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An Examination Of Transfer-Ribonucleic Acid And Transfer-Ribonucleic Acidacylase During Mammalian Cell Division And Cell Differentiation With Implications For Mammalian Aging

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An Examination Of Transfer-Ribonucleic Acid And Transfer-Ribonucleic Acidacylase During Mammalian Cell Division And Cell Differentiation With Implications For Mammalian Aging

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Content AN EXAMINATION OP TRANSFER RIBONUCLEIC ACID AND TRANSFER RIBONUCLEIC ACID ACYLASE DURING MAMMALIAN CELL DIVISION AND DIFFERENTIATION, WITH IMPLICATIONS FOR MAMMALIAN AGING by LEO ALEXANDER ANDRON II A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Cell and Molecular Biology) August 1972 INFORMATION TO USERS This dissertation was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted. The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction. 1. The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity. 2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. You will find a good image of the page in the adjacent frame. 3. When a map, drawing or chart, etc., was part of the material being photographed the photographer followed a definite method in "sectioning" the material. It is customary to begin photoing at the upper left hand corner of a large sheet and to continue photoing from left to right in equal sections w ith a small overlap. If necessary, sectioning is continued again — beginning below the first row and continuing on until complete. 4. The majority of users indicate that the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the Order Department, giving the catalog number, title, author and specific pages you wish reproduced. University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 A Xerox Education Company I I 73-4902 ANDRON II, Leo Alexander, 1944- AN EXAMINATION OF TRANSFER RIBONUCLEIC ACID AND TRANSFER RIBONUCLEIC ACID ACYLASE DURING MAMMALIAN CELL DIVISION AND DIFFERENTIATION, WITH IMPLICATIONS FOR MAMMALIAN AGING. University of Southern California, Ph.D., 1972 Biochemistry University Microfilms, A XEROX Company, Ann Arbor, Michigan UNIVER SITY O F SO U TH ER N C A LIFO R N IA TH E GRADUATE SCHOOL U N IV E R S IT Y PARK LOS ANG ELES, C A LIF O R N IA 9 0 0 0 7 This dissertation, w ritten by k®9 . . A s . ? * . .Andron II............ under the direction of /zls— Dissertation Com mittee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillm ent of requirements of the degree of DOCTOR OF PHILOSOPHY Dean ^ August 1972 D a te............ 'QitJM.'id... DISSERTATION COMMITTEE Chairman PLEASE NOTE: Some pages may have i nd i st i net p rin t. Filmed as received. U niversity M icrofilm s, A Xerox Education Company To Linda, Allison, and Alex To Leo and Mary Raymond and Marie ii TABLE OP CONTENTS Page LIST OF ABBREVIATIONS v LIST OP TABLES vii LIST OP FIGURES viii ACKNOWLEDGMENTS x ABSTRACT xi Chapter I. INTRODUCTION 1 Some Relationships Between Cell Division, Differentiation and Aging Discussion of Recent Evidence Concerning the Relevance of Translational Control to Cellular Metabolism Viral Systems Bacterial Sporulation Plants Invertebrates Amphibians Birds Mammals Other Regulatory Systems tRNA Modifications Preparation of tRNA Preparation of Enzyme Associated tRNA Preparation of tRNA Amino Acylases Protocol for Amino Acylation Chromatographic Procedures Tissues: Source and Preparation Data Representation II. MATERIALS AND METHODS 29 iii Chapter Page III. RESULTS....................................... 36 Regenerating Rat Liver System Embryonic and Adult Rabbit System Enzyme Associated tRNA IV. SUMMARY AND DISCUSSION................... . n3 LITERATURE CITED .................................... ll6 iv LIST OP ABBREVIATIONS RPC-1 Reversed Phase Chromatography, Type 1 DEAE Diethylamino-ethy1-cellulose ala alanine arg arginine asn asparagine asp aspartate cys cystine gin glutamine glu glutamate giy glycine his histidine ile isoleucine leu leucine lys lysine met methionine phe phenylalanine pro proline ser serine thr threonine try tryptophane tyr tyrosine val valine v tRNA tRM®®? OD260 OD280 transfer ribonucleic acid the third isoacceptor-tRNA for aspartate mili-micron (10“^ meters), a unit for measuring the wavelength of light optical density at 260 m^ optical density at 280 m r* LIST OF TABLES Table Page I. Graphical Representation of Literature Data on tRNA Variations.............................24 II. Relative Amounts of Seryl-tRNA in Fetal and Adult Rabbit Co-chromatographs ............. 81 III. Relative Amounts of Phenylalanyl-tRNA in Fetal and Adult Rabbit Co-chromatographs . . 100 IV. Relative Amounts of Aspartyl-tRNA in Normal and Regenerating Rat Liver....................66 V. Relative Amounts of Lysyl-tRNA in Fetal and Adult Rabbit Brain ......................... 105 vii LIST OP FIGURES Figure Page 1. Bio-gel plOO Profile of 100,000 xg Enzyme Supernatant................................. 39 2. Bio-gel plOO Enzyme-Kinetics ................ 41 3. Enzyme Activity on DEAE...................... 43 4. DEAE Enzyme-Kinetics ...................... 45 5. RPC-2 Co-Chromatograph of Seryl-Isoacceptors from Normal and Regenerating Liver .... 48 6. RPC-2 Co-Chromatograph of Glutamate-Isoaccep- tors from Normal and Regenerating Liver . 51 7. RPC-5 Co-Chromatograph of Glutamate-Isoaccep- tors from Normal and Regenerating Liver . 53 8A. RPC-2 Co-Chromatograph of Glutamate-Isoaccep- tors from Normal and Regenerating Liver . 56 8B. RPC-5 Co-Chromatograph of Glutamate-Isoaccep- tors from Normal and Regenerating Liver . 58 9A. RPC-5 Co-Chromatograph of Aspartate-Isoaccep- tors from Normal and Regenerating Liver . 60 9B. RPC-5 Co-Chromatograph of Aspartate-Isoaccep- tors from Normal and Regenerating Liver . 62 9C. RPC-5 Co-Chromatograph of Aspartate-Isoaccep- tors from Normal and Regenerating Liver . 64 10A. RPC-2 Co-Chromatograph of Aspartate-Isoaccep- tors from the Data of Jackson, Irving, and Sells (45)................................. 69 10B. RPC-2 Co-Chromatograph of Aspartate-Isoaccep- tors from the Data of Tidwell, et al. (100) 69 viii Figure Page IIA. RPC-5 Co-Chromatograph of Fetal and Adult Brain Serine-Isoacceptors.................. 72 IIB. RPC-5 Co-Chromatograph of Fetal and Adult Brain Serine-Isoacceptors.................. 75 12A. RPC-5 Co-Chromatograph of Fetal and Adult Kidney Serine-Isoacceptors ............... 77 12B. RPC-5 Co-Chromatograph of Fetal and Adult Kidney Serine-Isoacceptors ............... 79 13A. RPC-5 Chromatograph of Fetal Kidney Phe- Isoacceptors .............................. 84 13B. RPC-5 Chromatograph of Adult Kidney Phe- Isoacceptors ............................ 86 13C. RPC-5 Co-Chromatograph of Fetal and Adult Kidney Phe-Isoacceptors.......... 88 13D. RPC-5 Co-Chromatograph of Fetal and Adult Kidney Phe-Isoacceptors.................... 90 14A. RPC-5 Chromatograph of Adult Brain Phe-tRNA. 92 14b . RPC-5 Chromatograph of Fetal Brain Phe-tRNA. 94 14C. RPC-5 Co-Chromatograph of Fetal and Adult Brain Phe-Isoacceptors .................... 96 14D. RPC-5 Co-Chromatograph of Fetal and Adult Brain Phe-Isoacceptors .................... 98 15. RPC-5 Co-Chromatograph of Fetal and Adult Brain Lysine-Isoacceptors....................103 16a . RPC-5 Co-Chromatograph of Serine-Isoacceptors 107 16B. RPC-5 Co-Chromatograph of Leucine-Isoaccep- tors from Adult Rabbit Liver................110 16C. RPC-5 Co-Chromatograph of Leucine-Isoaccep- tors from Adult Rabbit Liver................112 ix ACKNOWLEDGEMENTS It is quite a pleasure to acknowledge the assist ance and guidance, so liberally extended to me, by my good friends Gerry Hirsch, Richard Nordgren, and Michael Bick. A special note of thanks is also due to the members of my dissertation committee, Robert Baker, Jim Birren, and Bernard Strehler. Without the special assistance of Bernard Strehler, my committee chairman, I would never have been introduced to the unique satisfactions of a career in scientific research in Gerontology. Special thanks are also due to Ruth Weg and the other members and administrative staff of the Gerontology Center. The Center has provided not only considerable financial assistance but also a certain supportive, en couraging, and healthful atmosphere from which my devel opment in the field of Gerontology has immeasurably profited. x ABSTRACT A brief discussion of some relationships between cell division, tissue regeneration, differentiation and aging is presented. Recent evidence is reviewed toward an evaluation of the proposition that translational con trol, involving tRNA and tRNA acylase, is a key part of those control systems operating during differentiation and aging. Evidence is taken from the following areas: procaryotic and eucaryotic viral systems exhibit various translational mechanisms involving tRNA and tRNA acylase. B. subtilis sporulation involves the loss of a unique lysyl-tRNA. Extensive differences have been seen in tRNA and tRNA synthetase between different plant tissues and during the senescence of specific plant organs. Marked developmental differences in translational compon ents occur in nematodes, meal worms and sea urchins. Several examples exist of similar variations in amphi bians, birds, and mammals. Steriod and polypeptide hormone systems involve variations in translational components. Several enzyme systems have been examined in which tRNA or aminoacyl-tRNA most likely acts as a co-factor in repression or derepression. Recent evidence, which has been obtained concerning the in vivo relevance of various tRNA alterations, is discussed. xi Two experimental systems have been examined with respect to quantities and kinds of acylated-tRNA's, for certain amino acids, which they possess. These are the regenerating rat liver and the developing rabbit. In the regenerating rat liver, evidence was ob tained for multiple, non-mitochondrial, seryl-and glutamyl- tRNA acylating enzymes. In addition, RPC-5 co-chromato- graphic profiles were obtained for glutamic acid and aspartic acid iso-acceptors from normal and twenty- three hour post-hepatectomized rats. For these experi ments acylase prepared from normal liver was used to charge tRNA prepared from normal liver and analogous preparations were obtained and treated similarly with regenerating liver tissue. In all cases the elution patterns were identical when the labels were reversed. The results indicate no gross differences for either amino acid within the present limitations for isolation of enzyme and tRNA and in vitro acylation. However, marked differences were seen in the quantity of one minor aspartyl-tRNA, the greater amount being present in re generating tissue. In the developing rabbit, kidney and brain tissues- were isolated from 25-day old embryos and compared to homologous tissues from adult (6-10 lb.) rabbits. Al though no major differences were seen between the two xii developmental stages for either seryl- or lysyl-tRNA profiles, the seryl-tRNA profiles of kidney were quite distinct from those of