Some aspects of the carbohydrate metabolism of Endamoeba histolytica

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Content S O M E ASPECTS O F TH E C A R B O H Y D R A T E M ETA BO LISM O F
E N D A M O E B A HISTOLYTICA
by
Joseph B. M ichaelson
A D is s e rta tio n P resented to th e
FACULTY O F THE G R A D U A T E SCH O O L
UNIVERSITY O F SO U TH ER N CALIFORNIA
In P a r tia l F u lfillm e n t of th e
Requirem ents f o r th e Degree
D O C T O R O F PHILOSOPHY
(B iochem istry and N u tritio n )
June 1955
UMI Number: DP21563
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Ph. o Bio 'SS M65.I
This dissertation, written by
Joseph B. M ichaelson
under the direction of.hi£.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 OF P H I L O S O P H Y
Dean
D ate
J&l H 3 c
Guidance Committee
t airman
ACKNOWLBDOfflHTS
I t is w ith sin ce re g ra titu d e th a t I acknowledge th e in v alu ab le
guidance and understanding in a l l re sp e c ts of D r. John W. M ehl.
I w ish to express my d eep est a p p re c ia tio n to D r. James N«
DeLamater w ithout whose a s s is ta n c e , encouragement, and advice none of
t h i s could have been accom plished.
I would lik e to express my thanks to the F a c u lty of th e
Department of Biochem istry and n u tr itio n fo r th e ir guidance d u rin g my
graduate career • A ppreciation is a lso extended to th e Department of
Defence, Armed Forces E pidem iological Board and to the N ational
I n s t i t u t e s of H ealth f o r t h e ir f in a n c ia l support of t h is re sea rc h
■ program, and to the A llan Hancock Foundation and th e C o llis P . and
Howard H untington I n s t i t u t e of M edical Research fo r th e f a c i l i t i e s th g t
were made a v a ila b le .
I w ish to thank Miss Frances Hallman, Mr. H erbert Blum enthal,
Mr. W illiam C. J . Yang, and a l l o th ers w ith whom I have been asso c ia te d
in t h i s work fo r t h e i r a s s is ta n c e and co o p eratio n .
M y s in c e re s t a p p re c ia tio n to my w ife, D olores, fo r her co n stan t
encouragement and in s p ir a tio n .
TABLE O P C O N TEN TS
C H A PTER PAGE
I . HISTORICAL D E V E L O PM E N T . ♦ . ............................................... 1
In tro d u c tio n . • • • • • .................................................... 1
The ro le of c u ltu re media in n u tr itio n a l
requirem ents .............................................................. • • • 2
D iphasic media ................................................ • • • • • 2
Liquid media • • • » • • • • ........................ • • • • 3
D efined or sy n th e tic media .................. 5
E lim in atio n of b a c te ria • • • « • # • • • • • « 5
Role of s t a r c h ............................................... 6
Growth f a c to rs and growth s t i m u l a n t s ......................... 7
General m etabolism • • • • • • • • • • • • • • • * 11
Other enzymes of p o ssib le m etabolic sig n ific a n c e • 14
I I . ST A T E M E N T O F THE PROBLEM A N D PLAN O F EXPERIM ENT . . 16
I I I . EXPERIMENTAL............................................. .................................... 20
Method fo r la c tic acid d eterm in atio n and p re p a ra tio n
of a standard curve ..................... 20
D e p ro te in iz a tio n . • • • •• •• •• •• •• •• 20
C o lo rim etric procedure • • • • • • • • • « . . . 20
Composition of c u ltu re medium and co n d itio n s of
c u l t u r e .............................. 22
Tissue p rep a ratio n s . . . • • • • • 26
iv •
C H A PTER PA(B
B uffer ............................................................... 25
P re p a ra tio n of whole c e ll suspensions • • • • • • 25
P re p a ra tio n of c e llu la r fra c tio n s • • • • • • • • 28
T issue in cu b atio n procedures • • • • • • • • • • • • 29
Ih o le c e lls under aerobic co n d itio n s ...................................... 29
Ififhole c e lls under anaerobic co n d itio n s • • • • • • 30
C ellu la r fra c tio n s « • • • • • • • • • « • • • • • 30
B a c te ria l ferm en tatio n of glucose • • • ............................. 32
IV . RESULTS A N D DISCUSSION.................................................................. 33
Aerobic in cu b atio n of whole c e lls » • • • • • • • • 33
Anaerobic in cu b atio n of whole c e lls • • • • • • • • 43
C e llu la r fra c tio n s • .................. • • • • • • • • • • • 43
B a c te ria l ferm en tatio n of glucose • • • • • • • • • 51
V . INTERPRETATION O F RESULTS .................................................................. 53
B a c te ria l d a ta ••••.•.•••••••••••• 5?
