundation and the Institute of Medical Re- 'search of the Collis P, and Howard Huntington Memorial Hospital for the facilities that were made available. My sincere appreciation to my wife, Diane, for her help, encouragement, and inspiration. TABLE OF CONTENTS i CHAPTER PAGE i i I, INTRODUCTION . ................................... 1 i II. HISTORICAL . . .......................... 3 Initial discoveries....................... 3 i , i Proof of pathogenicity . . . . . . . . . . . 4 j In vitro culture of Endamoeba histolytica , 4 ! i Diphasic media ..... .................. 5 Liquid media 6 ; Random modifications.............. 6 ■ ! Attempts at standardization . ........ 7 Defined media . . . . . . . . . . . . . . 8 j Attempts to eliminate bacteria ...... 9 j Preconditioned media .. ........ 9' Monobacterial cultures.............. • 10 Use of embryonic fluids and living tissues ........ ................ 11 Trypanosoma cultures .......... . 11 Stimulatory and essential substances . . . * 12 Cellular fractions .................... 121 Hole of proteins............................ 13 Hole of starch ........... 14 Lipids . . . . . . . . . . . . 15 General ..... .................... 15 Cholesterol.............................. 15 i V CHAPTER PAGE Vitamins............ • . . . . ............ 15 Inorganic requirements . . 16 Other substances . . . . . . . . . . . . . 17 Anaerobiosis and oxidation-reduction ■ potentials . . . . . . . . . . . . . . . . 17 Anaerobiosis ............... 17 Oxidation-reduction potentials ....... 18 Composition of Endamoeba histolytica ..... 19 i Enzymatic activity of Endamoeba histolytica. 20 ' Proteolytic enzymes • 20 ! | Phosphatases • • • . . . . . . . * • « • • 21 I Hyaluronidase ............... 21 Amylolytic enzymes ............... 22 Observations of significance in carbohy drate metabolism 22 f i III. METHODS . 28 ! Preparation of cultures............ . 28 Source of Endamoeba histolytica cultures . 28 | i Media and method of eulture ....... 28 i Preparation of tissues..................... 29 Washing fluids..................... 29 Washing procedure ......... 31 | General considerations ............... . 31 ! ..... Initial attempts to .devise a..washing________ j CHAPTER PAGE procedure . . . * ........ ..... 31 Pinal washing procedure ........ 34 Lysis procedures ............ 37 Counting procedures . . . . . . . . . . . 38 Assay procedure.......... 38 General considerations ................... 38 Procedure ......... ............. 40 Stock solutions ................. 40 Preparation and incubation ....... 41 Extraction of formazan ......... 42 Standard curve . 43 IV. EXPERIMENTAL AND RESULTS ' ........... 44 Succinic dehydrogenase • .......... 44 General................. 44 Whole c e l l s ......................... 44 Lysed cells . . . . . . . . . . . . . . . 47 Malic dehydrogenase.......... 49 General . . . . . . . . . . . . . . . . . 49 Whole c e l l s ................. 51 Lactic dehydrogenase ..... ............. 51 General . .......... 51 Whole cells . . . . . . . . . . . . . . . 53 Lysed c e l l s .......... 53 CHAPTER I I Triosephosphate dehydrogenase ......... . . . Fructose 1,6-diphosphate as the substrate , General . . . . . . . . . . . . . . . . . Whole cells .................... Lysed eells . . . . . . . . . . . . . . . Soluble starch as the substrate ...... General . . . . . . . . . . . . . . . . . Whole cells ..................... .. Glucose as the substrate ........ ........ General ......... ............. . Whole cells . . . . . . . . . . . . . . . Lysed cells ....... ............. . Glucose dehydrogenase . ........... General . . . . . . . . . . . . . ......... Whole cells ............... Glutamic dehydrogenase . . . . . . . . . . . . General . . . . . . . . . . . . . . . . . . Whole cells ...... ................... Alcohol dehydrogenase . . . . . . . . . . . . General....................... ............ Whole cells . . . . . . . . . . . ........ Beta-Hydroxybutyric acid dehydrogenase . . . . General . .. ... ... . ......... . . • Whole cells , _ . • • • • _. „..._. . viii CHAPTER PAGE V. DISCUSSION AND CONCLUSIONS................ 74 General . 74 Aerobic metabolism ................. 75 Anaerobic metabolism ..... .......... 75 Fructose 1,6-diphosphate as a substrate . 75 Embden-Meyerhof pathway ............... 75 Hexosemonophosphate shunt ............. 76 Glucose dehydrogenase . ............ 77 Soluble starch as a substrate ...... 77 Glucose as a substrate .......... 77 Lactic dehydrogenase ...... 78 Oxidation of reduced D P N ................... 80 Auxiliary dehydrogenase systems ........ 80 Glutamic dehydrogenase ................... 81 Some general conclusions .......... 82 VI. SUMMARY......................... 85 BIBLIOGRAPHY....................................... 87 LIST OP TABLES TABLE PAGE I. composition of Ringer's solution........ 30 II. Composition of Stone's Buffer ........ 32 III. Triphenyl Tetrazolium Chloride Reduction by Succinic Dehydrogenase at pH 7.2 46 IV. Triphenyl Tetrazolium Chloride Reduction by Succinic Dehydrogenase at pH 7.4 ..... 48 V. Triphenyl Tetrazolium Chloride Reduction by Succinic Dehydrogenase with Lysed Cells . . 50 VI. Triphenyl Tetrazolium Chloride Reduction by Malic Dehydrogenase.......... 52 VII. Triphenyl Tetrazolium Chloride Reduction by Lactic Dehydrogenase ..... ............. 54 VIII. Triphenyl Tetrazolium Chloride Reduction by Lactic Dehydrogenase with Lysed Cells . . . 56 IX. Triphenyl Tetrazolium Chloride Reduction with Fructose 1,6-Diphosphate as the Substrate , 59 X* Triphenyl Tetrazolium Chloride Reduction by Using Fructose 1,6-Diphosphate as the Sub strate with Lysed Cells . ............ , 61 XI. Triphenyl Tetrazolium Chloride Reduction with Soluble Starch as a Substrate . ........ 63 XII. Triphenyl Tetrazolium Chloride Reduction with Glucose as a Substrate 65 iTABLE I ! XIII. Triphenyl Tetrazolium Chloride Reduction with | Glucose by Lysed Cells . . . ............... XIV. Triphenyl Tetrazolium Chloride Reduction by | Glucose Dehydrogenase .... ............. XV. Triphenyl Tetrazolium Chloride Reduction by Glutamic Dehydrogenase........ ............ XVI. Triphenyl Tetrazolium Chloride Reduction by Alcohol Dehydrogenase ......... ...... XVII. Triphenyl Tetrazolium Chloride Reduction by Beta-Hydroxybutyric Dehydrogenase ........ LIST OF FIGURES FIGURE 1. The Relationship Between Number of Washes and Flora Removed and Amebae Destroyed......... 2, Standard Curve for the Reduction of Triphenyl Tetrazolium Chloride •••«. ............. PAGE 36 4 4 I CHAJPTER I i | j INTRODUCTION t Although this work will be concerned with the metab- I olism of Endamoeba histolytica, no introduction to the ;problem can be made without some reference to amebiasis, i |the disease it causes. | Amebiasis is an ubiquitous disease by no means con- i ifined to the tropics. In fact, it was first described by a; i ^Russian physician at St. Petersburg (1). ! In 1927, Craig said (2) that from 5 to 10 per cent I of the population of the United States suffered from ame biasis, a statement greeted with much skepticism. However, with more complete epidemiologic study, the truth of Craig*s statement became so apparent that by 1944 Paust (5) was able to write that, "Little by little, as epidemiologic studies were carried out, the discovery was made that ame biasis is practically cosmopolitan in its distribution," and Miller (4) had referred to it as the "subtle murderer.1 * Today, the accepted incidence in the United States is about 9,5 per cent (5), Despite the fact that E. histolytica has been known for so many years, no absolute drug therapy is known (2, 6), although recent drugs used in conjunction with anti biotics show great promise. With the advent of the Korean conflict, the United i States Army was faced with the task of coping with large numbers of amebiasis cases among its troops (7), One di rect result of this situation was an enlarged program of research on E. histolytica supported by funds of the Surgeon General1s Office, Such a project is that support- ! i ing the work described here, I ! The purpose of this work was to attempt to elucidate! i i the basic metabolism of E. histolytica. At the time this m m m...1 I project was started, virtually no work, other than cultural, ;had been done on either the enzymology or metabolism of this organism, ( ' j i The purpose of this study, therefore, was to design ! a series of experiments whose results would help outline the essential metabolic pathways of this organism. The ■ triphenyIt etrazolium technique of Kun and Abood (8) seemed ;admirably suited to this task. With it, a large class of ienzymes, namely the dehydrogenases, could be tested for. Since we were interested in activities per se, rather than i specific kinetic data, the semi-quantitative nature of this procedure was overshadowed by its simplicity and adapt- l ability to a large number of enzymes. CHAPTER II HISTORICAL Since, as stated in the introduction, work on the metabolism of Endamoeba histolytica is very scanty, a large part of this history will consist of cultural data from which some inferences may be drawn about the metabolism of this ameba, I. INITIAL DISCOVERIES The first mention of amebae in connection with a dysentery was made by Lb'sch in 1875 (1), He observed amebae in the stools of a patient and wrote a careful de scription. However, he did not believe that they were the causAtive agent, but that they delayed the healing of the intestinal ulcers. Koch, in 1883 (9) observed five cases of dysentery in Egypt, two of which were complicated by liver abscess. He was able to show amebae deep within the tissue abscesses and concluded that they had some relation to the disease. Kartulis, in a series of papers (10, 11), demon strated the presence of amebae similar to those described by Losch in both intestinal ulcers and liver abscesses following dysentery. ~ " ' 4 II. PROOF OF PATHOGENICITY la 1891, Councilman and La Fleur (12) studied four teen cases of amebic dysentery at the Johns Hopkins Hospital I i and concluded that this disease was a clinical entity char- iacterized hy definite lesions due to the amebae. By 1894, * iKruse and Pasquale (13) produced typical amebic dysentery : ! 1 in cats by rectal injection of bacteriologically sterile 1 pus from liver abscesses containing amebae. Kartulis, in 1904 (14), discovered amebae in a brain abscess following dysentery. He was also successful in producing dysentery in cats via rectal Injection of feces containing amebae. By this time, there was no longer any doubt that amebae, not bacteria, were the cause of these dysenteries. 111 • M VITRO CULTURE OF E. HISTOLYTICA Penfold et al, (15), as early as 1916, had claimed to have cultivated E, histolytica in medium containing nutrient broth and a pancreatic digest preparation. Yoshida (16), using one part horse serum, four parts Ringer*s solution and red blood cells, had claimed 72-hour survival of amebae. Cutler (17) also claimed to have cultivated amebae, but none of the results of these early workers could be confirmed, and credit for the first suc cessful cultivation of the amebae must go to Boeck and Drbohlav (18, 19), ' i Diphasic Media, j Earliest successful media for the culture of E. j histolytica were diphasic in nature. That is, they con- ! sisted of a solid portion usually slanted and a liquid ! portion which overlaid the solid slant. Thus, the medium j i of Boeek and Drbohlav, accidentally come upon when these j {workers found pathogenie amebae growing in a flagellate i ! • i culture obtained from infected stools (18, 19), was made up 1 ; of slants composed of coagulated whole egg suspended in the Locke’s solution and overlaid with a 1 per cent solution of; ■ i {inactivated human serum in Locke’s solution. Later, this j was modified by Drbohlav (20) who used crystallized egg 1 albumin instead of the human serum of the original medium. In 1928 Tanabe and Chiba (21) obtained excellent growth on :a medium consisting of slants of asparagine and agar in Ringer’s overlaid with Ringer’s solution containing 5 per cent non-inactivated rabbit serum. In England Dobell and Laidlaw (22, 23) modified the Boeck-Drbohlav medium by the , addition of starch, which they said was ingested by the amebae. Observing that amebae often grew in the livers of infected patients, Cleveland and.Collier (24) devised a very successful medium consisting of slants made of liver j infusion agar overlaid with 10 per cent serum in physio- I logical saline. Frye and Meleney (25) substituted Lilly . liver extract number 343 for serum, stating that this ! eliminated the tedious procedure of preparing inactivated, ^ sterile human serum. Nelson (26) cultivated amebae in a medium where the ! |slants were composed of