brain. Thus, these differences are established very early in the development of the embryo. An extensive series of profiles for fetal and adult kidney and brain phenylalanine isoacceptors were obtained. The results indicate a twofold increase in one isoac ceptor in the adult for both tissues. In the case of kidney, the source of this variation is in the tRNA preparation. Since there are two triplets assigned to phenylalanine, these results may reflect a one-quarter or one-half reduction in code reading capability. xiii CHAPTER I INTRODUCTION Some Relationships Between Cell Division, Differentiation and Aging It is a fundamental truism that all real systems that are not in their lowest free energy state tend to deteriorate spontaneously with time. Thus, mechanical systems deteriorate unless energy is expended, and the parts of the system are replaced at least at the rate at which they degenerate. Biological systems are of course subject to the same law. Thus, it follows that an under standing of tissue regeneration may afford key insights into the origins of the deteriorative aspects of aging in Intact organisms. Aging processes are most obvious in so-called non dividing cell types such as heart muscle cells and neurons. Even in dividing cell types, a recent study of cell pro liferation and renewal during the aging of mice draws attention to the fact that even though the potential for cell division may be present, the mechanism(s) that pro vides for continual cell renewal nevertheless deteriorates (18). Such alterations are especially important in 2 systems that exercise homeostatic control, such as the neuro-endocrine system. Indeed, there is a report cited in the above-mentioned study that a substantial decrease in mitotic activity in thyroid, parathyroid and adrenal cortex tissue occurs in the guinea pig between one month and three years of age. There are at least two ways in which cells can be renewed: (1) as a result of cell division, or (2) as a result of the complete turnover of each cellular component. However, cell division is not of itself an infallible mechanism for renewing tissues for at least the follow ing two reasons: (1) in some cases the stimulus to cell division may be defective, or conversely, (2) in some cases, the mechanism(s) which halt cell division tend to deteriorate. In fact, the age specific incidence of cancer, or the peak incidence of cancer (as measured by cohort analysis) may be one of the better objective meas urements of aging (47). Moreover, this age specific incidence of cancer correlates well with the rate of aging in organisms of different species such as mouse and man. Thus, tumor formation is closely correlated with the aging process and it seems likely that studies of fundamental differences between pathological and normal cell division will not only lead to an understanding of oncogenic processes but also undoubtedly contribute to an understanding of the underlying processes of aging. There is another factor which may be important in limiting tissue renewal through cell division; namely, the two cells produced at a given mitosis may not be functionally equivalent to cells produced during earlier mitoses of that clone even though available evidence on liver regeneration in rats of various ages indicates that several biochemical parameters of regenerated liver cells in old animals are indistinguishable from those of younger cells (1). Nevertheless, other regenerated cells in other tissues need not be necessarily altered with age. There is much evidence that the specific time patterns of deterioration in different biological sys tems are ultimately genetically determined. An organism's genome and the manner and rate at which that genome is expressed determines maximum life span in a given environ ment. For this reason it may be assumed that the patterns of development of an organism's parts ultimately deter mine precisely how long that organism will be able to function in a given environment. Like development, the patterns of tissue regeneration are species dependent, and therefore genetically determined. For example, func tional recovery after complete spinal cord section has been observed in the Japanese rice minnow (103). Also, return of vision after transection of the optic nerve has been observed, with histological evidence for nerve fiber regeneration, in larval and adult anurans (91). In contrast, there has been no evidence for regeneration in mammalian optic nerve or retina (90). Many models have been devised to explain dif ferentiation and development in biological systems. For example, translational models for developmental pro gramming have been elaborated in detail in several publi cations and specific implications of such mechanisms in aging have recently been discussed (9^). Briefly, a key postulate of such models is that the levels and kinds of acylated tRNA’s present in a cell determine which pro teins, unique to a cell’s physiological state, can be made. In the present paper the recent literature concern ing translational control will be reviewed so as to permit the proposition that translational control is a crucial part of the control systems operating during development and aging to be evaluated. Evidence will be drawn from the following areas: significant differences in tRNA and/or tRNA acylase complements have been demonstrated, with respect to various stages of development and differ entiation, in a number of species ranging from sea urchins to mammals. Chloroplasts and mitochondria possess unique tRNA and tRNA acylase components. Various hormones have been shown to alter tRNA profiles markedly. Examples will be given of several viral systems in which the virus codes for unique tRNA’s or tRNA modifying enzymes. Chromatography of tRNA's from many kinds of tumors reveal unique and altered tRNA profiles. Quite recently, an im portant role for tRNA as a co-repressor or co-activator has been demonstrated in at least three different enzyme systems. Discussion of Recent Evidence Concerning the Relevance of Translational Control to Cellular Metabolism Viral Systems Procaryotic and eucaryotlc viral systems have been shown to contain unique tRNA's. Some of these viruses code for specific tRNA modifying enzymes and some for acy lase modifying proteins. Hybridization methods have re vealed five phage coded tRNA’s that are produced by T4 infected cells and fourteen new species present in T5 infected cells (8l). Experiments employing pulse labeled tRNA from T4 infected cells indicate that phage tRNA syn thesis is an early function of the replicative process (80). This in addition to the fact that tRNA synthesis takes place in the presence of chloramphenicol indicates that tRNA syn thesis is independent of viral-induced protein formation. Most recently, these T4 specific tRNA's have been shown to respond quite differently than homologous host cell tRNA's in a ribosome binding assay (82). In addition, the T4 tRNA's were more efficient in a T4 mRNA primed protein synthetic system than host cell tRNA’s. Avian tumor virus has been shown to contain fourteen unique tRNA's (102). In addition, virions of this virus puri fied by conventional techniques were shown to exhibit acylase activity for arginine, tryptophan, cystine, and lysine (30). Substantial changes in tRNA methylation have been seen in vaccinia infected HeLa cells (56). Furthermore, in the same system, reversed phase chroma tography has revealed a quantitative change in aspartyl- tRNA and a quantitative and qualitative change in pheny- lalanyl-tRNA (24). After infection of E. coli with T2, the leucyl-tRNA which codes for CUG is degraded. Evi dently, this code word is untranslatable in infected cells, and it is significant that this codon is only rarely present in the T2 genome (50). In E. coli, T4 or T6 phage infection but not Lambda, T5, or S13 infec tion has been shown to result in the modification of host valyl-tRNA synthetase (70). However, mutant T4's, which upon Infection result in markedly reduced phage specific valyl-tRNA synthetase activity, develop normally in normal hosts. Therefore, this modified synthetase is not required for normal phage production. Bacterial Sporulation Early studies of B. subtilis had revealed a twofold variation in one of two valyl-tRNA types present in spores (52). However, the interpretation of these findings was made uncertain by later work which showed no difference in valine profiles when correction was made for a tenfold difference in amino acid concentration used in the earlier experimental protocols (40). Other early studies of B. subtilis indicated the appearance of an additional lysyl- tRNA upon sporulation (59). However, subsequent studies indicated that the presence of this tRNA was dependent upon growth rate and not unique for spores (60). More recently, stationary phase B. subtilis cells have been shown to exhibit an altered lysyl-tRNA profile when com pared to log phase cells for both wild type and asporo- genous mutants (107). Similar experiments have been re ported using E. coli in which early log phase cells were shown to contain nearly equal amounts of two tyrosyl- tRNA species, whereas late log phase cells produce only about one-half of the normal complement of an early eluting isoacceptor (36). Marked differences in lysyl- tRNA patterns in sporulating and non-sporulating B. subtilis cells have recently been reported which are not affected by the growth rates of the cells (23). Further more, the two lysyl-tRNA's involved respond differently to the lysine codons AAG and AAA in the Nirenberg and Leder assay. Plants Workers in several laboratories have obtained evi dence for extensive tRNA and synthetase differences be tween different plant tissues and during the senescence of specific organs. Anderson and Cherry demonstrated an organ specific deficiency in the complement of leucyl- tRNA (4). These same workers also reported a correspond ing organ specific difference in the leu-tRNA synthetase (51). Fractionation on RPC-2 columns revealed the presence of six species of leu-tRNA, whereas the hypocotyl contains only four of these species. In addition, hypocotyl tissue does not contain one of the three leucyl synthetases normally found in cotyledons. As might be expected, the synthetase lacking in the hypocotyl specifically acylates those leucyl-tRNA species which are also missing in hypo cotyl tissue. Significant tRNA differences have been observed between dividing and non-dividing cells in the roots of pea seedlings (105). Transfer-RNA was obtained from the rapidly dividing meristem of roots of pea seedlings and compared to tRNA from post-mitotic cells in maturing, non-elongating root tissue. The results indicate that a 9 larger percentage of the tRNA from dividing cells can be aminoacylated than the tRNA from non-dividing cells. Also, fractionation of the acylated tRNA’s demonstrated a significant decrease in the relative amounts of two of the three tyrosyl-tRNA species in non-dividing cells when compared to dividing cells. Significant