VI • S U M M A R Y ......................................................................................................... 59
BIBLIOGRAPHY................................................................................... 61
LIST O F T A B L E S
TABLE PAGE
I* Composition of C ulture Medium • • • • • « • • • • • • 24
II* Composition of M odified S to n e’s Buffer • ........................ 26
I I I * Micrograms of L aotie Acid Produced Per M i l l i l i t e r
of Medium by P o stin g Amebae C ells • • • • • • • • • 3 4 ;
17* Aerobic Form ation of L actic Acid From Sodium
Pyruvate • • • • * • • * • • • • ...................... ••• 35
V • E ffe c t of C ysteine on th e Aerobic Form ation of
L actic Acid ...............................* ..................... .... 36
VI* E ffe c t of C ysteine on th e Aerobic Form ation of
L actic Acid From Pyruvic A c i d ............................................ 37
V II • U tiliz a tio n of L actic Acid Added to Suspensions of
Amebae C ells and of B a c te ria l C e l l s ............................... 38
V III* Anaerobic P ro d u ctio n of L actic Acid From Sodium
Pyruvate .................................................... .... 39
IX* Anaerobic P ro d u ctio n of L actic Acid by Amebae
C e llu la r F ra c tio n s • • • • • • • • • • • 44
X* L actic Acid Form ation From Glucose by Amebae C ell
P a r tic u la te s * • • * • • ..........................• • * • • • • 48
XI* Per Cent I n h ib itio n o f L actate Form ation From Glucose
by 0*005 M Sodium F lu o rid e and 0*005 M * Io d o acetic
Acid • • • « •• •* ......................................................... 49
LIST O F FIG URES
FIGURE PA G E
1* Standard L a ctic Acid Curve ........................ 23
2« P roduction of L a ctic Acid From Glucose by B a c te ria
Obtained From Amebae C ultures ......................... 46
C H A PTER I
HISTORICAL D E V E L O PM E N T
In tro d u c tio n
For th e p ast 50 years th e re have been no r a d ic a l advances in
th e treatm en t of the d ise a se known as nam ebiasis", th e cau sativ e agent
of which is th e p a r a s itic protozoan, Sndamoeba h i s t o l y t i c a * Many
ex p lan atio n s fo r th is lack of progress have been o ffered * None is
adequate per se* However, when th e y are c o rre la te d and c a re fu lly
evalu ated th e re appears one fundam ental o b serv atio n , in h eren t in a l l ,
which accounts fo r most of the delay in e f fe c tin g an adequate cure
f o r th is d isease* Namely, v ery l i t t l e is known about the n u tr itio n a l
requirem ents and m etabolic a c tiv itie s of th is organism*
Most of th e in v e stig a tio n s in the f ie ld of am ebiasis have been
concerned p rev io u sly w ith c l i n i c a l , p a th o lo g ic a l, and epidem iological
aspects of th e d ise a se , i t s tre a tm e n t, and th e morphology and l i f e
cycle of th e organism which causes i t . Many stu d ie s have centered
around attem pts to c u ltu re amebae in v itr o fo r use in developing
chem otherapeutic a g e n ts . Some in v e stig a tio n s have attem pted to d is
cover and d efin e th e growth and m etabolic c h a r a c te r is tic s of amebae
sin ce the organism was f i r s t c u ltiv a te d by Boeck and Drbohlov (1) in
1925, but no one has succeeded in o b tain in g iri v itr o growth and
m u ltip lic a tio n in a com pletely-defined medium w ithout b a c te ria or
2 .
b a c te r ia l p ro d u cts, r ic e s ta rc h , or Trypanosoma cru zi fo r th e req u ired
long-term experim ents •
At th e p resen t tim e th e re is v ery l i t t l e inform ation concerning
th e in term ed iary m etabolism of amebae • The lit e r a t u r e on th is asp ect
of th e n u tr itio n and physiology of Endamoeba h is to ly tle a is to be
found in a v ery few w idely s c a tte re d p u b lic a tio n s in m edical jo u rn a ls
and jo u rn a ls of p a ra s ito lo g y . Hence, any d isc u ssio n of th e m etabolism
of amebae is lim ite d to a general d isc u ssio n of th e n u tr itio n a l
requirem ents of amebae and m ention of th e random observ atio n s on
amebic m etabolism .