solvent (ethanol, ether, chloro form or acetone) extracts of egg yolk suspended with agar in buffered saline, while the overlay was buffered saline. Nelson (27) also showed that alcoholic extracts of liver preparations from humans, calves, beef, guinea pigs, and cats would support amebic growth. Tsuchiga (28) prepared a simple medium consisting of nutrient broth and a mixture of starch and charcoal. The charcoal presumably adsorbed any deleterious gases such as ammonia and hydrogen sulfide. The use of diphasic media has one great advantage; the rich slant and the poorer overlay result in reduced bacterial growth. On the other hand, diphasic media suffer from a serious lack of uniformity (29), Liquid Media. Random modifications. Craig (30, 31) seems to have made the earliest attempts to produce an all-liquid culture medium. He simplified the medium of Boeck and Drbohlav by eliminating the slants and using Locke*s solution and serum! i only* Later Craig and St. John (32) stated that normal j j saline could be substituted for the Locke1s solution of I i their medium. However, this work has not been repeated, and it seems likely that survival was mistaken for growth. ; I | Later St. John (33) devised a medium consisting of ex- j tracted powdered heart muscle in a modif ied Locke’s solu- ! ! tion. i ! ! The search for a simpler medium continued in a I 1 random rather than organized fashion. Different substances were added on the basis of chance rather than systematic analysis of what might be of importance in the successful | ! i media already devised. Thus, El Kordy (34) reported ex cellent amebic growth in hydatid fluid. He also cultured E. histolytica in an extract of dried hydatid scolices in Ringer’s solution (35), as well as fresh tomato juice. Inoki and his collaborators (36) cultivated amebae in whole blood rather than serum. Attempts at standardization. Balamuth and Sandza (37) prepared a standardized fluid medium from an infusion of coagulated egg yolks made up in buffered salt solution. This medium was further improved by the addition of Wilson’s liver concentrate powder (38). This medium is wholly transparent and thus chance contaminations could be noted by the appearance bf turbidity. Hitchcock and Rawson (39) replaced the fresh egg of Balamuth1s medium with Difco, I [Bacto dehydrated coagulated egg yolk. This now eliminated ' variations due to the egg3 themselves since dehydrated egg i ! was quite stable and could be bought in large batch lots. A medium composed of commercial dried egg yolk, yeast ex tract, and sodium chloride was also described (40), | More recently Gieman and Becker (41, 42) applied a “perfusion jar” technique to the culture of E. histolytica. The amebae were placed inside a dialysis membrane which was j immersed in a liquid medium. The whole system was in equilibrium with a special gas mixture. These workers were ■ t ,able to grow amebae in sufficient numbers to analyze for the amino acid composition of amebic protein. However, this culture procedure is tedious in preparation and re quires special vessels as well as the special gas mixture. Defined Media. The term “defined” or “synthetic media,” although slightly optimistic, has been used in reference to a series of media in which all the components, except rice powder or a blood protein fraction, are clearly defined chemical entities. The first effort along these lines was that of Hansen and Anderson (43), who described a liquid medium containing twelve amino acids, ten synthetic vitamins of the B complex, nucleic acid, cholesterol, rice starch, and j trace minerals in buffered saline. These workers also found that liver extract used by previous workers could be i replaced by synthetic vitamins (44), Hallman et al, (45) , modified the medium of Hansen and Anderson, increasing the J number of amino acids and the amount of cholesterol, nucleic acid, and some of the trace minerals. They further added J i small amounts of glucose and glycogen to the medium, and j ! found they could now substitute purified human serum al bumin or mucoprotein for rice starch, I I | Attempts to Eliminate Bacteria, All of the media mentioned thus far will support j amebic growth only in the presence of growing bacterial populations. Since the presence of such bacteria obscures observations made on the amebae per se, numerous attempts have been made to eliminate them. Preconditioned media. In 1947 Jacobs (46, 47) re ported a medium in which there were apparently no viable 1 J bacteria present. He grew Escherichia coli on the usual egg slant-Locke‘s solution for 24 hours, after which he heat-killed the bacteria. He then added rice, powder and additional heat-killed bacteria to these cultures and in oculated them with amebae grown monobacterially with a penicillin-sensitive Clostridium perfringens. Penicillin j was then added to inhibit the latter, under these con ditions, the amebae grew with great difficulty and Jacobs i _ * himself stated that he had no proof they were entirely free! 1 ; i of viable baeteria, j In a series of studies, using a similar technique, \ Shaffer at al. (48-52) were able to grow amebae in the j presence of relatively few bacteria. They grew an anaero- j i ; bic Streptobacillus in fluid thioglyeollate-dextrose-rice ! i ' i i 1 flour medium for 24 hours and then spun down most of the i ; ! bacteria. The supernate of this culture, in conjunction ' i ' with a bacteria-free filtrate of HRS bacterial complex ; grown 24 hours in an egg medium plus normal horse serum and; i ! ^penicillin, was able to support the growth of the HRS strain of E. histolytica indefinitely, Monobacterlal cultures. Various workers have ap- , proached the problem of freeing amebae from their normal mixed floras and growing them monobacterially with a selected concomitant, Rees at al, (53) established the first monobacterlal culture of E, histolytica with the as- : sociate bacterium designated as organism Mt." Many methods of obtaining monobacterlal cultures have been used, among which are: washing of cysts with mercuric chloride (54), micro isolation (55), use of selected antibiotics (47) and, most recently, use of Trypohosoma cruzi cultures (56), ■Since the advent of monobacterlal cultures, E. histolytica jhas been shown to grow and multiply in the presence of at least eight distinct bacterial types (56). Additional bacterial species that will support amebic growth in mono- I • I ibacterial culture have been reported (57-61). i ( Use of embryonic fluids and living tissues. Sadum et al. (62) reported good growth of E. histolytica in chick i amniotic or allantoic fluids* They stated that the age of !the embryo was an important factor. However, they could i not get a culture to survive longer than 13 days. In a re port from the same laboratory, Everitt et al. (63) tried direct chick embryo, ehorio-allantoic, and yolk sac inocu lation using living embryos. In no case were the amebae living after 72 hours. In all cases, except amniotic in oculations, no living amebae were present after 12 hours• To Shaffer et al. (64, 65) mustggo the credit of having first grown sterile amebae. Using tissue culture techniques they were able to grow bacteria-free amebae in the presence of tissue suspensions from 4-5-day-old chick embryos. They noted (65) that fresh intact tissue cells were necessary for successful propagation of E. histolytica under these conditions. Trypanosome cultures. In 1950 (66) Phillips pub lished his, initial report on the culture of E. histolytica ! " ' 12 i in the presence of T. cruzi, This was the first mention of : the concomitant growth of E. histolytica with any organisms i i ■ •other than bacteria. Other workers had tried to grow amebae with yeasts (59) but had failed, Phillips had chosen T, cruzi as his first test organism because it cul- jtured at the same temperature as E. histolytica, and be- ! ' ' ' jcause it grew on a variety of media (67), He was fortunate ] in this choice since subsequent work showed that no other ■ trypanosome tested would support amebic growth (67, 68), IV, STIMULATORY AMD ESSENTIAL SUBSTANCES i Cellular Fractions, i The need for the concomitant growth of some sort of metabolizing system, either bacterium, trypanosome or living tissue, for successful culture of Endamoeba histo lytica points to a close metabolic interrelationship be tween the amebae and the accompanying cells. Since bacteria serve so well in this regard, some workers have logically assumed that they may be a source of a growth factor or factors. Thus, Jacobs (46, 47) cultivated amebae in the presence of heat-killed bacteria. One must assume that the dead bacteria provided some needed substance since Jacobs (47) also reported that dead yeast, yeast juices, ether- killed bacteria, and plasmolysates of bacteria had no growth-promoting activity for the amebae, Shaffer et al. (48-50), in maintaining cultures of amebae in the absence j of actively multiplying bacteria, added a bacteria-free I filtrate from a culture of four species of bacteria growing together (50). In this case the factor(s) were in the fil trate rather than the cells themselves, Karlsson (69, 70) reported on a growth-stimulating factor in dead bacterial cells which was labile to heat, alkali, and oxidation, but stable in the presence of formaldehyde. He stated the pos sibility of the presence of a labile metabolic intermediate (69). Sawada et al, (71) filtered freeze-thaw-lysed bac teria that had been grown for 24 hours in sodium citrate in Ringer*s solution. They reported that such filtrates of Staphylococcus aureu3 and Bacillus mycoides stimulated amebic growth while a Bacillus coli filtrate did not, Phillips et al, (72) have been able to grow amebae In cultures with T. cruzi where the trypanosomes had been heat-treated at 48°C. for 10 minutes. If, however, the trypanosomes were heated to 52°C., no growth of amebae could be demonstrated. Phillips (68) has also noted that amebae actively ingest trypanosomes. Role of Proteins. It has been reported (73) that hemoglobin and horse red blood cells when added to Dobell and Laidlaw<s medium had stimulatory action on amebic growth. As previously " ■ 14 mentioned, Hallman et al. (45) presented evidenee that the : protein component of rice powder was beneficial to amebae. j i |Role of Starch. ; i < ' The exact role of starch in amebic nutrition is not 1 known. In fact, there is still controversy as to whether j it is a requirement for optimum growth or not. In this j connection Spingarn et al. (74) made a careful study of thej |effects of rice powder on the growth of cultures of j ! Endamoeba histolytica. They concluded that the addition of j rice powder to cultures increased the amount and duration j of amebic growth. They noted that there was an optimum amount I ; | of rice powder for the most abundant growth of amebae, j i : > The question of what stimulatory factors are con tained in rice powder has been answered to some degree by the work of Hallman et al. (45). These workers were able to substitute a simple carbohydrate and one of a number of serum protein fractions for rice powder in their synthetic media and obtain good growth of amebae. They also showed that rice powder was really a complex of a number of sub stances, including protein, lipid, and many of the B vita mins as well as starch (75). The fact that the amebae ingest starch particles is clearly seen in culture; and Hopkins and Warner (76), in a careful study, reported the ! actual engulfment, breakdown, and digestion of starch ' 15 granules by E. histolytica, I j Lipids ; t General, In discussing the digestive processes of amebae, Hopkins et al. (76) mention the fact that the ground substance of the "digestive spherules" may be of a lecithin-like nature. Nelson (26, 27) was able to devise | a successful medium based on a lipid extract of egg yolks j I i or tissue. He stated (27) that such lipid extracts could 1 !substitute for serum in his medium, 1 ■ : ! Cholesterol. Snyder and Meleney (77) were the first I i to state that cholesterol was essential to the growth of I ' I E. histolytica. Following this Rees _et al. (78) found that' cholesterol plus eight B vitamins stimulated amebic growth on egg white media. Cholesterol or the vitamins alone had no effect. In egg yolk medium cholesterol or vitamins ; alone or In combination had no effect, Griffin and Mccarten j - - - (79) reported that with E« histolytica, while cholesterol alone could not substitute for serum in culture media, ; cholesterol oleate or cholesterol plus adequate amounts of oleic acid could do so. Cholesterol has been used as a component in defined media (44, 45). Vitamins. As. already mentioned a number of workers have stated that E. histolytica requires