changes in leucyl- and tyrosyl-tRNA's have been observed in soybean cotyledon during their senescence (8). The proportion of two of the six leucyl- tRNA's from cotyledons increases with age, between 2 and 15 days in this system, a system which undergoes complete senescence in 21 days. Tyrosyl-tRNA, which can be sep arated into three species, shows a sharp decrease in species 2 and a simultaneous increase in species 3 over the 15-day period. Equally striking changes in leucyl synthetases have b een observed during cotyledon senes cence (9). There is a significant reduction in the capacity of synthetase derived from 21-day old cotyle dons to acylate leucyl-tRNA*s species 1-4, while the capacity to charge leucyl-tRNA’s 5 and 6 remains high in the old tissues. This deficiency appears to involve an inhibitor of synthetase, present in the senescing organs. All the results in plant systems are complicated by the fact that tRNA's and synthetase derived from 10 chloroplasts and mitochondria are included in the prep arations. In soybeans, leucyl species three and four seem to be the major mitochondrial species (4). In Phaseolus, at least species three, four, five and six are chloroplastic; while species one and two seem to be con fined to the cytoplasm (15). Invertebrates An interesting example of tRNA and tRNA acylase mediated translational restriction has been reported by Ilan in the meal worm T. molitor (43) . In this system, adult cuticular protein, which exhibits a characteris tically high tyrosine to leucine ratio, is not made by one-day larvae; although the mRNA for such protein has been shown to be present in the one-day insect. Messen ger RNA derived from one-day pupae cannot be translated except in the presence of both tRNA and synthetase de rived from seven-day pupae. Juvenile hormone treated Insects yield tRNA’s and synthetases which act as one- day tRNA’s and synthetase in the cuticular protein syn thesizing system. The high ratio of tyrosine to leucine incorporated into seven-day cuticular protein was not obtainable using either tRNA or synthetase preparations from hormone-treated insects. However, incubation of tRNA from hormone-treated insects with nucleotidyltrans ferase and CTP resulted in a tRNA preparation which could 11 substitute for seven-day tRNA. Further examinations of the exact nature of the seven-day tRNA synthetase requirements were not made. However, later work in this system indicates a requirement for stage specific 1M KC1 salt wash factors from ribosomes for the formation of mRNA-ribosome initiation complexes (42). In nematodes, quantitative alterations in arginyl- and tyrosyl-tRNA's have been described at various stages of development (76). Several studies have indicated tRNA and/or acylase changes in developing sea urchins, although mitochondrial- tRNArs are included in all these studies. Taylor, et al., reports MAK column profile differences between unferti lized eggs and 24-hour blastula cells in leucyl-, seryl- and lysyl-tRNA’s: for leucine, a distinct quantitative difference; for serine and lysine, marked qualitative differences (98). These changes are true tRNA changes and not tRNA acylase changes since varying the enzyme source has no effect on the MAK elution profile. No changes were observed for arginine, tyrosine, valine, phenylalanine, or aspartate. Amphibians In the case of amphibians at least four major re ports have been published recently concerning alterations 12 in translational machinery during development. Because mitochondrial-tRNA's were not excluded in any of these studies, the possibility that the observed differences are due to changes in mitochondria specific tRNA species has not been ruled out. Marked shifts in both liver and kidney leucyl- tRNA chromatographic profiles were observed during spon taneous and triiodothyronine induced metamorphosis of bullfrog tadpoles (101). In this same study dramatic differences in tail and kidney leu-tRNA were also demon strated. In another study MAK column elution profiles for methionyt and arginyl-tRNA's from adult and larval bullfrog red blood cells were shown to be different (27). In both of these studies, the tRNA’s were charged in vivo. In Rana pipiens, marked changes in tRNA and/or tRNA acylase have been observed during early development (20). Using a mixture of amino-acids of uniform specific activity, Caston found that the enzyme preparation ob tained from unfertilized eggs and blastulae would acylate 20 percent of the tRNA isolated from unfertilized eggs. However, enzyme obtained from tadpoles could acylate 70 percent of the tRNA isolated from tadpoles. Moreover, tadpole enzyme could acylate about 100-fold more "stage 25" tRNA than could enzyme from unfertilized eggs or 13 embryos during cleavage stages. These differences oc curred progressively over several intermediate stages. A more direct test of tissue translating capacities in amphibians has been carried out (68). In these studies tRNA binding capacities of RNA codons were determined in trinucleotide binding assays. Amino-acyl tRNA from em bryo and adult Xenopus tissues showed no qualitative dif ferences for the twelve amino-acids tested. However, marked quantitative differences were detected, for example, in the AUA response of ile-tRNA and the GAG response of glutamyl-tRNA. Birds In birds, Lee and Ingram have demonstrated MAK and Freon column profile differences in methionyl-tRNA from 4-day old and adult chicken reticulocytes (61). In this case, too, however, mitochondrial components were included in the tRNA preparation. Portugal has examined the tRNA elution profiles for fourteen amino acids in the de veloping chick embryo (74). These comparisons were made between whole embryo and adult chicken liver preparations. Although no tRNA or enzyme changes were observed for arginyl-, glutamyl-, gly cyl-, leucyl-, lysyl-, meth- ionyl-, seryl-, alanyl-, histidyl-, phenylalanyl-, threonyl-, or valyl-tRNA's marked qualitative and quantitative differences in tRNA fractions were found in the case of both lysyl- and tyrosyl-tRNA. In the case of lysine, these differences were present when tRNA was isolated either in the presence or absence of par ticulate fractions. However, the generality of this finding is in doubt because no such differences could be detected for lysyl- and tyrosyl-tRNA’s isolated from duck embryos and liver. This species difference, how ever, may reflect the limitations in the resolving power of the reversed phase column system because qualitative changes were in fact seen using BD-cellulose chroma tography for chick lysine, embryo versus adult. BD- cellulose was not used to compare duck lysine or tyrosine profiles. Another possible source of uncertainty derives from the fact that CTP was not present in the charging reaction mixtures. In the absence of added CTP any tRNA present which lacked an intact CCA terminus could not be acylated. In this connection, it should be noted that several nucleotidyltransferase enzymes have been shown to be non-specific with respect to their tRNA-isoacceptor substrates (26). Therefore, since several chick and duck isoacceptor profiles have been shown to be identical, it is unlikely that the quantitative changes reported by Portugal are due to tRNA’s which lack terminal nucleotides. It is interesting to note that the observed change in the lysyl-tRNA profile (a quantitative increase in peak II) was also seen in several other adult tissues. Thus, the 15 reduction in amount of this tRNA could represent a general alteration In cell metabolism common to several different tissue types. In consequence, a decrease in the produc tion of certain long-lived molecules would perhaps result from the reduction in this particular tRNA. Mammals The differentiation of a number of mammalian tissues has been shown to be accompanied by marked alterations of isoacceptor tRNA’s. In fact, the first report of organ specific differences in translational components was made by Weavers, et al. who showed that calf spleen contains only one of two histidyl-tRNA's present in liver (113). Shortly thereafter, Strehler, et al. showed that rabbit reticulocyte acylases charge only two of three leucyl- tRNA's which liver enzymes charge (93). In the same study, rabbit kidney was found to lack an alanyl acylating activity present in liver. In this same period, Taylor et al. reported the existence of a seryl-tRNA in liver which was not present in kidney or muscle (98). More recently it was shown that embryonic mouse tissue contains a unique leucyl-synthetase (75). In other studies it was shown that the amount of prolyl-tRNA is much greater in rat granulation tissue than in rat liver (58). The amount of at least one isoaccepting tRNA species for aspartyl-, isoleucyl-, leucyl-, and lysyl-tRNA from 16 bovine lens and muscle exhibited quantitative differences as great as two to threefold (72). In this last study, alanyl-tRNA from muscle and methionyl- and tyrosyl-tRNA from lens contained one species that was almost completely absent in the other tissue. In all of these studies mitochondrial-tRNA was present and in most of them, mitochondrial acylases were also in the incubation medium. Recent work has shown that at least for phenylalanine, mitochondrial tRNA can be acylated by cytoplasmic enzyme and vice versa (10)(64). No studies have demonstrated compartmentalization of mitochondrial tRNA's. Thus, even if a given isoacceptor is shown to be mitochondrial, the possibility exists that such a tRNA participates in the extramitochondrial control of message translation. In this connection, an investi gation of nuclear, nucleolar, and cytoplasmic valyl- and leucyl-tRNA*s has appeared (77). The results indicate little, if any, compartment-specific tRNA's for these two amino acids in normal rat liver or in Novikoff hepa toma cells. Non-mitochondrial chromatographic differences in seryl-tRNA's have been reported in profiles from beef and rabbit liver and brain (38). In this study, codon responses were determined for the various isoacceptors. Quantitative, but not qualitative differences in triplet 17 binding were observed. In addition, a seryl-tRNA was discovered which responded to UGA, usually considered a non-sense triplet. Other Regulatory Systems Several recent papers have indicated that trnas- lational mechanisms are likely to be of key importance in various hormone mediated alterations in cell metabo lism. A marked quantitative variation in seryl-tRNA has been observed during estrogen induced phosphoprotein synthesis in rooster liver (7)(66). In these studies, acylase enzymes were isolated in such a way that mito chondrial enzymes were not eliminated. However, tRNA’s were isolated from a non-mitochondrial cell lysate. Non- mitochondrial, estrogen or diethylstilbestrol treated, chicken liver tRNA profile differences for serine, argi nine, and lysine have been reported (17). These studies were carried out using MAK columns. Recently a non- classical role for tRNA’s in steriod hormone mediated metabolic control has been suggested (21). The data reported