the presence of the B- eomplex vitamins and have devised several media based on this assumption (44, 45, 78). Nelson stated that in his ^gg yolk extract medium (26) ascorbic acid and calcium ^pantothenate caused definite stimulation of amebic growth. i I Inorganic Requirements. In almost all media devised for the cultivation of E. histolytica some sort of physiologic salt mixture is in cluded. However, determinations of the actual inorganic j ; requirements for this organism are few, Chang (80) re ported the optimum total salt concentration for growth in his medium was m/30 phosphate and 0.4 per cent sodium chloride. Nelson (81) stated that in hi3 medium phosphate buffer between m/40 and m/10 was required by the amebae; he further stated magnesium was also a requirement. Blumen- thal et al. (82) found that in whole egg media phosphate was not required as a buffer, and that successful growth could be obtained in the presence of alternate buffers such as amino acid buffers. These workers further reported (83) that although phosphate buffers per se were not necessary, phosphate appeared to be a definite requirement. They stated that the level of phosphate required by E. histo lytica is between m/10 and m/19. | ' 17 [ I ! Other Substances. A large number of miscellaneous substances has been brought forward as growth stimulants or absolute require- j : ments for E. histolytica. These run the gamut from di- | 1alysates or extracts of various complex substances to i ;simple organic molecules. DeLamater and Hallman (84) re- : j ported a heat-stable, dialyzable substance from the protein-- free fraction of human serum which they believed essential i to the growth of a strain of E. histolytica. Rees et al. |(77, 78) thought that egg white contained a substance I needed for the growth of E. histolytica. They reported the !disappearance of this'substance on aging of the media and suggested that it was rendered inactive by oxidation. Karlsson and Nakamura (85) found a growth stimulatory factor in fresh yeast cake which was highly unstable and lost upon Seitz filtration. Andrews et al. (86) reported ,a stimulatory factor in extracts of human feces. This ex tract was water soluble, passed through a Berkefeld filter, and was inactivated by heating at 6G°C. for 50 minutes. Recently Greenberg et al. (87) stated that glucosamine was a definite requirement for growth under their cultural conditions, V. ANAER OB I OS IS AND OXIDATION— REDUCTION POTENTIALS Anaerobiosis. Dobell and Laidlaw (23) had stated that in their i medium the amebae grew in the bottom of the culture tube. j 'Whether this was entirely a function of the specific j I gravity of the amebae or a direct requirement for an an aerobic environment was not known. Spyider and Meleney (77) i | considered anaerobiosis essential for growth. They offered indirect evidence (88) that marked reduction in oxygen tension and probably elevation in carbon dioxide tension were necessary for growth, Hartman (89) was able to reduce I ( the depth of his media to 1 centimeter as long as the ear- : bon dioxide tension was sufficiently increased. He ob served that since the carbon dioxide tension of host tissues is generally greater than that of the air, an increased C0S tension might be expected to prove beneficial to the amebae. Some authors have stated that anaerobiosis is essential to optimum amebic growth (41, 76, 90). Recently Balamuth (91) in carefully controlled studies has shown that oxygen ten sions as low as 0,1 per cent definitely hampered amebic growth, Oxidation-Reduction Potentials. Many studies of the proper oxidation-reduction po tential for maximum growth of E. histolytica have been made. Although results vary widely, they all suggest that a strongly negative (reducing) potential is most beneficial. Thus, although Chang (92) reported different potentials l i for different media, he found that the optimum range was | j strongly negative, being between -350 and -425 mv. Hopkins! I _ .i and Warner (76) found the optimum redox potential to be j well below -400 mv. Some workers (93, 94) have suggested i ! - | that one of the functions of bacteria is to reduce the | redox potential of the environment. It is well to note at j ' i i this point that the requirement for a reduced potential ; j is another manifestation of the need of the amebae for an i j i 1 | anaerobic environment. Such a theory would agree with the i j ; ! fact that low oxygen tensions and/or negative oxidation- i reduction potentials yield optimum growth, j j ' VI. COMPOSITIOIT OP BHDAMOEBA HISTOLYTICA Kofoid et al. (95) made a study of the cyst wall of E. histolytica and reported that it had the properties of a keratin. Lillie (96), using hlstochemical techniques, showed that E. histolytica often contained glycogen, or some similar material that was a Bauer-positive polysac charide digestible with malt diastase and ptyalin, present , in the cytoplasm. Hallman et al. (97), using a variety of histoehemical methods, also demonstrated the presence of a polysaccharide in the cytoplasm of the amebae. Becker and Geiman (42) harvested sufficient numbers 20 ; i , of amebae to do a total amino acid analysis of amebic j protein. They found that it contained no unusual amino ! I acids, and was quite high in glutamic acid, phenylalanine, and lys ine. i ! ! VII. ENZYMATIC ACTIVITY OP ENDAMOEBA HISTOLYTICA I \ • i !Proteolytic Enzymes. I ------- Craig as far back as 1927 (98) reported that E. histolytica contains an enzyme capable of digesting intes- itinal mucosa. Anderson and Hansen (44) and Reardon and i Bartgis (99) suggested the presence of an enzyme capable of attacking the gluten surrounding starch granules. Recently Rees est al, (100) demonstrated the liberation of starch granules from rice powder, a process which is proteolytic in nature, with E. histolytica grown monobacterially with seventeen different organisms. In the case of sixteen of these bacteria, such liberation was not apparent when they were grown without the amebae. These workers also stated that the liberated particles were microscopically indis tinguishable from starch granules liberated by either hydrolysis in dilute alkali or digestion with papain, ficin or crystalline trypsin. In this same paper, Rees and his co-workers (100) showed that amebae could liquify gelatine. Although they offered no evidence, they sug gested that this gelatinase activity was discrete from the Jproteolytic activity that resulted in liberation of starch ^ I particles. I l I ' * * ■ i Phosphatases. | In 1948 Carrera and Changus (101) reported a histo- , chemical demonstration of acid phosphatase in E. histo- I lytica. Balamuth (102), using a similar technique, was able to confirm this finding. Later Blumenthal et al. (103), I using an assay method based on the hydrolysis of p- j !nitrophenylphosphate, were able to demonstrate not only an acid phosphatase, but an alkaline phosphatase as well, Hyaluronidase. I - - - | In an effort to find the mode of tissue invasion by !*• histolytica, Bradin (104) investigated the possible hyaluronidase activity of E. histolytica. He reported that the detection of this enzyme was possible only after animal passage of amebae. He also noted that this activity dis appeared again after about the third to fifth subculture. DeLamater et al. (105) also examined E. histolytica for possible hyaluronidase activity. They tested for in vitro production of intra- and extracellular enzyme, and also production after animal passage or seeding with hyaluronic acid. In no case were they able to demonstrate enzyme activity. These workers suggest that the discrepancies be tween their results and those of Bradin (104) might be due 22 to strain variations or differences in stock cultures. Amylolytic Enzymes. i The early work of Hopkins £t al. (76) on starch in- j jgestion by E. histolytica suggested a possible amylase in ! i ! jthese organisms. Hallman and DeLamater (106) incubated I |amebae in penicylinders on blood agar plates in which 1 soluble starch was incorporated, and then flooded the plates ! I with Gram*s iodine solution, A clear zone about the peni- l !cylinders demonstrated that hydrolysis of starch had oc- i 1eurred and further that the hydrolyzing principle diffused a considerable distance from the cylinders. Recently Baernstein et al. (107) demonstrated the presence of amylase in E. histolytica by more quantitative methods. They measured the amount of glucose produced from starch after incubation in whole cultures, supernates of cultures, and extracts of acetone powders of amebae. They also showed the enzyme to be extracellular, with an optimum pH between 5 and 6. Observations of Significance in carbohydrate Metabolism. Rees at al. (100) measured total C0a production in a variety of cultures and have come to some interesting con clusions. When E. histolytica was grown monobacterially with either A. aerogenes or organism " t," the amount of C02 produced was greater than that produced by the bacteria grown alone. Using turbidity as a measure of bacterial numbers, tbese workers found that the presence of the amebae resulted in increased bacterial growth. However, • the ratios of C0S produced to turbidity were also higher for the amebae plus bacteria, since they found that when glucose was the substrate the ratio of C0S to turbidity was dependent on the amount of substrate available, they con cluded that the increased ratio of C0S to turbidity when j starch was the substrate, must be due to a greater avail ability of starch for bacterial C0Z production caused by ; the liberation of starch particles due to amebic activity. In the case of £l» perfringens, which can utilize rice I • flour quite well* there was no difference in either C0S production, turbidity values or the ratio of C02 to turbidity for the amebae plus bacteria or bacteria alone, Baernstein et al, (107) carried the work of Rees et al, (100)na step further when they seeded monobacterial cultures of E. histolytica with glucose and measured not only C0S> production, but glucose utilization, and lactate production. They found that the amount of C0S produced was far in excess of the glucose utilized and lactate produced. These workers suggested that either starch utilization or diffusion of glucose from the solid phase of their medium could account for this large excess of C0a. Using washed amebae cultures, Nakamura et al, (108) ! 24 ) jmeasured anaerobic glucose utilization. They found that (with monobacterial cultures, whe.re starch was still present in the amebic residue, there was considerable-acid produc tion as measured by C02 ‘ evolution from a bicarbonate buffer. I However, when amebae were grown with Trypanosoma cruzi as 1 the supporting organism, a system where starch is not 'present, they observed no C0S evolution from glucose, Bradin and Kun (109, 110), using amebae grown with a mixed flora in the presence of starch and a similar incubation technique, reported production of C0g from a number of sugars, including glucose, mannose, and galactose. These 'workers noted the evolution of hydrogen sulfide in the presence of cysteine. They stated that in starved cultures, where endogenous substrates were exhausted, an interdepend ence between C02 and H2S production occurred, and that glucose utilization without cysteine was not possible. With both glucose and cysteine they showed G02, HsS, and lactate production, it was noted that only 10 per cent of the GOa could be accounted for in terms of lactate. Entner and Anderson (111), working with washed mono- bacterial cultures where residual starch was present, re ported endogenous production of lactic acid. The addition of maltose did not enhance the production of lactate and addition of glucose seemed to inhibit lactate formation. Where T. cruzi-amebae cultures were used, glucose stimulated lactate production, whereas added maltose or starch had no effect or even depressed lactate production. These workers i also reported that in the case of monobacterial cultures a • small amount of succinate was produced. However, this sue- 1 i * cinate production could be demonstrated only when the gas- I i sing mixture contained C0a. If nitrogen alone was used, | l mo succinate was formed. The authors suggested that any j I i t succinate formation was due to a condensation of a 3- and l-carbon fragment. Hallman et al. (112) studied the dis appearance of glucose in washed monobacterial cultures and ■concluded that there was no utilization evident. However, 1 I :these experiments were run aerobically and in the presence of large amounts of antibiotics designed to suppress the floral metabolism. Kun and Bradin (104), in a recent letter to the editor, elaborated on their theory of a coupled carbohy drate oxidation-cysteine reduction (109, 110) in the me tabolism of E. histolytica. They state that, anaerobically, there is an equal C0S production,from glucose, fructose or ! ; mannose with a concomitant release of HsS from cysteine, and that both cysteine and a sugar must be present. With pyruvate as a substrate, C0S production is two-thirds that of glucose in unlysed preparations and equal to glucose in ■ lysed preparations. In the ease of pyruvate utilization, J no HaS is produced. In the case of glucose plus cysteine in lysed preparations, there is no gas production unless a : j heat-stable extract of rat liver acetone powder is added, | On addition of this liver fraction gas evolution is equal , i j to or greater than that occurring in unlysed preparations, J I The liver preparations cannot be replaced by Coenzyme I or : II, diphosphothiamin, pyridoxine plus adenosine triphos- | phate, or eoenzyme A, Arsenate inhibits the COg production I . ! from glucose but not the HaS production from cysteine. A variety of chelating agents inhibits production of both C0a i ! and HsS from these substrates, i ; The authors then postulate a coupled system wherein j i triose phosphate oxidase transfers hydrogen to the sulfur ' atom of cysteine. However, it is interesting that the molar ratio of C0a to HaS is 10-20 moles of C0a. produced to 1 mole of HaS, They rationalize this large difference by suggesting that thiol radicals formed in the course of the reaction are used up mainly in the interaction, “with each other or other intermediates,’ * However, it is difficult to see how thiol radicals which did not ultimately form HaS could account for all the hydrogen liberated in the triose oxidation step, secondly, one would expect that any thiol free radicals formed would come wholly from the cysteine itself. It seems that a more simple, and equally plausible, explanation would fit the data presented; namely, that 27 cysteine is indeed,important, but mainly as a protector of SH groups on the dehydrogenase molecule and that E. histo- lytica contains a desulfurase system the importance of i which is unrelated to the triose oxidase. The statement of i these authors that arsenate, while inhibiting glucose , j utilization, does not alter HgS production, would also lend| credence to this hypothesis, | The metabolism of succinic acid by E, histolytica j i was briefly mentioned by Seaman (114), who was studying the I inhibition of succinic dehydrogenase in a number of para- ! sitic protozoans by arson© and phosphono analogues of sue- i • - • ' ! j ! einic acid. He reported that acetone powders of E. histo- ' I ! lytica contained a succinic dehydrogenase which was in- i hibited by these analogues. However, he ran no bacterial controls, but merely stated that observation of the arneba suspensions through a phase microscope showed that there was only “one bacterium per 75-120 amebae,” It has been i our experience that no visual comparison of bacterial numbers is at all possible, due mainly to the adherence of numerous bacteria to both the starch and the amebae. The use of an acetone powder which would also contain bacterial enzymes might therefore account for a large part, if not all, of the activity observed. CHAPTER I I I METHODS I. PREPARATION OP CULTURES Source of Endamoeba Histolytica cultures. Trophozoites of the DKB strain of Endamoeba histo- j lytica with a mixed flora—^ were used in all these studies,j The DKB strain is of human origin and was isolated by ! Clifford Dobell in England, November, 1924 (115), It was j I obtained by us through the courtesy of Dr. Fred Ryden, ' I Vanderbilt University School of Medicine, Nashville, ' i i !Tennessee. ! | ; i Media and Method of Culture. Amebae were grown in Pyrex culture tubes (15 x 125 ram.) with cotton plugs, on slants of Difco, Bacto Endamoeba Medium (24), overlaid with approximately 3 ml. of 10 per ^ cent (v/v) of human, normal inactivated serum in Ringer's solution. The composition of the Ringer's solution is ""Dr. Quentin M. Geiman of the Harvard School of Public Health has recently made a partial analysis of this flora. He states that there are possibly six different species of bacteria present. He has definitely identified Alcaligenes faecalis, Escherichia coli, and Pseudomonas aeruginosa. There is a gram-positiverod that would either be B. subtilis or B. pumilis and a gram-positive coccus thal appears to belong to tKe genus Enterococcus. There is another gram-negative rod that is a possible variant of Pseudomonas aeruginosa. shown in Table I, Both the slants and the Ringer's solu- j ! tion were sterilized by autoclaving for 20 minutes at 20 j pounds. Serum was sterilized by Seitz filtration and in- j activated by heating in a water bath for 1 hour at 56°C. \ Before inoculation, 20-25 mg. of a purified Belgian rice starch (Stein Hall Company, Hew York, N, Y.) were added to each tube. The rice starch was sterilized by heating in an oven for 1 hour at 180°C. Tubes were inoculated with 0.5 ml. of the sediment from a growing culture. Transfers were i routinely made at 48-hour intervals. If a particular set j of culture tubes was to be used for an assay, the sediment !of these tubes was thoroughly broken up and mixed by i ! I |pipette after 24 hours of incubation (29). Such agitation j ,of growing cultures resulted in increases of ameba popula- tion ranging from 50 to 100 per cent. i II. PREPARATION- OP TISSUES 1 Washing Fluids. Several modifications of stone's buffer (116) were used to wash the amebae free of flora. They were: j a.. the complete Stone's buffer, b. the complete stone's buffer.made 0.001 M with respect to cysteine.• HCl, c. Stone's buffer with the phosphate salts I eliminated (Stone's salts), j TAB.EE I COMPOSITION OP RINGER'S SOLUTION Compound Grams Molarity Nael 9.0 0.1146 GaClSs 0.2 0.0018 KCl 0.2 0.0027 Pinal volume equals 1 liter. . . _ - - I d. Stone’s salts made 0,001 M with respect to j i cysteine • HCl, I jThe composition of Stone’s buffer is shown in Table II, Itj will be noted that the Ca++ content of Stone’s buffer is j high enough to result in the precipitation of calcium acid J phosphate. In use, this sediment was not disturbed, and J i only the supernatant solution was used, i i Washing Procedure, i I : I General considerations, Endamoeba histolytica, in j culture, exists in the sediment at the bottom of the slants, ! Here, it is intimately mixed with residual rice starch, the : flora, and a mucilaginous substance elaborated by either \ the accompanying flora, the amebae, or both. The flora are distributed throughout the overlay fluid, and large numbers of them are adsorbed on both the starch residue and the i amebae of the sediment. Ideally one would wish to wash the amebae free of all these factors. However, since the mass of the ameba is very close to that of a rice starch parti cle, washing with aqueous salt solutions does not com pletely separate the amebae from either the rice starch residue or the bacteria adsorbed thereon. Initial attempts to devise a washing procedure. A number of washing procedures based on possible differences TABIE II COMPOSITION OP STONE’S BUFFER Compound Grams Molarity CaCla 0,2 0.0018 KCl 0.2 0.0027 ' MgCls 0.01 0.0001 NaHC03 0,4 0.0048 NaCl o . CO 0.1368, NaaHP04 2.0 0.0132 kh2pg* 0,3 0.0022 Final volume equals 1 liter. Final pH was 7.8. in the mass of the ameba and the starch particles was at- ! I I tempted. Saturated solutions of agar, gum arabie, gelatin, j viscarin, and sodium alginate were made, and their ability ! to float particles of rice starch observed. It was found ! that approximately three-fourths of the starch particles would float in the sodium alginate, while in the other ; solutions virtually all the atarch settled to the bottom V ( of the tubes. Taking advantage of the high viscosity of tbe ' alginate, sediments of cultures were mixed with saturated alginate solutions or overlaid on top of such solutions, ' The solutions were then allowed to come to equilibrium with gravity, at which time it was found that the starch, as ; ' I : well as the amebae, was distributed throughout the solu- » tions. Evidently, In culture, many of the starch particles were broken up with the result that a wide range of parti cle sizes was formed, Slow-speed centrifugation of sedi- I , ment-alginate suspensions resulted in the sedimentation of most of the starch. However, the amebae were now dis tributed throughout the centrifuge tubes. Attempts to concentrate such amebae either by dilution of the alginate solution and slow-speed centrifugation, or centrifugation of the supernatant alginate solutions at high speeds, re sulted in a great loss of amebae due to lysis and manipula tion, Next, various concentrations of alginate solutions were layered upon one another and the sediment layered on ! j ' 34 top of these. Such tubes were then centrifuged at dif ferent speeds in an effort to concentrate the amebae in a ' narrow band of alginate solution, while most of the bac- i teria floated above and the starch settled to the bottom, i * Unfortunately, the amebae are unable to tolerate passage through an interface. They were either fragmented or j appeared dumbbell-shaped and distorted. Since separation of the starch residue from the amebae seemed to offer little immediate promise, emphasis , was shifted to a washing procedure that would remove most of the bacteria, and at the same time leave as many intact, and morphologically sound, amebae as possible. Pinal washing procedure. In early experiments Stone*s buffer was used as the washing fluid. In later experiments it was found that Stone’s salts gave essen tially the same results without the formation of the troublesome precipitate of calcium acid phosphate present in the complete stone’s buffer. A preliminary study of the relationship between number of washings and removal of bacteria and amebic lysis was made. It was found that after the third wash the amount of flora removed became small and almost constant, and that after the fifth or sixth wash there was a sudden acceleration of amebic lysis. Thus the ideal number of washes lay at the point where the bacteria removed per ad- j ditional wash was low and the amount of amebic lysis was | i also low. Figure 1 shows these relationships in the typi- I cal wash curve. Although initial numbers vary, the generalj shape of the curve is quite constant from run to run. It I was also observed that the smaller the volume of wash fluid,j the less lysis of amebae. With all these factors in mind, l I the following procedure was finally worked out, 1 i The sediments of groups of eight or sixteen culture tubes, depending on the number of amebae required, were pipetted into four clean culture tubes, mixed thoroughly by j 1 pipette and centrifuged at 500 SPM for 5 minutes. The J supernates of these tubes were discarded and 5 ml, of the \ washing fluid added to each tube, mixed, and centrifuged as before. This wash was repeated once more. The sediments of the four tubes were then pooled into a 50 ml., conical centrifuge tube, using 3 ml, of wash fluid. An additional 2 ml. of wash fluid were used to rinse out the four t est tubes. The conical tube was spun as above and the super- nate discarded. The sediment was brought up to whatever Volume would be required by the assays being done, mixed, and centrifuged as before. The final supernate then served as a flora control. The sediment, containing the amebae, was also brought up to volume, and this suspension comprised the amebic enzyme preparation used in subsequent ; 2 , 1 0 0 * 60 1 . 0 ■< 40- uj < 0 . 6 « 20- 0. 4 n - oc LU 0. 