indicate that progesterone, estradiol, testo sterone, and 5-dihydrotestosterone bind to acylated but not to unacylated tRNA. The binding of the steriods was shown to alter poly (U) stimulated peptide synthesis in vitro. Poly (U) does not itself bind steriods and the experiments were carried out with yeast phenylalanyl-tRNA 18 (which does bind steriods) and E. coli phe-tRNA (which does not bind steriods). Furthermore, such inhibition was observed only when the tRNA concentration was rate- limiting . With regard to hormone-controlled systems it is noteworthy that both "energy charge" and cyclic-AMP have been shown to specifically affect components of the trans lational machinery. The cyclic nucleotide has been shown to alter the function of some acylases but not others when in the presence of non-saturating concentrations of ATP -2 -3 and at 10 to 10 M cyclic AMP, concentrations consider ably above physiological concentrations (67). Histidyl-, arginyl-, valyl-, and lysyl-tRNA synthetases of Salmonella exhibit inhibition by ADP and AMP and are therefore sensi tive to energy charge (12) . Consistent with a transla tional role in the synthesis of steriods, is the fact that puromycin, cycloheximide, or cholramphenicol, but not actinomycin-D block the steroidogenic affect of ACTH (67). Several systems have been studied in which tRNA or amino-acyl-tRNA plays a crucial role as a "co-factor" in the repression or de-repression of various enzyme activi ties. Evidence has been presented for the existence of a special class of regulatory RNA (so-called chromosomal RNA, or cRNA) which is intimately associated with 19 chromatin (25). The properties of this interesting class of RNA are controversial; however, several reports have indicated that cRNA from a variety of sources contains 7 to 10 percent dihydropyrimidine; a characteristic similar to tRNA molecules (41). Duda, et al. have sug gested that the biosynthesis of phenylalanine in E. coli is controlled by pheynylalanyl-tRNA which is shown to bind to the first enzyme in the phenylalanine biosynthetic pathway (28). In Salmonella, a multivalent model for repression of the ilv (ADE) operon has been suggested based upon the observation that in vitro assembly of L- Threonine deaminase from its constituent parts results in the formation of an inactive enzyme which specifically and reversibly binds leucyl-tRNA (39). The native, catalytically active form of the enzyme does not bind leu-tRNA. The authors suggest that the immature holo- tetramer-leucyl-tRNA complex functions as the active holorepressor by acting directly upon the DNA either at the level of transcription or at some point in the trans lation of mRNA into protein. An extensive series of papers has appeared concerning the role of histidyl-tRNA in regulation of histidian biosynthesis in Salmonella. An examination of six classes of regulatory mutants of the histidine operon revealed a direct correlation between the in vivo concentration of charged histidyl-tRNA and repression of the operon (63). The authors suggest that most of the mutants are derepressed for histidine bio synthesis since they contain a lesser amount of charged histidyl-tRNA. More recent work has indicated that histidyl-tRNA from four of these mutants can be normally acylated and that the mutant hisT-tRNA, which is known * to be structurally different from homologous wild type tRNA, binds to histidyl-tRNA acylase as efficiently as wild type histidyl-tRNA under several assay conditions (13). Although it is suggestive that the affinity of the acylated tRNA for the acylase is extremely high, the exact mechanism of histidine operon regulation is not yet firmly established. Gallo, et al. have reported qualitative and quanti tative differences in asparaginyl-tRNA isoacceptor pro files between two human tumors, one of which is sensitive to L-asparaginase (32). These authors suggest that the unique Asn-tRNA found in the L-asparagenase sensitive tumor is a corepressor at some point in the asparagine biosynthetic pathway. In Drosophila, it has been shown that a specific isoacceptor of tyrosyl-tRNA functions as an inhibitor of the enzyme tryptophan pyrrolase obtained from a vermilion mutant (104) . Further studies have sug gested that the mutant contains an altered tRNA matur ation enzyme (46). Other systems have been reported in which tRNA's may play non-classical roles in the control of cellular 21 activities. Several amino acid incorporating systems have been reported which require acylated-tRNA but do riot require ribosomes or mRNA. These reactions have been described in rat liver (48)(49), rabbit liver (89), and sheep thyroid (88). Many cellular membrane and cell wall components in procaryotes appear to be involved in similar non-classical amino acid additions— reviewed by Lengyel and Soil (62) . Such studies as those discussed in the above paragraphs suggest that quantitative or qualitative changes in isoacceptor profiles may exercise control of synthesis through mechanisms other than the modulation of codon usage. tRNA Modifications An extensive and significant literature has accumu lated concerning qualitative and quantitative alterations in the state of methylation of tRNA and other nucleic acids. Such alterations in tRNA methylation have been correlated with biological changes such as differenti ation and morphogenesis (84), neoplasia and viral trans formation (31)(56), and hormone induced transformations (55). Specific functional changes, to be discussed in the following paragraphs, have been shown to correspond to such alterations in tRNA methylation. In similar studies, yeast phenylalanyl-tRNA has been shown to lose about 50 percent of its acceptor capacity when modified 22 by kethoxal, a substance present in yeast (65). Observa tions have also been reported which indicate marked changes in 2° and 3° structure of tRNA upon alkylation (73). Along this same line, a group of methylase inhib itors have been isolated from slime molds and from various mammalian tissues. In slime molds, a non-dialyzable, heat and trypsin-sensitive inhibitor of tRNA methylase is produced about eight hours after the onset of morpho genesis (84). An inhibitor of tRNA methylases of normal adult mammalian tissues has been fractionated into a high molecular weight protein and a low molecular weight (< 700) component (54). It may be highly significant that the high molecular weight component is absent in rapidly mitosing systems such as embryonic and tumor tissues. Nicotinamide has been found to act as a co factor together with a non-dialyzable tRNA methylase inhibitor in normal adult rat liver (69). Further work has shown nicotinamide capable of acting as an inhibitor of methylase activities derived from several human tumor tissues but not of methylase from corresponding normal tissues (14). Table I represents a summary of some of the recent data on tRNA variations under various cell-physiological conditions. Two particularly significant studies which appear in this table were reported by Yang (115), and Gallo and Pestka (33). Yang has reported the 23 TABLE I Graphical representation of literature data on tRNA variations This table represents a collection of the recent data reporting tRNA differences in organisms in various physiological states. A plus indicates a qualitative or quantitative change was observed; a minus indicates such a change was not observed; and a blank indicates no observation was made of that amino acid. The exact systems studied are indicated in the titles of the works listed under Literature Cited. - = r — — -frO L C C i 4 <tl 4 4 — 4 4 ■ — 4 * 4 4 4 4 4 4 4 4 4 h i . 4- LO L 4 4 4 h 4 4 4 4- 4 - t - 4 4 4 4 4 — 4 * 4 ~t" SOT - h 4 4 4 - 4 “ 4 4- 4 4 4 4 — 4 4 18 4 " 4 4 8tT ■ — 4 —■ 4 - 4 4 4 — L6 4 LUL"._" • — — 4 ~ 4 — Ly 4 '— 4- LZ — 4 ~ — 4 —. - h 4 4 4 — — —■ 4 - — 4 zL 4 ~ 8 t — — - — — —- — - — — ■ — — — '— UU L 4 btr — 4 — * — ■ — — 4- — — — — ~ -— t 7 L ■ — 4 - 4 —■ — 4 yu l 4 - 4 4 4 S o — — ■ — 4 — — 4 - — — 4 4 4 — — — 4 4 — — - ^8 — ■ — — ■ — 4 — ...Ttt — + — 4 - H- — —• ■ —- 4 — — — —■ 4 — — — — — bb — — — — — 4" 4 - 4 4 — 4 -H 4 — — 4 4 Q ■+* 6Z 4 4 - - g - y j - — — — — — — 4 — 4 — —• —■ t 7 l l 4 - 9b + — 9L / * A4 / 0 -/U l o - f e /* 7 d&UI S V < 1 n ? l 9ll s 'IH * 1 3 "10 4s\j vsy ^ - » v J ? ! V / X "siaV-L fractionation of rat liver tyrosyl-tRNA into six iso acceptor peaks. Adult rat liver contains three of these isoacceptors, while fetal liver and hepatoma cells contain these three and three more. Gallo and Pestka have examined all twenty amino acids with respect to the reversed phase co-chromatographic profiles of their tRNA-isoacceptors in normal and leukemic lymphoblasts. Pronounced differences in tyrosyl and glutaminyl-tRNA's were found with smaller, but reproducible differences in leucyl-, seryl-, threonyl-, and prolyl-tRNA. The source of the glutaminyl-tRNA pro file difference was shown to be the enzyme preparation and not the tRNA preparation. Unfortunately the in vivo functional significance of such variations observed in vitro have rarely been directly measured, although a few such attempts have been made. Among these is the report by Hatfield and Portugal, already mentioned (38). A similar study has been reported by M. W. Taylor (96). Both of these studies were designed to evaluate differences in triplet binding capabilities for acylated tRNA's, but, in Taylor's study, the protein synthetic capabilities of four different phe-tRNA prepara tions was examined as well. One of these preparations was derived from normal rat liver, one from Ehrlich as cites tumor, a third from normal chicken liver, and the fourth from E. coli. The results indicated no significant differences, dependent upon differences in these tRNA’s, in either poly (U) or poly (UC) stimulated binding to coli ribosomes or in TCA precipitable peptides in a poly (U) or poly (UC) stimulated E. coli protein synthetic system. It should be emphasized that significant tech nical problems exist in such experiments and indeed, in all triplet binding and protein synthesizing experiments involving mammalian tRNA. Mammalian ribosomes cannot be used in such triplet binding studies, because they do not give clear-cut results in these systems. This could be due to a requirement for special protein "factors," such as the one recently described by Gaisor and Moldave (3*0, in mediating tRNA binding to mammalian ribosomes. The use of E. coli ribosomes in a synthetic mRNA primed, in vitro protein synthetic system is subject to similar objections with the additional complication that mammalian initiation, termination, and elongation factors probably interact quite differently than analogous bacterial fac tors with mammalian tRNA’s. In comparable studies, Anderson has used an in vitro protein synthetic system from E. coli to demonstrate that the rate of protein synthesis could be regulated by the concentration of arginyl-tRNA in the reaction mixture (3). Thus signif icant quantitative variations in specific tRNA-isoacceptors can markedly affect the rate of protein synthesis. 