2$? Z3 ~ Z L NUMBER OF WASHES FIGURE I THE RELATIONSHIP BETWEEN NUMBER OF WASHES AND FLORA REMOVED AND AMEBAE DESTROYED. A IS AMEBAE. B IS FLORA. experiments. In this way the amebae were washed a total of four times. In the case where fructose 1,6-diphosphate was used as a substrate, a slight modification in wash procedure was j i introduced in experiments 4-7 for whole cells and in all | lysed cell experiments. Since positive activity was noted J i with this substrate, the need for a flora control with ! i flora numbers equal to or greater than those in the amebae , i t sediments was required. It was found that if the first and I 'second wash were pooled and centrifuged at 1,500 RPM for 10 i iminutes, the resulting sediment gave the desired flora counts when brought up to volume, since the supernate of the last wash was no longer required, this last wash was eliminated so that the total number of washes for both pro cedures was the same. This washing procedure was also used for the experiments where soluble starch or glucose in the presence of adenosine triphosphate served as substrates, Amebae whole-cell suspensions were rarely incubated for periods longer than 1 hour, since beyond this point autolysis occurred. Lysis Procedures. In some experiments lysed amebae rather than whole cells were used. The ehoice of the lysis procedures was based on the desire to-obtain maximum amebic lysis with little or no lysis of accompanying flora, Tb.us, acetone I ;powders, homogenization and trituration, all of which are |known to lyse bacteria, could not be used. Two different ! !procedures of a sufficiently mild nature were finally used, llhe first was simply the incubation of amebae suspensions 1 at 41°c. for 90 minutes with shaking at 10-minute intervals. i !The second procedure was the immersion of amebic suspen- t sions in a dry-ice solvent bath for 1 minute followed by a ' thaw at 37°c. for 1 minute, Lysed preparations were never incubated longer than 3 hours in order to avoid bacterial attack of the amebic proteins• Counting Procedures, Flora were counted using a water dilution plate count technique (117), Plates were made of nutrient agar and incubated for 48 hours at 37°c., at which time colonies were counted. The amebae were counted using an improved leubauer hemocytometer by the technique of Paulson (118), III. ASSAY PROCEDURE General considerations. The assay procedure used in all these studies is essentially that of Kun and Abood (8) with some modifica tions designed to accommodate the system being used. These workers found that 2,3,5-triphenyl tetrazolium chloride i (TTC) would accept the hydrogens released in the conversion; I j of succinate to fumarate under the aegis of succinic de hydrogenase. The reduced TTC formed a red, insoluble pre- ■cipitate (formazan) which could be quantitatively extracted I jwith acetone, and such formazan-acetone solutions obeyed I ! I Beer’s laws with respect to concentration of reduced tetra- : i jzolium vs, optical density. Later workers (119, 120) sug gested that the oxidation-reduction potential of the TTC- formazan system (Eo^ = -0.08 v) (121, 122), was such that a large group of diphosphopyridine nucleotide (DPU)-requiring enzymes would be oxidized in the presence of TTC, with the formation of the red formazan of reduced TTC. Such an explanation, though plausible in the case of a large number of dehydrogenases whose oxidation-reduction potentials lie considerably below that of the tetrazolium system (120), has been disputed on the grounds that isolated enzymes in the presence of DPH often lack the ability to reduce TTC (123, 124), Kuhn (125) has stated that the action of TTC was upon a flavoprotein, and Brodie and Gots (126) have offered convincing evidence that TTC reduction is really due to a coupled reaction whereby a flavoprotein, in this case diaphorase, will quantitatively reduce TTC with simul taneous oxidation of a molar equivalent of reduced DPH. The advantages of the tetrazolium method for the detection of dehydrogenases are: J a, it requires simple equipment and a minimum of manipulation, an important factor in working with pathogens, b. it does not require anaerobiosis since the for- j rnation of an insoluble end product prevents reversible oxi-j dation by molecular oxygen.ia/ j The disadvantages of the tetrazolium method are: j a. the formazan is adsorbed by the tissues and I • i i starch residue and is very difficult to extract quantita- j i tively, b. the method is less sensitive than some spectro- ' photometric and manometric techniques. I f Procedure. ! Stock solutions. All solutions were made up fresh daily except for the wash solution, which was made weekly; 1 stock solutions of 0,1 M dibasic sodium phosphate and mono basic potassium phosphate, which were kept no more than 1 : month; and stable substrates, which were kept up to 4 days. I All reagents were obtained from commercial sources and were: Several authors (119, 123) claim a partial de crease in TTC reduction in the presence of 0S. They sug gest this is due to competition for the hydrogens of the reduced substrate between the molecular oxygen and the TTC. 41 of analytical grade unless otherwise stated. Solutions were stored in the cold until used. Only deionized water, the equivalent of triply distilled water, was used. All glassware except the actual culture tubes were cleaned by soaking overnight in sulfuric acid-bichromate solution and then rinsed at least four times in distilled water after the cleaning solution had been removed with tap water. Buffer solutions were made by titrating 0.1 M Na2HP04 with 0,1 M KH8P04, until the desired pH had been reached, with a Beckman Model G pH meter. The constituents of other solutions will be de scribed as they occur in the experimental section. Preparation and incubation. All incubations were carried out in 15 ml. conical centrifuge tubes in a water bath at 37 t 0,5°C. All reagents were added to the assay tubes and allowed several minutes to equilibrate with respect to temperature, at which time the enzyme suspen sions were added and the timing of the assays begun. All tubes were brought to a final volume of 3 ml. Two types of blanks were run. One was a blank to show endogenous tetra- zolium reduction. This varied from the exogenous activity tubes only in the substitution of an equal volume of water or buffer for the substrate solution. The second blank was identical to the exogenous assay tubes except that it was 42 inactivated at zero time. Such a blank was compared with an endogenous blank inactivated at zero time to see whether ;he substrate contributed to the optical density of the assay tubes. For each substrate tested the concentrated flora, as taken from the overlays of growing cultures, was used as a positive control. In the case of such concentrated floras, no attempt was made to correct for starch adsorption. Therefore, all values listed for the concentrated flora, although valid in a relative sense, are not valid in an absolute sense. Extraction of formazan. Assays were stopped by the rapid addition of 5 ml, of cold acetone and immediately shaking the tubes, using a rubber finger cot to cap the tubes. This served a threefold purpose: inactivating the enzyme, denaturing and precipitating protein, and extract ing the formazan. The tubes were then sealed with parafilm- covered cork stoppers and centrifuged at 1,500 RPM for 20 minutes in a refrigerated centrifuge at 0-5°C, After centrifugation, 6 ml, aliquots were removed from the assay tubes and pipetted into Klett colorimeter tubes and stop pered with parafilm. Color density was read with a Klett- Summerson colorimeter using a blue filter Humber 42 (8), A green filter Number 54 gave parallel but lower results (127)* A1& assays were rim in duplicate and the results of both tubes averaged. Standard curve, A standard curve was constructed using a freshly prepared stock solution containing 300 X of TTC/ml, of 0,1 M phosphate buffer at pH 7*.4, Bach tube contained 1,5 ml, of a serial dilution of the stock TTC prepared with buffer, plus 1,5 ml, of water. Each dilution was run in duplicate. The TTC was then reduced with sodium hydrosulfite and treated with acetone in the exact manner ! described for regular assay tubes. Color density was read as described in the assay procedure. This procedure was repeated three times using freshly prepared TTC standard, A standard curve (Figure 2) relating of TTC reduced to Klett values was constructed by averaging the results of the three standard assays. 44 ✓ ✓ 70- 100- 80- 60- 40- Y TRIPHENYL TETRAZOLIUM CHLORIDE FIGURE 2 STANDARD CURVE FOR THE REDUCTION OF TRIPHENY1 TETRAZOLIUM CHLORIDE CHAPTER IV EXPERIMENTAL AND RESULTS I. SUCCINIC DEHYDROGENASE General. Succinic dehydrogenase catalyzes the conversion of succinate to fumarate (128), Although it requires neither coenzyme I nor II, it has been suggested that it can be j linked to the diaphorase system through an unidentified | j substance termed the "Slater factor” (119), In this way i the reaction of any substance that will accept hydrogen j I from the diaphorase system can be used to indicate enzy- | i matic activity, Kun and Abood (8) were able to demonstrate the presence of this enzyme in tissues by the use of the tri phenyl tetrazolium technique. Since Seaman (114) had stated that this enzyme was i ; present in the amebae, and since its presence in the amebae 'is at variance with the glycolytic and anaerobic nature of j ■ this organism, this was the first enzyme studied. j j Whole Cells. Amebae were washed as described under "Methods," Stbne's buffer was used as the washing fluid except for the i last wash, where stoned salts was used. Assays were run J at two pH levels, and in one group of experiments, even though DPF has been reported to inhibit activity (130), it i j was added on the chance that the amebic enzyme might show a I ! I ! unique requirement for this substance* j , In the first group of experiments, assay tubes con- I i tained the following: , i 1 ml. of an amebic or floral suspension, j 1 ml. of a 1 mg./ml. solution of TTC in Buffer, j 1 ml. of a 0.02 M solution of sodium succinate in ! buffer for exogenous activity or 1 ml. of buffer for endogenous activity. The buffer used was 0,1 Id phosphate ! |at pH 7.2. I | The results of these experiments are shown in Table ! i i III. Prom Tabie III, It can be seen that there is ap parently no dehydrogenase activity present in the amebae, and also that the activity of the concentrated flora is much higher, per unit cell, than that for the flora ! intimately associated with the amebae. i In the second group of experiments, assay tubes con-! tained the following: ml, of amebic or floral suspension, 1 ml. of a 2 mg./ml. solution of TTC in buffer, il ml, of 0.2 M solution of sodium succinate in water for exogenous activity or a ml. of water for endogenous activity. TABLE III TRIPHENYL TETRAZOLIUM CHLORIDE REDUCTION BY SUCCINIC DEHYDROGENASE AT pH 7.2 Exp, Amebae!/ Flora control!/ Concentrated flora N°* Amebae/ Flora/ t f TTC Flora/ * TTC Flora/ Time V TTC Unit£/ tube (xlO®) tube (xlO®) reduced tube reduced (xlO®) tube (xlO6) (min.) reduced activity 1 1.5 48 0 i i i j o i i 1 i i ! 3 1 H 1 1 2 1.4 24 0 7 0 3 1.3 99 4 31 0 600 30 64 3.6 4 0,8 134 12 10 0 400 30 58 3.8 Assay tubes contained; 2 x 10“5 moles of sodium succinate, 1 mg, of TTC, 2 x 10“4 moles of phosphate. Total volume was 3 ml. Tubes were incubated at 37°C. Incubated for 60 minutes, 2/ unit activity equals % TTC reduced/100 x 10® bacteria/10 minutes. ; ...... " 47 i | ^ ml. of 0,1 M phosphate buffer at pH 7.4, and 1 i | al, of a 2 mg./ml. solution of DPN in water or j g ml. of water only. The results of these experiments are shown in Table I ; IV. They are in essential agreement with Table III in that ! : there is apparently no amebic activity shown, and that the I !concentrated flora again have greater activity than do the l flora associated with the amebae. It can be seen that the presence of DPH in the system had no effect on the activity. | ,Lysed cells. Amebae cells were washed as previously described for whole cells, except that the washing fluid was Stone*s salts made 0.001 M with respect to cysteine. Both heat and freeze-thaw lysis procedures were used. In Experiment 1, lysates were prepared by freeze- thawing the washed amebae three times. After lysis the suspension was centrifuged for 5 minutes at 1,500 RFM. Assays were run on the supernate and on the sediment brought up to the initial volume. Assay tubes were pre pared as described’ for whole cells at pH 7,2, In Experiment 2, lysates were prepared by freeze- thawing the washed amebae one time. An assay was done on the total lysate only. Assay tubes were set up as described for whole cells at pH 7,4, TABLE IV TRIPHENYL TETRAZOLIUM CHLORIDE REDUCTION BY SUCCINIC DEHYDROGENASE AT pH 7,4 Exp. No. Amebaei/ Flora controli/ Concentrated flora Amebae /tube (xl0 6) Flora TTC /tube reduced (xlO*>) No DPN DPN Flora /tube (xlO6) t f TTC reduced No DPN DPN Flora / tube (xlO6) Time (min,) # TTC reduced No DPN DPN Unit activity2/ No DPN DPN 1 0.8 48 0 0 9 0 0 2 0.7 2.5 0 0 9 0 0 100 10 33 34 33 34 3 0.7 400 37 38 102 0 0 100 10 26 26 26 26 Assay tubes contained: 1 x 10~4 moles of sodium succinate, 2 mg, of TTC, 1.5 x 10“6 moles of DPN, 1.5 x 10~4 moles of phosphate. Total volume was 3 ml. Tubes were incubated at 37°C, 1/ incubated for 60 minutes, 2/ unit activity equals TTC reduced/100 x 106 bacteria/10 minutes. 49 In Experiment 3, heat lysis was used and the lysate was again separated into a sediment and supernatant frac tion, In this case separation of sediment and supernate was achieved by centrifugation at 500 RFM for 5 minutes. Assay tubes were prepared as described for whole cells at pH 7.4, | The results of these experiments can be seen in j i Table V. Prom the data in Table V, it is evident that I there is no amebic reduction of tetrazolium due to succinate by lysed amebae cells, ! i I II. MALIC DEHYDROGENASE i I i ; ; General, i j i I The apparent absence of a succinic dehydrogenase in j E. histolytica seemed to indicate the lack of an aerobic metabolism embodying the classical reactions of the tri carboxylic acid cycle. A number of workers (103, 111) have :suggested that E. histolytica is purely glycolytic in jnature. As a further test of this hypothesis, experiments l !designed to show the presence or absence of malic dehydro- i genase were run. Malic dehydrogenase catalyzes the oxidation of 1- • rnalate to oxaloacetate by DPN (131). Its presence in tissues has been demonstrated by several workers (119, 120), using the tetrazolium technique, l i -. J TAB IE V i TRIPHENYL TETRAZOLIUM CHLORIDE REDUCTION BY SUCCINIC DEHYDROGENASE WITH LYSED CELLS ' i Before lysis After lysis • CiXpSP « No. i Amebae Flora/ Amebae Flora Flora % Time V TTC reduced /tube (xl0 6) tube (xlO6) /tube (xlO®) sed. (xl0 6> sup, (xl0 6) amebic lysis (min.) Sus- Super. Sed. pension iiA®/ 0.9 27 0.005 2.51/ — 94 60 0 0 gi/,S/ 1.6 a * , m * 0 0,25^/ — >99 180 0 ! s5/.6/ 1.2 12 0.08 5.5 9 93 18© 0 0 Total volume was 3 ml. J : j Tubes were incubated at 37°C. I l i Pinal pH was 7.4, 1/ Amebae freeze-thawed 3 times. ! I 2/ por contents of assay tubes see legend for Table III. ! 3/ Flora count in total lysed suspension. j 4/ Amebae freeze-thawed 1 time, j 5/ For contents of assay tubes see legend for Table IV. j i 6/ Amebae heat-lysed at 41°C. for 90 minutes. * © Whole Cells, j . i Amebae were washed using stone’s buffer as the wash- j ing fluid except for the last wash, where stone’s salts was ; iused. Assays were done on the amebae and a flora control. The concentrated flora was used as a positive control. Assay tubes were identical to those used in whole cell as- j says for succinic dehydrogenase at pH 7,4 except that 0,2 M j I I sodium malate was substituted for the sodium succinate, j i The sodium malate was obtained by dissolving 1-malic acid I in approximately 90 per cent of its final intended volume, and then titrating to pH 7,4 with dilute sodium hydroxide, !after which the' solution was brought up to its final volume.; j ! I Results of these experiments are shown in Table VI* j i I Prom Table VI, it can be seen that, as in the case of suc cinic dehydrogenase, the amebae have no activity and the flora of supernates of amebic cultures has more activity, per unit cell, than does the flora intimately associated with the amebae. III. IACTIC DEHTDROGENASE j . General. With the apparent absence of aerobic activity in E. histolytica indicated by the lack of succinic and malic ; dehydrogenase activity, it was decided to investigate some anaerobic dehydrogenases• TAB IE VI TRIPHENYL TETRAZOLIUM CHLORIDE REDUCTION BY MALIC DEHYDROGENASE Amebaei/ Flora controli/ Concentrated flora Amebae Flora X TTC Flora % TTC Flora Time V TTC Unit ~ ~ ~ /tube /tube reduced /tube reduced /tube (min.) reduced activity^/ (xlO6) (xl06> jjo (xl0 6) no (xlO6) No Wo DPN DPN DPN DPN DPN DPN DPN DPN 1 0.7 17 0 0 3,5 0 0 250 10 6 20 2.4 8.0 2 0.5 10 0 0 4,0 0 0 200 10 5 17 2.5 8.5 3 0.6 324 15 18 40.0 0 0 4 0.7 15 0 0 3.0 0 0 Assay tubes contained: 1 x 10'* moles of sodium malate, 2 mg. of TTC, 1.5 x 10“6 mdes of DPN, 1.5 x 10“4 moles of phosphate. Total volume was 3 ml. Tubes were incubated at 37°C. Final pH was 7.4, 1/ Incubated for 60 minutes, 2/ xjnit activity equals TTC reduced/100 x 10® bacteria/10 minutes. . _ _____ 53 Lactic dehydrogenase catalyzes the oxidation of lactate to pyruvate by DPH (132). It has been demonstrated in tissues by several workers (119, 133) by the tetrazolium technique. i i 'Whole Cells. •1 " l - " * ’ " ■ 1 Amebae were washed as previously described. Stone’s buffer was used as the washing fluid except for the last j wash, where Stone’s salts was used. Assay tubes were i I ; ■ (identical to those used for succinic dehydrogenase whole i I ! jcells at pH 7.4, A 0.2 M sodium lactate solution was sub stituted for the sodium succinate. Concentrated flora was i i I used as a positive control, j Prom Table VII it can be seen that the amebae show 1 no lactic dehydrogenase activity. The concentrated flora shows good activity and is considerably more active, per unit cell, than is the flora associated with the amebae. i Lysed Cells. I Amebae were washed as usual. The washing fluid in Experiment 1 was stone’s buffer 0,001 M with respect to cysteine except for the last wash which was done with Stone’s salts 0.001 M with respect to cysteine. In Experiments 2 and 3 the washing fluid, throughout, was Stone’s salts with 0,001 M cysteine, Amebae were lysed either by freeze- thawing or heat lysis at 41°C. Sediments were separated TABLE VII TRIPHENYL TETRAZOLIUM CHLORIDE REDUCTION BY LACTIC DEHYDROGENASE Amebaei/ Flora controli/ Concentrated flora Exp. No. Amebae /tube (xl0 6) Flora /tube (x1Q6) * TTC reduced No ------ DPN DPN Flora /tube (xl0 6) * TTC reduced No DPN DPN Flora /tube (xlO6) Time (min,) if TTC reduced No DPN DPN activity?/ No DPN DPN 1 1 50 0 0 22 0 0 2 0.7 115 6 6 2 0 0 150 60 160 248 17.8 27.6 3 0,9 115 6 6 23 0 0 4 0.9 45 0 0 11 0 0 275 10 20 45 54 86 — 16.4 19.6 16.6 — Assay tubes contained: 1 x 10-4 moles of sodium lactate, 2 mg. of TTC, 1.5 x 10-6 moles of DPN, 1.5 x 10-4 moles of phosphate. Total volume was 5 ml. Tubes were incubated at 37°C. Pinal pH was 7,4, 1/ Incubated for 60 minutes. j?/ Unit activity equals s TTC reduced/100 x 106 bacteria/10 minutes. ___ _ . . 55j from the lysis supernatants by centrifugation at 500 RPM for! 5 minutes. The assay tubes were set up as described for I I whole cell experiments, J Results of these experiments are shown in Table VIII , j Prom the data in Table VIII it can be seen that there is no J i amebic laetie dehydrogenase activity evident, j IV. TRIOSEPHQSPHA.TE DEHXDROGEMSE Fructose 1,6-Diphosphate as the Substrate. General, The inability to demonstrate a lactic de hydrogenase in E. histolytica, coupled with the apparent : absence of aerobic metabolism, suggested that its metabo- i . ilism might be entirely unique, bearing little resemblance to usually accepted metabolic schemes. To further test this conclusion, it was desired to assay the amebae for the ipresence of the triose dehydrogenase system, i Triosephosphate dehydrogenase (phosphoglyceraldehyde dehydrogenase) catalyzes the conversion of 3-glyceraldehyde i • • ! phosphate, DPN and inorganic phosphate to 1,3-diphospho- j glyceric acid and reduced DPN (134, 135), It has been demonstrated with the tetrazolium technique by Jensen et al. (12Q)• Since 3-glyceraldehyde phosphate was not available, ifructose 1,6-diphosphate was Used as the substrate. This j TAB IE VIII t TRI PHENYL TETRAZOLIUM CHLORIDE REDUCTION BY LACTIC DEHYDROGENASE WITH LYSED CELLS Before lysis After lysis i i Exper. No, Amebae /tube (xl0 6) Flora/ Amebae ] / tube (xlO6) Flora Flora % Time y rpipC reduced tube (xl0 6) sed. (xl0 6) sup. (xlO®) amebic lysis (min.) Sus- Super, Sed.j pension & 1.3 50.0 0.1 35 15 92.2 120 4 0 0 2i/ 1.0 3.5 0.15 1.8 2.8 85.0 180 §S/ 0 0 0 0 85/ 1.4 15.0 0.01 0.5 1.8 99 180 0 0 0 For the contents of assay tubes in Experiment 1, see the legend of Table III. Table For the IV. contents of assay tubes in Experiments 2 and 3, see the legend of Total volume was 3 ml* Tubes were Incubated at 37°C« Final pH was 7,4* Lysed by incubation at 41°C. for 90 minutes with shaking at 10 minute Intervals. 2/ Lysed in 0,2 M lactic acid instead of stone*s salts plus 0.001 M cysteine hydrochloride. 3/ Lysed by freeze-thawing 3 times. " " ~ 57* was prepared from the dicalcium salt in the following manner* A weighed amount of the calcium hexose diphosphate j was dissolved in a minimum of cold water, in an ice bath, by acidifying with a few drops of a dilute solution of HCl* |The volume of this solution was approximately one-half the ! ! i final desired volume. This solution was then chromato graphed, in the cold, on a column of Nalcite HCR (Dowex-50), in either the sodium or potassium form. The dimensions of the column were 10 x 120 mm. and the flow rate was 2 drops per minute. The eluate from the column was collected directly into a volumetric flask and the column was washed with water until the volumetric flask was filled to the mark. Whole cells, Amebae were washed as described in the "Experimental" section. In Experiments 1-3 the washing fluid was stone*s salts with 0,001 M cysteine. In Experi ments 4-7 the washing fluid was Stone's salts with 0.001 M cysteine except that the final sediment was brought up to volume with 0.1 M phosphate buffer (pH 7,4) which was 0,003 I M with respect to cysteine (details of this alternate wash ing procedure are described in chapter III). In Experi ments 4-7 flora controls were obtained by pooling the supernates of three tubes from the third wash, spinning this at 1,500 RHJ for 10 minutes, discarding the supernate, . . . , - - _ _ _. 5 8 - and bringing the sedimented flora to volume with 0,1 M phosphate buffer at pH 7,4 and 0,003 M with respect to J cysteine. It was found that such a flora control usually i contained slightly more flora than was present in the ameba > I sediment, i For Experiments 1-3 the assay tubes were set up as j for succinic dehydrogenase at pH 7.4, except that a solu- i i tion of 0.2 M sodium hexose diphosphate was used instead of; i |succinate, in Experiments 4 and 5 the substrate was 0.003 ; M and in Experiments 6 and 7 it was 0.06 M with respect to l the hexose. In Experiments 4-7, all reagents except the : i |substrate were made up in phosphate buffer with 0.003 M ; i . . . . , I i ! cysteine. In Experiments 4-7 where iodoaeetic acid was ! used, 0,08 ml. of a 0.0077 M solution of iodoaeetic acid (IAA) in water was added to the assay tubes, bringing the final concentration of IAA to 0,0002 M. Except for Experi ment 4, where the IAA was used as supplied by the manu- ; facturer, all IAA was recrystallized i ' The results of these assays can be seen in Table IX. ; From Table IX, it can be seen that the amebae show an ap- i ‘ preciable reduction of tetrazolium and also that this re duction is inhibited 80 per cent or more by 6.16 x lO’ "^ 3/ Obtained through the courtesy of Dr. J, L, Webb, TABLE IX TRIPHENYL TETRAZOLIUM CHLORIDE REDUCTION WITH FRUCTOSE 1,6-DIPHOSPHATE AS THE SUBSTRATE Amebae Flora control Exp. Time Amebae/ Flora/ /TTC reduced Unit!/ Per cent Flora/ /TTC | No. (min,) tube tube No activity inhibition tube reduced ' (xl0 6) (xl0 6) IAA IAA (xl0 6) 1 32/ 70 1 20 6 M mm 5.1 2 0 22/ 46 1.7 26 6 -- 4.6 __ 15 0 90 1,5 16 9 — 4.0 — 17 0 43/ 60 1.2 3.5 9 2 7.5 78 4.5 0 1 53/ 60 1.0 3.3 6 0 6.0 100 7 0 61/ 60 1.7 4.5 7 0 4.1 100 7.5 0 7i/ 60 2.0 9.7 20 4 10,0 80 13.7 0 All assay tubes contained: 2 mg. of TTC and 1.5 x 10“6 moles of DPN. Experiments 1-3 contained 1.5 x 10“4 moles of phosphate. Experiments 4-7 contained 2.4 x 10-4 moles of phosphate. The concentration of iodoacetate was 6.16 x lO”? moles/tube. Tubes were incubated at 37°C. Final volume was 3 ml. Final pH was 7.4. 1/ Unit activity equals / TTC redueed/l x 106 amebae/60 minutes, 2/ substrate equals 1 x 10“4 moles of fructose 1,6-diphosphate (sodium salt).! 3/ Substrate equals 1.5 x 10“6 moles of fructose 1,6-diphosphate (potassium salt). 4/ Substrate equals 3 x 10“6 moles of fructose 1,6-diphosphate (sodium salt). . _ . _ ~ g g j moles of IAA, thus suggesting the presence of an active triosephopphate dehydrogenase. i Lysed cells. Amebae were washed as described in J I Experiments 4-7 for whole cells. Lysis was achieved by I jincubation at 41°C. for 90 minutes. The contents of assay j tubes were the same as described for whole cell Experiments ! 6 and 7* j j Results of these assays can be seen in Table X. J | Prom Table X it can be seen that although amebic reduction j . of the tetrazolium is still detectable its magnitude was greatly reduced when compared to whole amebae cells. i Soluble Starch as the Substrate. ! General, since triosephosphate dehydrogenase ac tivity could be demonstrated in the amebae, the question of what substances could act as a precursor to hexose diphos phate formation was investigated using tetrazolium reduc tion at the triose level as an indicator. Since rice starch is an accepted nutrient for E. histolytica its role : as a precursor to the hexose was studied. Whole cells. Amebae were washed and flora controls obtained, in the manner described for hexose diphosphate whole cell Experiments 4-7, Assay tubes were identical in content to those used for the hexose diphosphate whole cellj TABLE X TRIPHENYL TETRAZOLIUM CHLORIDE'REDUCTION BY USING FRUCTOSE 1,6-DIPHOSPHATE AS THE SUBSTRATE WITH LYSED CELLS Whole cells Heat-lysed cells Amebae/ Flora/ TTC reduced Amebae/ I Flora/ Time TTC reduced W ""VVV 1 ■Va ' a ^ n___4- _ /__* „ \ ■ ■ ■ ■ 1 I tube. (xlO6) tube (xlO®) No IAA iaA, tube (xlO®) lysis tube (xlO6) (min.) No IAA IAA 1 ! 