27 Shugart, et al. has reported a striking restoration of amino acylation activity of undermethylated tRNA by in vitro methylation (85). In a mixture of undermethylated tRNA’s, isolated from a methionine starved E. coli RCre^‘ mutant, all four amino acids tested (phe, leu, tyr, and his) showed decreased amino acylation activities relative to normal tRNA’s. After in vitro methylation, an in crease in amino acylation activity occurred. With phe and his, essentially complete restoration of acylation capacity was observed. Similarly, Stern, et al. have reported a much lower efficiency of binding of methyl- deficient phe-tRNA in the Nirenberg, Leder assay (86). At least two important technical difficulties have hindered progress in evaluating tRNA and tRNA acu;ase alterations in higher organisms: (1) tRNA and enzyme preparations are seldom isolated from homogeneous cell populations, and (2) tRNA’s acylated in vitro may not accurately reflect in vivo conditions. Recent reports indicate that both of these difficulties can be overcome. Chou and Johnson have used a NaI0/| oxidation procedure to aid in the determination of in vivo levels of acylated tRNA in developing mouse brain (22). Other methods for the separation of acylated from unacylated tRNA’s are available; and, ideally, would be used in conjunction to cross verify experimental results. Furthermore, techniques have been recently developed for the separation of neurons from other brain cell types (37) (87). The use of these and similar experimental approaches would likely result in highly significant data relevant to tRNA and tRNA acylase mediated control mechanisms. CHAPTER II MATERIALS AND METHODS The following methods have been adapted with indi cated modifications from those employed by Yang and Novell! (116). All operations were done at 4° C and in the following manner unless specifically stated otherwise. tRNA Preparation To prepare tRNA, fresh tissue was excised, minced and then homogenized in 2.5/1 (weight/volume) buffer A (. 4Msucrose, .OlMNaCl, .4 Mg/ml Bentonite, and . 02M Mercapto-ethanol). The homogenate is then centrifuged at 34,800 x g for fifteen minutes. The pellet (contain ing mitochondria, nuclei, membrane fragments and other cellular debris) is discarded and the supernatant is di luted with an equal volume of buffer B (.15 NaCl, . 01M tris. ph7.4, .001 M EDTA, 4 Mg/ml Bentonite, and .02 M Mercaptoethanol). This solution is then extracted with an equal volume of reshly distilled phenol. The nucleic acids in the aqueous phase are precipitated with ethanol- acetate. The precipitate is dissolved in 10 ml per 5 gm. 29 30 starting tissue in buffer C (.05M NaCl, .01 M MgCl2 .001 M EDTA, .4 Mg/ml Bentonite, and .02 M Mercaptoe- thanol). Enough 5M NaCl is added to make the solution 1M WRT NaCl and high molecular weight nucleic acids precipitate for two hours in an ice bath. The precip itate is collected by centrifugation (12,000 xg for 15 min.) and washed with buffer D (1M NaCl, .01M MgCl2, .001 M EDTA, and .02M Mercaptoethanol). Wash and super natant fractions are combined and nucleic acids are precipitated with ethanol-acetate. The pellet is dis solved in .3M tris, ph. 8, and incubated at 37° C for 20 minutes. Nucleic acids are again precipitated with ethanol-acetate, dissolved in buffer E (.01M tri, ph. 7.4, .001M DTT, .02M Mercaptoethanol, and .001MEDTA) and stored at -20° C. Preparation of Enzyme-Associated tRNA For the experiments involving enzyme-associated tRNA, tRNA was prepared as follows: fresh liver tissue was homogenized in 2/1 SI buffer (.25M sucrose, 15 percent glycerol, .01M tri ph. 7-5, 1M KC1, .006M MgCl2, .02M- Mercaptoethanol) containing .4 Mg/ml Bentonite. The homogenate was centrifuged at 34,800/g for 15 min., and the pellet was discarded. The supernatant (containing .5M KC1 and .001M EDTA) was centrifuged at 100,000 xg for two hours. The top 4/5ths of the supernatant was 31 poured into dialysis bags and dialyzed for two hours vs. SII (see above section for composition) containing 50 percent glycerol. Fifty ml. of this dialysate was loaded onto a large Bio-gel plOO column (6cm. dia x 44cm. height) previously equilibrated with and then chromatographed with SII buffer. The first one-half of the void volume was collected and mixed with an equal volume of phenol. The aqueous phase was separated and nucleic acids were precipitated with ethyl alcohol-potassium acetate. The pelleted precipitate was suspended in .3M tris, ph8 and incubated for 20 min. at 37° C, after which nucleic acids were again precipitated with alcohol-acetate and pelleted. The pellet was taken up in buffer E (see above) and stored at -20° C. Preparation of tRNA Amino Acylases To prepare acylase enzymes, fresh tissue was ex cised, minced and homogenized in 2/1 buffer SI (see above section). The homogenate was then centrifuged (34,800 xg for 15 min.) and the pellet discarded. The usual protocol, at this point, calls for the centrifugation of the low speed supernatant at 100,000 xg for two hours to pellet ribosomes which are then discarded. However, evidence has recently accumulated that tRNA acylase may exist in a particulate form in vivo (6)(35)(78)(106) or bound to ribosomes (44). In either case acylase activity 32 would be lost in the high speed pellet by usual methods. The work mentioned above indicates that, after sedimen tation in . 001M EDTA and . 5M KC1, ribosomes are washed free of most acylase activity and any high molecular weight aggregation of acylase enzymes would be dis rupted. Thus, at this point the low speed supernatant was brought to .5M in KC1 (with KC1) and .001M EDTA (with .1M EDTA) and then centrifuged (two hours at 100,000 xg). The top four-fifths of the high speed supernatant is dialyzed versus SII (made to 50 percent in glycerol) for two hours. This solution was then stored at -20° C and used within two months. Before use, a portion of the enzyme solution was run on a Bio-gel plOO column equili brated and run with SII. The active fractions were then pooled and made . 3M in KC1. Approximately 10ml of washed, packed DEAE (previously equilibrated in SII made .3M in KC1) was stirred in and the slurry was gently mixed for three to five minutes. This slurry was then centri fuged (12,000 xg for 15 min.) and the supernatant was poured through glass wool into dialysis tubing. After dialysis for two hours versus SII buffer the enzyme was used immediately. Amino Acylation Procedure Amino-acylation was performed according to Yang and Novell! (116). In each case a small aliquot of 33 reaction mixture was mixed with enzyme and kinetics run. The reaction characteristically reaches equilibrium in 10-20 min. and stays on a stable, tRNA limited, plateau for at least fifty min. The reaction was scaled up to 1-2 ml and run until plateau was reached. Acylated tRNA was then recovered from the reaction mixture by phenol extraction and ethanol-acetate precipitation. The pellet was then taken up in running buffer just prior to chro matographic analysis. Chromatographic Procedures Chromatography was performed according to Kelmers and Hea.therly (53) (RPC-5) or according to Weiss and Kelmers (112) (RPC-2). Gradients, fraction sizes and other pertinent details are indicated in the section on results. Our RPC-5 columns are ,9cm x 29cm. for a total volume of 18.5 ml. Normal procedures are to run a 200 ml. linear gradient, collect 1 ml. fractions, and add . 3mg. of carrier tRNA to each sample. The carrier tRNA. was prepared as described above, from adult rabbit liver. Tissues: Source and Preparation Mammalian liver (in this case rat liver) has been an excellent system for experiments designed to investi gate normal cell division. The procedure is as follows: 150 to 200 gram, male Long-Evans rats are subjected to surgical partial hepatectomy under ehter anesthesia. The two major lobes (median and left ventral) are tied off and excised. This eliminates about two-thirds of the wet weight of the normal liver. All operations were performed between 9 and 11 a.m. About eighteen hours after surgery a diffuse wave of cell division begins in parenchymal cells. Since non-parenchymal cells constitute about 40 percent of the total hepatic population, but only about 10 percent of the cellular volume, and lag about 24 hours behind the hepatocytes in initiating mitosis, this wave of cell division which peaks at about 25 hours represents a reasonably homogeneous cell population con sisting of mitotic hepatocytes. In these experiments both tRNA and enzyme were freshly prepared from both normal and regenerating liver. The specific combinations used and other details are indicated under the figure legends and in the following section on Results. Studies of fetal and adult tissues were carried out with tissue obtained from 25-day post-conception and adult (6-10 lb.) New Zealand white rabbits. Kidney and whole brain neural tissue, surgically removed and rinsed in ice cold SI buffer, were used immediately to prepare enzyme and tRNA. Specific combinations of enzyme and tRNA used in the various experiments are indicated in the figure legends. 35 Data Representation The chromatographic data was quantitated in the following manner. For each chromatograph, a background o 14 value Is determined for both Hb and C . All counts above background under each peak are totaled and the entire activity above background for all peaks for each label is determined. The total activity above back ground in each peak for a given label is then divided by the total activity above background for all peaks for that label and multiplied by 100 to give percent. CHAPTER III RESULTS Regenerating Rat Liver System Figure 1 is a plot showing a typical Bio-gel plOO column profile when enzyme supernatant from the 100,000 xg centrifugation is chromatographed. The same general profile is obtained with supernatant derived from adult 'brain, liver, or kidney. The OD 260/OD 280 ratio for peak tubes is about 1:1 for peak I and about 2:1 for peak II for all three tissues. Peak I and the first one- third of peak II fall within the void volume of the column. The broken line in Figure 1 indicates tRNA-acylase activity. The same activity profile can be seen for all amino-acids thus far tested (tyr, leu, ser, asp, phe) when these fractions are incubated with and without added tRNA. Figure 2 is a typical plot of enzyme kinetics ob tained when normal rat liver tRNA is acylated with normal rat liver enzyme. In this instance, as in all cases in these studies with rat liver, enzyme was prepared without the addition of salt and EDTA prior to 100,000 xg centri fugation. This may mean that some enzyme was lost with 36 37 the ribosomal (5)(6) pellet as indicated by Bandyopodhyay and Deutscher. The figure shows that about three-fourths of the counts incorporated at plateau, in the presence of added tRNA, are also incorporated in the absence of tRNA. This data, taken in conjunction with the chromatographic data in Figure 16 indicates that these counts are, for the most part, due to endogenous tRNA in the sephadex