1 1.3 1.8 — mm mm 0.09 95 13.0 180 29 0 2 i 1,3 32.8 10 0 0.2 85 31.8 60 2 0 ! ; 3 1.9 9,8 — — 0,3 95 10.0 60 6 0 t ; 4 2.5 14.5 mm mm 0.09 99.4 14.5 60 6 0 Assay tubes contained: 2 mg. of TTC, 1.5 x 10-6 moles of DPN, 2.4 x 10-4 moles of phosphate, 5 x 10-5 moles of fructose 1,6-diphosphate (sodium salt). The concentration of iodoacetate was 6,16 x 10-7 moles/tube. Tubes were incubated at 37°G. Final volume was 3 ml. Final pH was 7,4. 62 Experiments 4-7, except that a solution of 2 mg. of soluble starch^/ per ml. of water was used as the substrate and the disodium salt of adenosine triphosphate (ATP) was incor- j porated into the DPN stock solution. In Experiment 1, the stock solution contained 24 mg, of ATP/ml. and in Experi ments 2-4, the stock solution contained 12 mg. of ATP/ml. j The results of these assays are shown in Table XI. J From Table XI, it can be seen that the amebae show definite j i reduction of TTC with soluble starch as substrate and that J i such reduction is greatly inhibited at low concentrations j of iodoacetate. i glucose as the Substrate. i general. There have been a number of reports of glucose utilization by E, histolytica. However, no evi- : dence of the mechanism of utilization has been reported, i i : If a glucokinase (136) were present in the amebae, then its i j presence could be inferred if glucose plus ATP resulted in , I TTC reduction that could be inhibited at the triose de- ; : . j hydrogenase level, with these factors in mind a study of ! glucose utilization by E. histolytica was done. Whole cells. The washing procedure used was identi cal to that used when soluble starch served as the l ! iJ Obtained through the courtesy of J, B. Miehaelsonj TABLE XI TRIFHENYL TETRAZOLIUM CHLORIDE REDUCTION WITH SOLUBLE STARCH AS A SUBSTRATE Exp, No, i Time (mins.) Flora control Amebae 1 Flora/ tube (xl06) *TTC reduced Amebae /tube (xlO®) Flora/ tube (xlO®) YTTC reduced Uo IAA IAA % inhi bition Ameb,- . flora-/ Unit p .; activity—/ i i j 1 30 21 3 1.9 53 13 — 5.5 5.8 2 40 18 3 1.25 31 9 3 66.7 5.0 6.0 | 3 30 20 2 1,65 40 9 1 88.9 5.0 6.1 ; ,4 40 34 4 1,6 5 7 2 71.4 7.0 6.5 ; 5 30 30 3 1,0 40 6 1 83.3 2.0 4.0 Assay tubes contained: 2 mg. of TTG, j 1,5 x 10~6 moles of DPN, 1 1 mg, of soluble starch, j 2 x 10*"4 moles of phosphate, j Experiment 1 contained 1,9 x 10“5 moles of ATP, ! Experiments 2-5 contained 9,6 x 10*“® moles of ATP, [ The concentration of iodoacetate was 6,16 x 10”7 moles/tube. Tubes were incubated at 37°C. Pinal volume was 3 ml, Pinal pH was 7,4, j 1/ This value represents amebic activity, in terms of ^TTC, that has been ! corrected for flora activity. I ! I [ 2/ unit activity equals ^TTC reduced/l x 10® amebae/60 minutes. o>j 64 substrate* Assay tubes were identical to those used in Experiments 2-5 for soluble starch, except that a stock solution of 0.2 M glucose in water was used as the sub strate. Results of these assays may be seen in Table XII. Prom Table XII, it can be seen that there is considerable reduction of TTG in this system, and that this reduction is i again inhibited by low concentrations of iodoaeetate, j i Lysed cells. Cells were washed with Stone’s salts made 0,001 M with respect to cysteine. Lysis was achieved ! ! by both freeze-thawing and heating at 41°c. i i j Assay tubes were set up as described for whole cells ! except that the DPH solution contained 24 mg. ATP/ml. Amebae were lysed by both freeze-thaw and heat lysis meth ods. Results of these assays are shown in Table XIII. ; These data show no tetrazolium reduction by the amebae, in- i dicating that the enzyme systems involved are quite sensi tive to the lytic procedures employed. j . V. GLUCOSE DEHYDR06EM.SE ' ' 3 General. Since triosephosphate dehydrogenase activity was indicated by reduction, using fructose 1,6-diphosphate, glucose and soluble starch as substrates, the question of TABLE XII TRIPHENYL TETRAZOLIUM CHLORIDE REDUCTION WITH GLUCOSE AS A SUBSTRATE Flora control Amebae Exp. No. Time (min,) Flora/ tube (xlO®) J f TTC reduced Amebae /tube (xl0 6) Flora/ tube (xlQS) ^TTC reduced No IAA IAA inhi bition Ameb. n / floray activity^/ 1 30 14 2 1.6 8 7 — 6 7.5 2 40 1 0 1.3 12 5 0 100 3 3.45 3 30 28,5 4 1.2 75 14 5 64,3 3,6 6.0 4 30 15 3 1.5 15 10 2 80 7 9,3 Assay tubes contained: 2 mg, of TTC, 1.5 x 10“6 moles of DPN, 1 x 10”4 moles of glucose, 2,4 x 10“4 moles of phosphate, 9.6 x 10“6 moles of ATP. The concentration of iodoacetate was 6.16 x 10”? moles/tube. Tubes were incubated at 37°C. Pinal volume was 3 ml. Pinal pH was 7,4, 1/ This value represents amebic activity, in terms of TTC, that has been corrected for flora activity. 2/ unit activity equals S ' TTC reduced/l x 106 amebae/60 minutes. ^ or ! TAB IE XIII t j TRIPHENYL TETRAZOLIUM CHLORIDE REDUCTION WITH GLUCOSE BY LYSED CELIS 1 I Exp. j No, Before lysis After lysis Amebae/ tube (xlO6) Flora/ tube (xl0 6) Amebae/ tube (xlO ) Flora/ tube (xl0 6) % amebae lysed Time (min.) Y ttc TyseT" " reduced Unlysed ! & 2.1 5 0 2.0 >99 180 0 ... i 2?/ 1 0.9 0.5 0.05 2.5 94 180 0 0 Assay tubes contained: 1 x 10”4 moles glucose, 2 mg. TTC. 1.5 x IQ”® moles DPN, 1,9 x 10“5 moles ATP, 1.5 x 10-4 moles of phosphate. Tubes were incubated at 37°C. Total volume equals 3 ml. Final pH was 7.4, 1/ Freeze-thawed one time. 2/ Heat lysed 90 minutes at 41°C. ^whether or not glucose acts as a substrate for glucose de hydrogenase was investigated. Glucose dehydrogenase i catalyzes the DPN-dependent conversion of glucose to glu- leonate (137). Its activity has been demonstrated, by (several workers (138, 139), using the tetrazolium technique. ! Whole Cells, i ! Amebae were washed as described in the "Methods” Isection. The washing fluid was Stone’s buffer except for the last wash, where Stone’s salts was used. Assays were set up as described for succinic dehydrogenase whole cells at pH 7,4, except that 0.2 M glucose was substituted for the succinate. Results of these assays can be seen in Table XIV. Prom Table XIV, it will be noted that the amebae ex hibit no glucose dehydrogenase activity. The concentrated flora, on the other hand, exhibits tremendous activity, with the tubes containing no DPR being consistently more active than those with DPN. This occurrence may be ex plained by a possible inhibition due to DPN. Nakamura (140), using bovine liver glucose dehydrogenase, has stated that this enzyme is either DPN or TPN dependent. It may be that, in the case of the concentrated flora, we have a TPN- dependent system that is inhibited by the presence of DPN. VI. GLUTAMIC DEHYDROGENASE TABLE XIV TRIPHENYL TETRAZOLIUM CHLORIDE REDUCTION BY GLUCOSE DEHYDROGENASE Amebae.!/ Flora control!/ Concentrated flora Exp, Amebae Flora TTC Flora y TTC Flora Time ^ TTC Unit No, /tube /tube reduced /tube reduced /tube (min,) reduced activity^/ (xlO6) (xlO6) Ho (xlO6) No (xlO6) No Ho DPN DPN DPN DPN DPN DPN DPN DPN 1 0.6 15 0 0 4.5 0 0 150 5 140 108 ^ 186 144 2 0.7 37 0 0 12.0 0 0 125 5 114 87 182 139 3 0.6 5 0 0 2.5 0 0 200 5 193 174 193 174 Assay tubes contained: 1 x 10“4 moles of glucose, 2 mg. of TTC, 1.5 x 10~6 moles of DPN, 1.5 x 10~4 mole3 of phosphate. Total volume was 3 ml, j Tubes were incubated at 37°C. ! i I Final pH was 7,4, i Incubated for 60 minutes, , 2/ unit activity equals i TTC reduced/100 x 106 bacteria/10 minutes. j o>i OOj 69 General. Since triosephosphate dehydrogenase was present in the amebae and lactic dehydrogenase could not be demon strated, a number of dehydrogenases was tested in efforts to explore the means by which the amebae regenerated re duced DPN. Glutamic dehydrogenase (141) catalyzes the con version of L-glutamate to alpha-ketoglutarate by DPN. It was demonstrated, using the tetrazolium technique, by BeBarardlnis (133)• Whole Cells. Amebae were washed, as described in the “Experi mental" section, with Stone's buffer as the washing fluid ! ! except for the last wash where .Stone's salts was used, ! Assay tubes were prepared as described for succinic de hydrogenase whole cells at pH 7.4. The results of these assays can be seen in Table XV. Prom this table, it can be seen that the amebae show no I ‘ t !activity and that the concentrated flora is considerably more active than flora closely associated with the amebae. j ! VII. ALCOHOL DEHXDROGENASE • General. Alcohol dehydrogenase catalyzes the oxidation of ethanol to acetaldehyde by DPN (142). Jensen et al,. (120) ; . . . . . . . . . J TABLE XV TRIPHENYL TETRAZOLIUM CHLORIDE REDUCTION BY GLUTAMIC DEHYDROGENASE Amebaei/ Flora eontroli/ Concentrated flora ,Exp. Amebae Flora YTTC Flora YTTC Flora Time 'V TTC Unit !"°» /tube /tube reduced /tube reduced /tube (min.) reduced activity^/ ; (±106) (xlO®) No (xlO6) No (xlO®) No No------- DPN DPN DPN DPN - DPN DPN DPN DPN 1 0.65 107 10 12 32.5 3 4 350 10 36 43 1D.3 12.3 2 0.8 56 0 0 11.0 0 0 3 0.9 77 1 1 12.0 0 0 300 10 31 36 10, Q 12.0 4 0.65 156 16 34 34.0 3 3 Assay tubes contained: 1 x 10“4 moles of glutamate, 2 mg. of TTC, 1.5 x 10“6 moles of DPN, 1.5 x 10~4 moles of phosphate. Total volume was 3 ml. Tubes were Incubated at 37°C. Final pH was 7.4, 1/ Incubated for 60 minutes, 2/ unit activity equals STTC reduced/100 x 106 bacteria/10 minutes. . . . 71 were able to demonstrate this enzyme msing a tetrazolium technique, Whole Cells, Both washing and assay procedures were identical with those used for the assay of glutamic dehydrogenase. Results of these assays are seen in Table XVI, Prom Table XVI it can be seen that the amebae show no activity, ! ! VIII. BETA-groROXYBOTIRIC ACID DEHTOROGEHASE 1 j i General, | This enzyme catalyzes the conversion of L-beta- j ... - - - - - I Ihydroxybutyric acid to acetoaeetate by DPX (143), Its ac- J : . ' • i i tivity has been demonstrated by use of the tetrazolium ! te chnique (120), Whole Cells, i Amebae were washed and assay tubes set up as ! described for glutamic dehydrogenase, • Results of these assays can be seen in Table XVII, Prom Table XVII it can be seen that the amebae have no activity and that the activity in the concentrated flora is quite low. .J TABLE XVI TRIPHENYL TETRAZOLIUM CHLORIDE REDUCTION BY ALCOHOL DEHYDROGENASE Amebaei/ Flora controll/ Concentrated flora i * Amebae Flora TTC Flora ' t f TTC Flora Time T tTC Unit * /tubg /tube reduced /tube reduced /tube (rain,) reduced activity—/ (xlO6) (xlO6) lo----- (xl0 6) lo------- (xl0 6) lo------ lo------ I I I I DPN DPN DPN DPN DPN DPN DPN DPN 1 0,6 1 0 0 1.5 0 0 275 10 3 3 1.1 1.1 20 49 59 9.0 10.7 2 0.6 1.5 0 0 2.0 0 0 100 20 21 24 10.5 12.0 3 0.7 17.0 0 0 9.0 0 0 Assay tubes contained; 1 x 10**4 moles of ethanol, 2 mg, of TTC, 1.5 x 10“6 moles of DPN, 1.5 x 10-4 moles of phosphate. Total volume was 3 ml, ; Tubes were incubated at 3 7 °c , J Final pH was 7 ,4 , J > 1/ Incubated for 60 minutes, 2/ unit activity equals tfTTC reduced/100 x 106 bacteria/10 minutes, ! " " to, TABLES XVII TRIPHENYL TETRAZOLIUM CHLORIDE REDUCTION BY BETA-HYDROXYBUTYRIC DEHYDROGENASE Amebaai/ Flora controli/ Concentrated flora Exp, No, Amebae /tube (xlO®) Flora /tube (xlO6) S TTC reduced No DPN DPN Flora /tube (xlO6) V TTC reduced No DPN DPN Flora /tube (xlO6) Time (min.) XTTC reduced No DPN DPN Unit „ . activity—/ No DPN DPN 1 0.6 3 0 0 12.5 0 0 100 20 0 20 0 10.0 2 0.9 19 0 0 6 0 0 250 15 0 0 • 0 0 3 1.0 24 0 0 4 0 0 Assay tubes contained: 1 x 10“4 moles of beta-hydroxybutyrate, 2 mg. of TTC, 1*5 x 10~6 moles of DPN, 1.5 x 10“4 moles of phosphate. Total volume was 3 ml. Tubes were incubated at 37°C• Final pH was 7.4, 1/ Incubated for 60 minutes. 2/ Unit activity equals UTTC peduced/100 x 106 bacteria/10 minutes. C BA PTER ? ; DISCUSSION AND CONCECSIONS ! I : I. GENERAL I • t j If the simplest interpretation is made of the re- j suits reported in this dissertation, then a set of definite 1 t conclusions relating to the intermediary carbohydrate me tab-; olism of Endamoeba histolytica may be expounded. Since a ' large part of this work has yielded negative results, a j ' degree of uncertainty concerning these data still exists, ! Thus, one might argue that the amebic enzymes could be in- ; sensitive to TTC, or that variations in such factors as trace minerals, pH, cofactors, and incubation temperatures, to name a few, might have yielded positive results. These criticisms can in part be answered with the following argu ments; first, in the case of some enzyme systems positive amebic activity was found, indicating that at least one flavoprotein capable of donating hydrogen to TTG is present in the ameba, and that all other factors mentioned must have been adequate in this case, and secondly, in all cases of negative amebic activity the concentrated flora could be used as a positive control. Thus, within the framework of the methodology employed, the conclusions drawn from this work may be considered valid. ! 