enzyme. Figure 2 also shows the unusual kinetics ob tained when tRNA is added, in excess, to this crude enzyme system. It is unlikely that these curves are due to enzyme adenylate complex formation since they are not seen in an identical assay of DEAE-treated enzyme as shown in Figure 3• To ensure stable plateaus and that only added tRNA is acylated, it was found necessary to subject the enzyme to treatment with DEAE. In the case of rat liver, this was accomplished with the standard techniques de scribed by Yang and Novelli (116). Figure 3 shows the activity profile in the DEAE eluant for aspartate and leucine acylating enzymes from normal rat liver. These enzymes clearly possess different properties. The figure illustrates the fact that column eluants must be assayed for each amino-acid if all of the relevant enzyme ac tivity is to be recovered. Figure 4 is a typical plot of the kinetics obtained using DEAE-treated enzyme. The 38 FIGURE 1 Bio-gel p-100 profile of 100,000 xg supernatant. The sample was dialyzed against SII buffer made to 50 percent in glycerol. About 5 ml. of sample was slowly loaded and run on a column (2.7 cm x 30cm) previously equilibrated in SII buffer. One ml. fractions were collected and the optical density at 260 my and 280 my determined. The solid line indicates OD280 and the dashed line indicates tRNA acylase activity. Identical profiles were obtained from liver, kidney, and brain enzyme prep arations. In addition, the ^ 2 6 0 ^ ^ 2 Q 0 ra^ - ' - os ^or peaks I and II were very nearly identical for all three tissues. o p 2.80 loo - Iooo - 2.00 3 0 5 0 /o U) VO 40 FIGURE 2 Bio-gel plOO enzyme-kinetics. Fractions contain ing high tRNA acylase activity from the plOO eluant were pooled and the kinetics of charging of tRNA were deter mined, as described under Materials and Methods, for several different initial concentrations of tRNA. Note the lack of a stable plateau under conditions of added tRNA. UlUI 09 z m i w °n M N ^ XI VNMt *7 v w XS 0007; 000-b 0 0 0 9 vjJdD FIGURE 3 Enzyme activity on DEAE. Fractions from p-100 column containing peak I and the first half of peak II were combined, loaded and run on a DEAE column as de scribed by Yang and Novelli (116). Eluting fractions were assayed for tRNA acylase activity for the amino acids indicated. cpm 5o o o ' 7oqo looo o T" 1 0 H ?- Asp- CPffl . Zooo L e u - 30 - l o o o FR.AC.t- FIGURE k DEAE enzyme-kinetics. Fractions from DEAE column containing high acylase activity were pooled and the kinetics of charging were determined. The reaction characteristically reaches a stable plateau by about 10 min. Extra tRNA was added at points indicated by arrows. The resulting increase in charging and the stable plateau indicates the reaction is tRNA limited. c. pm -Cr VJ1 4 - 46 reaction attains a plateau value within fifteen min. and is clearly tRNA limited. In general, isoacceptor profiles may be quite useful as indicators of acylase enzyme differences, es pecially when such differences cannot be determined by directly chromatographing the enzyme itself. One method used here involves an isolation of partially acylated tRNA as follows: kinetics are obtained for enzymes from two physiological states (e.g., normal and regenerating liver) in order to determine time to plateau (or equilib rium). The two enzymes are then incubated separately, in one case with C^, in the other with . In both cases the reaction is stopped before reaching plateau; the isoacceptors are isolated and co-chromatographed as usual. If either enzyme charges an isoacceptor at a rate significantly different from the other enzyme (e.g., due to different Km for the tRNA), differences will occur in chromatographic elution patterns. Figure 5 shows the results of one such experiment. Peak I is charged relatively rapidly when regenerating liver enzyme is used. However, peak II is charged much more rapidly when normal enzyme is used, all other conditions being equal. Although the re-assays have not been carried out with the labels reversed, it is unlikely that the results obtained are due either to artifacts in the label preparation or to differences in amino acid concentration. FIGURE 5 RPC-2 co-chromatograph of seryl-isoacceptors from normal and regenerating liver. Normal, adult rat liver tRNA was charged to one-third the plateau level by regen- erating rat liver acylase and HJ - serine. The same tRNA was charged to the same extent in a separate mixture m with normal rat liver acylase and C -serine. The labeled tRNA's were then isolated and run on an RPC-2 column as described under Materials and Methods. - looo - Zoo SO loo 15 O Fm \cA.^ F,,.5 The former is unlikely because some of the co-chroma- togrames obtained with these preparations did not show any differences. The latter is unlikely because: (1) the lowest amino-acid concentration used was 10“^M in the final reaction mixture (enzyme Km’s are on the order v -8 of 10“' - 10“ ), and (2) using the same enzyme prepar ation, a tenfold variation in concentration of amino acid has very little, if any, effect on chromatographic profiles. Figure 6 illustrates a similar experiment for glutamate. Here glutamate enzyme, which eluted as a broad peak from DEAE, was divided into two fractions termed "early" and "late." At one-half plateau, the late enzyme appears to have charged the first isoac ceptor to a greater extent than early enzyme. The glutamate peaks in Figure 7 appear to be charged to the same extent at equilibrium by the two enzymes. This figure also illustrates the higher resolving power of RPC-5 chromatography. Chromatographic profiles in the regenerating rat liver system have been obtained for glutamic acid and aspartic acid isoacceptors using both RPC-2 and RPC-5 systems. Regenerating liver enzyme and tRNA were pre pared from 23-hour post-hepatectamized rats. For gluta mate, isoacceptors in a normal rat liver tRNA preparation 50 FIGURE 6 RPC-2 co-chromatograph of glutamate-isoacceptors from normal and regenerating liver. Normal rat liver enzyme eluant from a DEAE column was divided into early eluting and late eluting fractions of glutamate tRNA acylating activity. Separate reaction mixtures contain ing oppositely labeled glutamate and aliquots from either early or late enzyme fractions were incubated to one- half the plateau level. Labeled tRNA’s were then isolated and run on RPC-2 as described under Materials and Methods. 51 cpfn Goocr iooo z o o o O true &c\o Ut£ ( H V I I /oo /JO KoO Fry.G Fa^ci. # 52 FIGURE 7 RPC-5 co-chromatograph of glutamate-isoacceptors from normal and regenerating liver. Enzyme was isolated as indicated under legend for Figure 6. However, in this case the reaction was allowed to reach plateau. Also, note the increased resolution of the RPC-5 system. 53 (SfutAHnic. K c i d 3000 2,000 1 000 160 140 o )Z0 F,j.7 were charged with normal liver enzyme and compared by co-chromatography to isoacceptor tRNA's present in a regenerating rat liver, which were charged with the same enzyme but with the converse label. For aspartate, iso acceptors in a normal rat liver preparation were charged with enzyme from a 23-hour post-hepatectomy, regenerating rat liver preparation and compared by co-chromatography to isoacceptors in a regenerating (23 hour) rat liver preparation charged with the same enzyme but with oppo sitely labeled amino acid. Figures 8a and B, and 9A, B, and C illustrate the results of these experiments. Two isoacceptor peaks can be seen for glutamic acid with no qualitative or quantitative differences between normal and regenerating liver. The aspartic acid profile re veals three peaks. There are no apparent qualitative differences in the two profiles. There is a slight quantitative difference in peak I and a much larger quantitative difference in peak III. These results are collected in Table IV. This third peak represents about 6.6 percent of the total activity for regenerating liver tRNA but only about 3.8 percent of the total for normal liver tRNA. These differences are reproducible and are also apparent when the labels are reversed as seen in Figure 9 C. These results are controversial. With RPC-2 columns, Jackson, Irving, and Sells have observed 55 FIGURE 8A RPC-2 co-chromatograph of glutamic acid iso acceptors from normal and regenerating rat liver. These were charged to a tRNA limited plateau with enzyme from normal rat liver and opposite labels. c p m I& Q O IOOO JOO IB 0 57 FIGURE 8B RPC-5 co-chromatograph of glutamic acid iso acceptor tRNA's from normal and regenerating rat liver. These were charged to a tRNA limited plateau with enzyme from normal rat liver and opposite labels. In this case, tritiated glutamic acid was used to acylate normal rat liver tRNA. Thus, this profile is the reverse, with respect to label, of the profile shown in Figure 8A. cppv\ -3 X 10 58 43 %o - 14-0 160 FP^t- F15. 68 59 FIGURE 9A RPC-5 co-chromatograph of asparate-isoacceptors from normal and regenerating liver. Enzyme prepared from 23-hour post-hepatectomized rat liver was used to acylate tRNA's prepared from normal and regenerating (23 hour) rat liver. The solid line indicates activity in isoacceptors from normal rat liver. The box indicates the fraction of the total activity under each peak. RPC-5 chromatography was done according to Kelmers and Heatherly (53); with a linear KC1 gradient from .*15 to .75M, followed by a 1.5M KC1 wash. All three peaks eluted between . 45M and . 75M KC1. Less than 1 percent of the total activity eluted in the 1.5M KC1 wash. Recovery was 90 percent for H3 and 85 percent for C13. 60 CPlT) Go oo 4 o o o Zqoo H 3 IZ.G 83.6 3.8 C * //■o az.5 C > ' ( o r,,9 A A cid X it m 4(r e G£a/-) pR/Hi-i.# 61 FIGURE 9B RPC-5 co-chromatograph of aspartate Isoacceptors from normal and regenerating liver. The sample and the gradient are identical to that used to generate the profile in Figure 9A. However, in this case the plaskon bed is freshly poured. The 1.5M KC1 wash was begun at tube #179. Thus, peak III is the high salt-eluting O peak. Recovery was 91 percent for H and 82 percent for C13. Ho 63 FIGURE 9C RPC-5 co-chromatograph of aspartate isoacceptors from normal and regenerating liver. This sample is the reverse, with respect to label, of the sample used to generate Figures 9A and 9B. The column bed is the same as the one used for the run shown in Figure 9B. The gradient is linear from 0-.60M in KC1 (50ml total) and linear from .60-1.0M in KC1 (200ml total) with a 1.5M KC1 wash. All three peaks eluted in the .60-1.0 gradient with less than 1 percent of the total activity coming off in the high salt wash. Recovery was 90 percent for h3 and 84 percent for . CPKh * ID'3 5' 4 - 5 - 2 - I - 64 //O 9 o 70 F. 3 • 3C TABLE IV Relative Amounts of Aspartyl-tRNA in Normal and Regenerating Rat Liver This data is taken from the boxes in Figures 9A and 9C. This data in Figure 9B is nearly identical to that in Figure 9A and so it was omitted from the Table. t A&L E N $ -4 I 7 •+J <c or a) — jdJ *<5t t Q «u >r o<c <C_ __ <C SC 5 +i o O 4*«1 2-p 3 *. of 0 a . 3"» +* <* <£ CL •9 *C ' 5 I ' l ' » d H r o c i •^H H C O C O 0 O o 0 H - « % ■ N - V < 0 . J N i ^ 5 j C < c < •*3 ^ * c t : 1 ~p ^ <d _ n - < c - j > * » i : r c o ^fo qualitative and quantitative differences in these iso acceptors in a similar system (*15)(Figure 1 0 k ) . They report a marked quantitative variation in a peak which corresponds to our third peak. In addition, they have resolved what corresponds to our first peak into two peaks and have seen a peak, unique to regenerating liver, that lies between the first two major peaks, and a minor peak, unique to normal liver, which elutes at the very end of the gradient. Although the differences we report likewise indicate an increase in the third major peak for regenerating liver aspartyl-tRNA, this increase is not as pronounced as the one reported by Jackson, Irving, and Sells. In a few instances we have resolved the first peak into two peaks; however, we have not been able to reproduce either of the two unique peaks. Using RPC-2 chromatography and tissue from mouse tumors, Yang, et al. have observed three aspartyl isoacceptor peaks which elute in a pattern quite similar to those which we have observed (117). In contrast, Tidwell, et al. (100) have only been able to resolve two isoacceptor peaks for aspartate on RPC-2 in both normal and regenerating rat liver (Figure 10B). Kelmers has recently reported that small peaks of isoacceptors can be irreversibly bound in the absence of sufficient carrier tRNA (53). Thus Tidwell's results may be explained by the fact that relatively large amounts 68 FIGURE 10A RPC-2 co-chromatograph of aspartate-isoacceptors from the data of Jackson, Irving, and Sells (^5). FIGURE 10B RPC-2 co-chromatograph of aspartate-isoacceptors from the data of Tidwell, et al. (100). UJ-CPW Asp. 6000- - IC,oo 800 - Goo - 4oo ' ZOO 3 0 (A F tE R J a c U son m d S eI I s) cPm /5<50o Asp. 10000 ■ 100 150 (AFtfR Tiowell.SiL.aL) CTi vo of purified tRNA must usually be added as carrier in the RPC-2 system. While Tidwell et al. don’t report the amount of tRNA added as carrier, Jackson and Sells report adding 20 OD units and we normally add 100 OD units on a per-unit-bed-volume basis. Moreover, we have been unable to observe the third aspartate peak on RPC-2 columns in the absence of carrier tRNA. Furthermore, resolution is generally better in RPC-5 systems, although resolu tion can be highly variable in both RPC-2 and RPC-5 systems. Embryonic and Adult Rabbit System All chromatographic profiles of isoaccepting tRNA's in the embryonic and adult rabbit system were done with RPC-5 columns as described by Kelmers and Heatherly (53). Profiles of fetal and adult seryl- and phenylalanyl-tRNA’s have been obtained for both kidney and brain tissue. Figure 11A shows co-chroma- tographic profiles of serine isoacceptors from fetal and adult rabbit brain. Enzyme and tRNA were isolated as described under Materials and Methods. Fetal tRNA was acylated with fetal enzyme and adult tRNA with adult enzyme and the opposite label. The first peak, which appears unique to adult tissue, is actually an artifact. This peak is generated by osmotic shock when the sample is applied and washed with running buffer (containing 71 FIGURE 11A RPC-5 co-chromatograph of fetal and adult brain serine isoacceptors. Enzyme from fetal and adult tissues was used to acylate homologous tRNA*s. Box indicates the fraction of the total activity under each peak. Chromatography was done as reported in Materials and Methods; with a linear KC1 gradient from .45 —.75M and a 1.5M KC1 wash. Recovery was 97 percent for h3 and 80 percent for C1^. All peaks eluted in the .45-.75 gradient with less than 1 percent of the total activity eluting in the high salt wash. 72 ti r o <n tl oQ Cl. no eluting salt) and the gradient is started immediately after washing with running buffer containing .^5M eluting salt. The peak is not observed when a small amount of gradient, from 0-.45M in eluting salt, is run directly after sample washing and just prior to the . 45-.75M running gradient. Figure 11B is a reversed label profile of Figure 11A. Also, the running gradient in Figure 11B has been altered to improve resolution. In this profile the artifactual first peak has been eliminated. In com paring the data from these two sets of curves we notice five conspicuous peaks with no major qualitative differ ences. The quantitative differences are slight, but re peatable; peaks I and II representing a slightly larger fraction of the total activity for fetal tissue, while peaks IV and V are greater in the adult. Since all the results for seryl-tRNA in this fetal and adult system are for homologous charging, the differences seen could be due to enzyme differences, tRNA differences or both. Figure 12 shows the results for fetal and adult rabbit kidney. Here, the data indicates neither qualitative nor reproducible quantitative differences for the two developmental states. The quantitative data for both kidney and brain is shown collected in Table II. The elution patterns at both stages for kidney are clearly different from those for brain. This suggests that a study of stages of development earlier than 25 days FIGURE 11B RPC-5 co-chromatograph of fetal and adult brain serine isoacceptors. Enzyme from fetal and adult tissues was used to acylate homologous tRNA's. Box indicates the fraction of the total activity under each peak. This graph is the reverse with respect to label of the graph in Figure 11A. In addition a linear gradient (from 0-.5M in KC1) totaling 50ml. was run before a second gradient was run which totaled 200 ml. and whose salt concentration ranged from .5 to . 75M. All peaks eluted in the .5-.75 gradient and less than 1 percent of the total activity eluted in a final 1.5M KC1 wash. Recovery was 80 percent for and 70 percent 75 o Lu T T -V a £ . O \ S> «vi L-f 0 e* rJ £ O. CJ 8 o o o V0 O O f ' i cn U 76 FIGURE 12A RPC-5 co-chromatograph of fetal and adult kidney serine isoacceptors. Enzyme isolated from fetal and adult tissues was used to acylate homologous tRNA's. Box indicates fraction of total activity under each peak. Gradients were run in exactly the same way as indicated in the legend of Figure 11B. Recovery was 98 percent for and 80 percent for . Peak I eluted in the 0-.5 fractions, peaks II-V eluted in the .5-.75 fractions with negligible activity eluting in the 1.5M wash. CPtfl "5000 Z O O o ' / OOo■ 78 FIGURE 12B RPC-5 co-chr*omatograph of fetal and adult kidney serine isoacceptors. This sample is the reverse, with respect to label, of the one used for Figure 12A. The column bed is the same as that used for Figure 12A but the gradient is changed to 0-.65 and .65-.75 with a 1.5M wash. Again, peak I elutes in the O-.65 region, while peaks II-V elute in the .65-.75 region. Recovery • 3 n 2 i is 90 percent for and 79 percent for C . 77T 36 f5 COO • VO 80 TABLE II Relative Amounts of Seryl-tRNA in Fetal and Adult Rabbit Co-chromatographs This data is taken from the boxes in Figures 11A- 12B. The data is listed as percent of the total activity under each peak, which is calculated as described in Materials and Methods. F indicates fetal; A, adult. H L i i J. cD -c +f-S >-3 <43 +» f a ^ ~2 U. o -U I®* U- 43 2 Q> - • d < <£ <D <0 V H 10 52^ oOco 4- < v 2 oi c 4 H n'cO ■4-4* c O V o M 4- ro T O T O N <Vioi \ \ o o loro o iV O — . - d C < 0 Q - H c 4o O c y ? •*- in oi 0 4 o O V O to t o oi. < x ? V O T O H < 4 W c xiro *4- o • « O — < <4 u ) u g s UJ1 ? Li_< < li. U .< O ) L L . < a ) Z Q J x ' 82 Post conception would reveal major alterations in elution profiles. In the next series of experiments, phenylalanyl- isoacceptors from fetal and adult tissues were examined. The results of these experiments are collected in Table III. In both, brain and kidney peak I always eluted in the low salt (0-.i l5 and 0-.65) gradients. Since the ac tivity elutes in a broad erratic peak and at low salt, it is likely that it represents tRNA fragments. In kidney, a relatively large quantitative differ ence has been observed for peak II. A marked increase in peak II tRNA is seen for adult tissue from Figures 13C and 13D. In comparing all four figures for kidney, it can be seen that fetal enzyme charges fetal tRNA to a slightly greater extent than adult enzyme. Thus, there are both enzyme and tRNA differences for phenyl alanine in 25-day fetal versus adult tissue. In brain, a similar, large quantitative difference can be seen in phenylalanine peak II for homologous charging as shown in Figures l^A and 14b . The reverse label experiment was not done in this case; however, the results are validated according to the same arguments presented earlier for the seryl-tRNA data in the regen erating liver system. Figures 14C and l^D show the results of experiments in which adult brain enzyme was used to acylate fetal and adult brain tRNA. Here, only 83 FIGURE 13A RPC-5 chromatograph of fetal kidney phe-iso- acceptors. In this case, fetal rabbit kidney enzyme was used to acylate fetal rabbit kidney tRNA. Sample was loaded and washed with 80 ml. of running buffer. This was followed by a 100 ml. linear gradient (from running buffer plus no salt to running buffer plus .45M KC1). Then a linear gradient was run from .45 to 1.5M KC1. Recovery was 98 percent. Peak I eluted in the 0-.45 fractions and peaks II and III eluted in the 0-1.5 fractions. C P W 213 }?% 5 7 5 l o o o ' 30 r«j. 13 ^ oo J r 85 FIGURE 13B RPC-5 chromatograph of adult kidney phe-iso- acceptors. These were acylated with adult kidney enzyme. The sample was washed onto the column and a linear gradient from 0-.45 (100 ml. total) was run followed by a linear .45-.85 (200 ml. total) gradient and a 1.5M KC1 wash. Peak I eluted in the 0-.45 fractions, peak II eluted in the .45-.85 fractions and peak III eluted in the 1.5M wash. Recovery was 98 percent. CPtY\ IT T T j r J H L IZ.Z ZS-0 5 -3.4 1000 * 300 50 87 FIGURE 13C RPC-5 co-chromatograph of fetal and adult kidney phe isoacceptors. Here, enzyme isolated from adult rabbit kidney was used to acylate tRNA prepared from fetal and adult rabbit kidney. After washing the sample onto the column, a linear gradient from O-.65 (50 ml. total) was run followed by a .65-.75 linear gradient (200 ml. total) and a 1.5M KC1 wash. Peak I eluted in the O-.65 fractions and peaks II and III eluted in the .65-.75 fractions. Less than two percent of the label eluted in the 1.5M wash. Recovery was 92 percent for and 82 percent for C-^. m i+ looo' 30 4o 140 FR.RCt . F 1 3 . 89 FIGURE 13D RPC-5 co-chromatograph of fetal and adult kidney phe Isoacceptors. This sample Is the reverse, with respect to label, of that used for Figure 13A. Sample was loaded and washed with 80 ml. of running buffer. This was followed by 60 ml. of running buffer made to .45M KC1. Fractions were collected beginning with the latter wash. These washes were followed by a 300 ml. linear KC1 gradient (from .*15 to . 95M) and a 1.5M KC1 salt wash beginning at fraction 300. Recovery was 107 percent for and 100 percent for C^. 