75 I ! II, AEROBIC METABOLISM f I i The fact that no malic or succinic dehydrogenase : activity could be demonstrated for the ameba suggests that ; the usually accepted scheme for oxidative metabolism by way of the tricarboxylic acid cycle is either absent or greatly altered in E. histolytica, such a conclusion would be in line with the evidence that E. histolytica is an obligate anaerobe (41, 76, 90), and that molecular oxygen in minute amounts is toxic to this organism (89, 91), III. AHAEROBIC METABOLISM In the case of anaerobic metabolism, the amebae seem to possess all of the enzymes necessary to metabolize starch or glucose as far as the triosephosphate dehydrogen ase step. Fructose 1,6-diphosphate as a Substrate, When fructose 1,6-diphosphate is used as a substrate, there are a number of metabolic routes that might lead to dehydrogenase, and hence, tetrazolium reducing activity, Embden-Meyerhof pathway, The first such route is the Embden-Meyerhof pathway directly to the triosephosphate dehydrogenase step. The fact that the reductive activity generated by the hexose diphosphate is strongly inhibited by iodoaeetate, which has been shown to be a fairly spe cific inhibitor in low concentrations (144), indicates that such activity is, indeed, a measure of triosephosphate de- I hydrogenase, Hexosemonophoaphate shunt. A second possibility is that the hexose diphosphate is transformed to glueose-6- i ' phosphate by the Embden-Meyerhof mechanism, and by this means enters into the hexosemonophosphate shunt. The ;triosephosphate generated by the shunt might then be acted I upon by triosephosphate dehydrogenase (145). Since the j . dehydrogenase systems of the shunt mechanism are TPN spe- ■eific in other organisms, and the successful use of TTC with a TPN dehydrogenase system has not been reported, the inhibition of this system by low concentrations of iodo acetate does not rule out the presence of the shunt mechanism in the ameba. It seems likely, however, that even though the shunt mechanism has not been eliminated as a possibility, the fact that its operation in this instance would require the presence of the anaerobic scheme from fructose 1,6-diphosphate to glucose-6-phosphate would also indicate an operative Embden-Meyerhof mechanism. If this were true, then the most direct route between hexose di phosphate and the triose dehydrogenase step would seem the most likely. I Glucose dehydrogenase. Still another possibility is I 'that the hexosediphosphate is transformed to glucose-6- phosphate, and then by phosphatase action to glucose, and finally to gluconic acid by the action of glucose dehydro genase which would thus result in the presence of reduced TTC* Glucose dehydrogenase, however, has been tested for and found lacking. Soluble Starch as a Substrate. It has been demonstrated that soluble starch follows a similar pathway involving the triosephosphate dehydrogen ase, Although amylolytic activity has been demonstrated for the amebae (106, 107), the mechanism by which the glucose moieties of the starch are phosphorylated to enter the anaerobic scheme is not known. One could envision a direct phosphorylation mechanism analogous to the phos phorylation of glycogen in the classical Embden-Meyerhof cycle, or a preliminary breakdown of the starch to smaller units which would then be phosphorylated (146), Either scheme would eventually lead to a phosphorylated glucose that could enter into the reactions of the anaerobic cycle. Glucose as a Substrate. The fact that glucose without added ATP gives no TTC reduction and glucose with ATP does yield reduced TTC, and that such reduction is easily inhibited by iodoaeetate 78 points to the presence of a hexokinase in the amebae* Lactic Dehydrogenase. Beyond the triosephosphate dehydrogenase step, the I anaerobic metabolism of E. histolytica appears to differ radically from the accepted mammalian pathways. Lactic j dehydrogenase was tested for, and its presence could not be ^ f demonstrated with the tetrazolium technique. On the sur- i ' ' !face, such a result would be in direct opposition to the l i ; I reported lactate production by E, histolytica from a number j i !of substrates including glucose and starch (107-111). How- : i . 1 I ever, there is some evidence that this difference in the 1 ! I anaerobic metabolism of E* histolytica, from the triose ! I - 1 step, does, indeed, exist. Thus, Yang (147), using growth and multiplication of the amebae as a criterion of effect iveness, could demonstrate no inhibition of amebae by ■ fluoride, and Michaelson (148) was able to show that in .washed amebae preparations lactate production from soluble starch, glucose, and pyruvate was inhibited only 30 per cent by fluoride in concentrations as high as 0.005 M. If i amebic enolase were, in any way, similar to the mammalian counterpart, a much higher degree of inhibition could be expected (149, 150), since at such high concentrations of fluoride, not only enolase, but a large number of other important enzymes including glucose-6-phosphatase (151), ; adenylic kinase (152), and pyrophosphatase (153) are af- »• i feeted. l The apparent conflict between lactate production on the one hand, and the absence of a lactic dehydrogenase on the other, can be reconciled if we accept a lactic de hydrogenase that is not DPN dependent and/or will not couple with flavoproteins• Thus, the lactic dehydrogenase of some yeasts does not require DPN and does not interact with flavoprotein (154, 155), and the lactic dehydrogenase of B. coli has been reported to require DPN, but to act in the ! - - absence of flavoprotein (156), In this connection it might be well to point out that in bacterial fermentations, depending on the organism involved, either the D or L isomer of lactate is produced and in the case of some organisms like Lactobacillus pentoaceticus a racemic mixture is formed (157), However, in this work a racemic lactate was utilized as the sub strate so that difficulties due to optical specificity need not apply. Recently Hill and Mills (158) working with Bacterium tularense were able to show that this organism contained all the enzymes of the anaerobic scheme plus an active lactic dehydrogenase which was not DPN dependent. They further showed that, when an enzyme preparation that was capable of reoxidizing the reduced DPN generated at the !triose dehydrogenase step was added, glycolysis proceeded j !rapidly. These workers suggested that for B. tularense, ( — ----------- 1 in the wild state, this reoxidation of reduced DPN was normally a function of concomitant bacteria. t ! I IV. OXIDATION OP REDUCED DPN l ' Auxiliary Dehydrogenase Systems. Since triosephosphate dehydrogenase is present in E. |histolytica, and a DPN dependent lactic dehydrogenase ap parently is not, there is the important question of how the i IDPN reduced at the triose step is reoxidized. There are two apparent answers to such a question. The first, and most apparent, is the presence of a dehydrogenase system that could couple with the triose enzyme and, in this way, continually reoxidize the reduced DPN. The second, is the solution offered by Hill and Mills (158) for B. tularense, that the reoxidation of reduced DPN is normally a function of the accompanying flora. In order to check the first of these hypotheses, a number of dehydrogenase systems that are not actual members of the Embden-Meyerhof or Krebs schemes was tested. Using the tetrazolium technique, the following enzyme systems were found lacking in Endamoeba histolytica: glucose de hydrogenase, beta-hydroxybutyric dehydrogenase, alcohol dehydrogenase, and glutamic dehydrogenase. There are still ... _ . 81 a number of enzymes which were not tested for that might i serve in this capacity, such systems might be alpha- | glycerophosphate dehydrogenase (159); formic dehydrogenase (160), the DPN dependence of which has been demonstrated only in plants; diacetyl reductase, which irreversibly con verts diacetyl to acetoin (161); and butylene glycol de hydrogenase, which converts acetoin to butylene glycol (162). Finally, Nisman (163) has suggested that the Stlek- land reaction can act in a modified form so that the re- i ;duced DPN of a dehydrogenase system can be coupled to amino 'acids as hydrogen acceptors. i Glutamic Dehydrogenase. A parasite as highly specialized and evolved as E, histolytica appears to be, might not be expected to have many of the common dehydrogenase systems; however, the absence of a glutamic dehydrogenase presents a problem of more far-reaching magnitude. Glutamic dehydrogenase is of special significance, since this system is considered the primary means of ammonia fixation and hence, via trans aminations, of amino acid synthesis. Thus, the absence of glutamic dehydrogenase or a similar system in the amebae, would indicate that E. histolytica requires amino acids or proteins in a readily available state in order to survive. Although the nutritional data afforded by defined media . . : Q-g (43-45), point to just such a need, the absolute amino acid or protein requirements of E. histolytica have never been investigated. On the other hand, failure to demonstrate a glutamic\ i l j i I dehydrogenase in this work need not eliminate such a j i ' . i i mechanism for amino acid synthesis, from amebic metabolism. } ! I [Thus, it has been shown in bacteria, yeast, and plants thatj j i glutamic dehydrogenase may often be TFN dependent (164-166 ) , j i i jand as such not be detectable by TTC. A second possibility; is that aspartic acid may also act in an ammonia-fixing system. The synthesis of aspartate by fixation of ammonia |with fumarate (167, 168) has been demonstrated in a number j i ' ; |of bacteria. The reaction is easily reversible, and ape- 1 cific for aspartic acid and fumarate. It is, however, difficult to see where fumarate or alpha-ketoglutarate would arise from amebae unless this were a function of ac companying flora. Finally, there is the possibility that amebae may have a limited capacity for amino acid synthesis, or none at all. V. SOME GENERAL CONCLUSIONS From data presented in this dissertation, two un answered questions about amebic carbohydrate metabolism stand out in particular. The first, namely the regenera- \ ' tion of DPN, has already been discussed. The second , question, how E, histolytica obtains its required energy, is still largely unanswered. If the Embden-Meyerhof path way from starch or glucose to the triose dehydrogenase step is the only energy-generating system involved, the result is a net gain of only one high energy phosphate bond per glucose molecule utilized. It is difficult to see how so small an energy yield could support the growth and multi plication of this organism. It is possible, therefore, that some auxiliary, energy-yielding system, is present in E, histolytica. One such system might be the Stickland re action (169-171) which has been mentioned previously in connection with DPN oxidation. The stickland reaction is # normally characteristic of anaerobic, proteolytic bacteria, of the family Clostridiae, and involves a DPN-dependent oxldation-reduction between two amino acids acting as a couple with a resultant gain of two high energy phosphate bonds (163), Thus, the presence of this system would serve a twofold purpose; first as an energy generator and second, as a means of regenerating oxidized DPN, If the hexose- monophosphate shunt mechanism were operating in the ameba, it is possible that the transfer of the hydrogens released in the two dehydrogenase steps, from TPN to a carrier system, might yield high energy bonds. There is, of course, the possibility that the primary energy-yielding system for the ameba is one as yet entirely unknown, or it may be that^ 84 the gain of only one high energy phosphate, per unit glucose; [utilized, is all that the ameba requires* j CHAPTER VI SUMMARY i A survey of some of the dehydrogenase systems of Endamoeba histolytica has been made using the triphenyl tetrazolium technique, Eo malic or succinic dehydrogenase activity could be demonstrated, indicating that these enzymes of the tricarboxylic acid cycle are either absent or radically different from those of mammalian tissue. 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Creator Blumenthal, Herbert (author)

Core Title Some aspects of the metabolism of Endamoeba histolytica

Degree Doctor of Philosophy

Degree Program Biochemistry and Nutrition

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-603285

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