6000. X □ r u r w3 /S.? /o.4 65.1 C I4 /.8 15\(o & 0.5 4ooo- Z o o o Fij. ‘3° klDA/ey c ^ A b u L t ) * K&Ct 91 FIGURE l4A RPC-5 chromatograph of adult brain phe-tRNA. Enzyme from adult brain was used to acylate homologous tRNA. Sample was loaded and washed with 80 ml. of running buffer followed by 60 ml. of buffer made to . 45M KC1, in turn followed by a 200 ml. linear gradient from .45 to .95M in KC1 and an 1.5M KC1 wash. Recovery was 97 percent. Peak I eluted in the 0-.45 fractions and peaks II and III eluted in the .45-.95 fractions. / ' - / n i , Q9l 0-H O il O - f ? oZ O J K . 26 93 FIGURE 1*JB RPC-5 chromatograph of fetal brain phe-tRNA. Enzyme from fetal brain was used to acylate homologous tRNA. Sample was loaded and washed with 80 ml. of running buffer followed by 60 ml. of buffer made to .45M in KC1, in turn followed by a 200 ml. linear gradient from .*15 to 1.5M in KC1. Recovery was 99 percent. Peak I eluted in the 0-.45 fractions and peaks II and III eluted in the .*15-1.5 fractions. c P W & 0 0 0 - Aoqo- Z C O O ' £>RA|N PKe. (Fe-fcAl) r iar 3<3.6 7^.2 54:<D 40 50 IOO mo 130 140 cPm “ 3 o o o F ' 3 ' m 'ZOO 0 - 1000 F(U\Ci.-4= 95 FIGURE 14C RPC-5 co-chromatograph of fetal and adult brain phe-isoacceptors. Here, adult brain enzyme was used to acylate adult and fetal tRNA preparations. After washing the sample onto the column a linear gradient was run (60 ml. total, 0-.65M) followed by a .65-.75 linear gradient (200 ml. total) and a 1.5M wash. Peak I eluted in the 0-.65 fractions and peak II eluted in the .65-.75 fractions. Recovery was 95 percent for H° and 82 percent for C i Oil ' E , J . OOOf Z9 ■ooft 97 FIGURE 1*JD RPC-5 co-chromatograph of fetal and adult brain phe-isoacceptors. This sample is the reverse, with respect to label, of the one used for Figure 15A. Here, peak I elutes in the 0-.65 fractions, peak II elutes predominantly in the .65-.75 fractions and slightly in the 1.5M wash fractions. CO CX\ PH <w1 091 031 O f I OXI O f o Z -000/ Z S TABLE III Relative Amounts of Phenylalanyl-tRNA in Petal and Adult Rabbit Co-chromatographs t a b l e 100 4> _ n _a N -y O y 2 < • < £ -u a ) o Li- v-.. °Z ^r/ Z ^ o > I o>__ *7 ^ a l " 2 . o- < £ 3"> io io vS><0 r^oo < CD O i . cO co 0007 rk < o ^ o <; O vS s O '* — cC H o* C Q to to oo ta a? ^ C > * " o c v 2 c3 JO'sJ- q ) u $ 3 ^ o U1 °7 C l L « « u 4 a) * 2 2 3 o£ § -t><3 < U- U _ < < .U - u . < li_ C <c _1 M P O ^ 3 : to^. 3 ^ 0 roS: ^ c o ro rn to T zro roi 3 : 0 a ) 3 to < 0 V- < t C ( C L S 1 7. a JZ 101 two peaks were resolved and there are no reproducible qualitative or quantitative differences. These last two samples were run on a fresh column bed and it is possible that this particular bed gives poor resolution for the two phe peaks observed in Figures 1*JA and 1*IB. Indeed, these two peaks have always been poorly resolved. Therefore, the quantitative difference seen in Figures 1*1A and 14B must be due to an enzyme difference. That is, the fetal kidney does contain the same amount of peak II phe-tRNA as the adult kidney; but the fetal enzyme cannot charge this tRNA to the same extent that adult enzyme can. Figure 15 shows the results for brain lysyl- tRNA. Here, adult brain enzyme was used to acylate fetal and adult brain tRNA. The quantitative data for this column and for the reverse label experiment are shown in Table V. This data indicates no significant differences in lysyl-tRNA isoacceptors between fetal and adult brain. Enzyme Associated tRNA Figure 16a shows the serine isoacceptor profile on RPC-5 chromatography for tRNA isolated from enzyme as described under Materials and Methods and charged with tritium versus total tRNA isolated from liver cytosol and charged with . Both samples were acylated 102 FIGURE 15 RPC-5 co-chromatograph of fetal and adult brain lysine isoacceptors. Here, adult brain enzyme was used to acylate fetal and adult tRNA preparations. After washing into the column, the sample was eluted with a linear gradient (50 ml. total, 0-.65M KC1) followed by a .65-1.5M KC1 linear gradient. Peak I eluted in the 0-.65M fractions and peaks II and III eluted in the .65-l*5M fractions. Recovery was 92 percent for h3 and 80 percent for . /000' 60 < 3 0 ;oo Hu-t. Fij. 15 103 104 TABLE V Relative Amounts of Lysyl-tRNA in Fetal and Adult Rabbit Brain This data is taken from two figures, one of which is not shown, and the other is Figure 16. The two figures are identical except that one is the reverse, with respect to label, of the other. The gradients were run exactly the same way and the peaks eluted in similar fractions- for both. TM^Le. _c _ a <C •S’ +j ± 3 | s <£ I f * h O u_ u_ -M <* <J IX- O -P «i I CO < < i ) £ L o i i n v S l o i n i o H < v 3 c o f O T O H no <S.oQ s i * 2 . o J i l « q) c ^ S - H ° 2 < C t i L L < C ~ o _ £ » C _ J r o ~ ■ n : c ) t o : t =co 106 FIGURE 16A RPC-5 co-chromatograph of serine-isoacceptors from adult rabbit liver. Enzyme associated tRNA was prepared as described under Materials and Methods. Both tRNA samples were acylated with adult rabbit liver enzyme but with oppositely labeled amino acids. Recovery was 80 percent for and 50 percent for . Sample was applied and washed with 80 ml. of running buffer followed by a 200 ml. linear gradient from .5 to .75M in KC1. Peaks I-VI eluted in the .5-.75M fractions. Peak VII eluted partly in the . 5-.75M fractions and partly in the 1.5M wash fractions. 107 CD - o <o C L . <£ . C9 c o Lx_ 108 with adult rabbit liver enzyme. The data for leucine is shown in Figures 16B and 16C. Figure 16C shows some marked quantitative variations which were not reproduced with the reversed label experiment shown in Figure 16B. In general, this data indicates the presence of each leu and ser isoacceptor in the enzyme fraction. Furthermore, each isoacceptor appears to be in the same relative pro portion in the enzyme fraction as in the whole-cell fraction. 109 FIGURE 16B RPC-5 co-chromatograph of leucine-lsoacceptors from adult rabbit liver. Enzyme associated tRNA was prepared as described under Materials and Methods. Both tRNA samples were acylated with adult rabbit liver enzyme but with oppositely labeled amino acids. Sample was applied and washed with 80 ml. of running buffer, followed by a 50 ml. linear gradient (0-.45M in KC1), in turn followed by a 200 ml. linear gradient from .45 to .75M in KC1 and a 1.5M KC1 wash. Recovery was 96 percent for and 60 percent for C1^. Peaks I- III eluted in the .45-.75 fractions. Peak IV eluted in the 1.5M wash fractions. cpm mo- 1000- Goo- l o o - T m J E T I D T H3 55-/ 2/.S 7.3 11.7 c ' + 13.6 8-3 8.4 I T /20 pRAC-t.^ . /6B Ill FIGURE 16C RPC-5 co-chromatograph of leucine isoacceptors. This column is the reverse with respect to label of that seen in Figure 16B. The chromatographic condi tions for these two columns were the same. c pm Zooo looo A X 2L H L XZT //* 7 36 2 8 15 // c * 50 2 2 17 // l 4 o J50 J&o IGC 7?0~ ■ b : i /SO FftAC*. 112 CHAPTER IV SUMMARY AND DISCUSSION Prom the work presented here and that of Tidwell, et al. (100) and Agarwal, et al. (2), it is clear that, within the limitations of the present methodologies for tRNA and acylase preparation and separation, there are no large-scale changes in non-mitochondrial isoacceptor tRNA profiles during liver regeneration. It is possible that such changes do occur earlier in the period after hepatectomy than 18 or 23 hours. One handicap which has delayed study of these early time points is the difficulty in obtaining sufficient amounts of tissue for preparation of enzyme and tRNA. It is also possible that tRNA’s charged in vitro do not accurately reflect in vivo profiles. This would be especially important if certain mammalian acylase enzymes were shown to be affected by, as yet speculative, inhibitors. Prom the work reported here and that of Jackson, et al. (45), it is clear that there is a marked quanti tative change in a minor isoacceptor peak for aspartic acid. In the absence of triplet binding studies, the 113 11H functional significance of such an alteration must remain unclear. Among other possibilities, such an alteration could be the result of: (1) a marked increase in the synthesis of mitochondrial tRNAa^p in the regenerating liver, (2) an increase in regenerating liver precursor tRNAasp or a decrease in normal liver precursor tRNA~E5, H I I I I * (3) an alteration in the rate of transcription of tRNAa^p cistrons, or (4) a sequestering of tRNAa|^ in the mitochondria-nuclei-membrane pellet. With respect to future experimentation, it would be most interesting to compare the abilities of normal liver enzyme and regenerating liver enzyme to acylate tRNAj®^. In the embryonic and adult rabbit system, seryl- tRNA from kidney exhibited essentially no developmental differences. However, in brain there were slight, though reproducible, quantitative changes. In this case, a four percent increase in fetal peak II and a three percent increase in adult peak V tRNA. The generally higher relative percentage of peak V tRNA in Figure 12A is likely due to the poor resolution in this particular chromatograph. One Important point to notice in these experiments is the striking tissue difference in elution profiles between kidney and brain seryl-isoacceptors. Since kidney peak I elutes at very low salt and con stitutes such a small fraction of the total counts It 115 is likely that this peak represents degraded seryl- tRNA. Thus it is clear that there are only four major isoacceptor peaks for kidney seryl-tRNA while there are five major peaks for brain. These tissue differences must have developed, in the rabbit, at some point before the 25-day stage as has been discussed in the chapter on Results. For phenylalanine several interesting quantita tive alterations have been seen. For brain, in the case of homologous charging, three peaks were resolved. The second peak represented three times as much activity in the adult tissue as in the fetal. When chromatographs were run with fetal and adult tRNA acylated with adult enzyme only two peaks were resolved and the quantitative variations were not reproducible. From this data it appears that the difference observed in the case of homologous charging was due to an inability of fetal enzyme to acylate fetal peak II tRNA. For kidney, there is a clear twofold difference in peak II tRNA, again the adult tissue containing more of this particular tRNA. In addition, there seems to be a slightly greater ability for fetal acylase than for adult acylase to charge fetal peak II. Brain lysyl-tRNA isoacceptors were also examined. The results indicate no major quantitative or qualitative differences in the three peaks observed. LITERATURE CITED 116 LITERATURE CITED Adelman, R. C. 1970. The Independence of Cell Division and Age-Dependent Modification of Enzyme Induction. Biochem. Biophys. Res. 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Creator Andron, Leo Alexander, Ii (author)

Core Title An Examination Of Transfer-Ribonucleic Acid And Transfer-Ribonucleic Acidacylase During Mammalian Cell Division And Cell Differentiation With Implications For Mammalian Aging

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