The Role of C ulture Media in H u tritio n a l Requirem ents
D iphasic M edia. As e a rly as 1916, Penfold e t a l (2) olaimed
to have c u ltiv a te d Endamoeba h is to ly tic a in a medium co n tain in g
n u trie n t b ro th and a p a n c re atic d ig e s t. Yoshida (3) rep o rted th a t
amebae liv e d fo r 36-72 hours in v itr o in a medium c o n s is tin g of one
p a rt of horse serum, fo u r p a rts of R in g er’s s o lu tio n , and red blood
c e lls • However, th e work of th ese e a r ly in v e s tig a to rs could not be
confirmed by p ro to z o o lo g ists, and th e c r e d it fo r th e f i r s t su c c e ssfu l
c u ltiv a tio n of th e amebae must be given to Boeck and Drbohlav (1 , 4) •
T heir medium beeame known as Locke-egg-serum (L . E» S .) medium.
This b ein g th e f i r s t su cc essfu l c u ltiv a tio n of Endamoeba h i s to ly tic a ,
th e re re s u lte d a co n sid erab le number of stu d ie s on th e c u ltu ra l
behavior of amebae.
Most of th ese in v e stig a tio n s on th e c u ltu ra l behavior of
Endamoeba h is to ly tic a were concerned only w ith e f fe c ts produced on
amebic growth by a d d itio n of new fa c to rs to th e medium of Boeck and
Drbohlav# The E nglish in v e s tig a to rs , D obell and Laidlaw (5 -6 ),
m odified th e medium by in tro d u c in g r ic e s ta rc h in to c u ltu re s , s ta tin g
th a t s ta rc h p a r tic le s can be in g ested by amebae and u tiliz e d as a
carbohydrate source# S everal years l a t e r Cleveland and C o llie r (7)
having noted th a t amebae could invade liv e r tis s u e rep laced th e whole
egg s la n t of th e Boeck and Drbohlav medium w ith a liv e r in fu sio n agar
s la n t # In a d d itio n th e y found th a t th e Locke-serum o v erlay phase of
th e medium could be rep laced by a serum -saline so lu tio n to which r ic e
s ta rc h could be added# i f t e r th e se m o d ificatio n s a few a d d itio n a l
v a ria tio n s were made in d ip h asic media# Frye and Meleney (8) found
th a t a liv e r exbract p re p a ra tio n , No • 543 as prepared by L illy and
Company, could be s u b s titu te d fo r th e serum p o rtio n of th e o rig in a l
medium# Whole blood has rep laced serum su c c e ss fu lly ( 9 ) . Today,
however, th e L# E# S# medium of Boeck and Drbohlav and the liv e r
in fu sio n agar medium of Cleveland and C o llie r are th e prim ary d ip h asic
media used in th e c u ltiv a tio n of Endamoeba h i s t o l y t i c a .
L iquid M edia, C oncurrently w ith th e development of d ip h asic
media th e re was a tre n d to develop media co n tain in g in g re d ie n ts not
used in o th er media# This led to th e development of liq u id media of
which one of the f i r s t was th e serum-Locke's medium developed by
4 •
Craig (10-11) • Another medium c o n siste d of e x tra c te d powdered h e a rt
muscle in a m odified Locke’s s o lu tio n (12) • S u ccessfu l c u ltiv a tio n of
amebae has been claim ed to occur in h y d atid f lu id (1 3 ), in media made
of e x tra c ts of d rie d h y d atid s c o lic e s in R in g er’s s o lu tio n (1 4 ), as
w ell as in fre s h tom ato ju ic e (1 5 )• Another medium s ta te d to be
s p e c ific fo r Endamoeba h is to ly tic a co n sisted of s t e r i l e n u trie n t broth
and a m ixture of s ta rc h and charcoal (16) •
Although th ese media supported th e growth of amebae th ey were
q u ite complex and were elab o rated by s u b s titu tio n s of components
which might stim u late or m aintain th e growth of amebae w ithout
c o n sid e ra tio n of th e c u ltu ra l requirem ents of th e amebae# From t h i s
tim e on in te r e s t tu rn ed to th e development of stan d ard ized media w ith
more emphasis on s p e c ific n u tr itio n a l requirem ents of the amebae#
Balamuth and Sandza (17) prepared a stan d ard ized f lu id c u ltu re
medium from an in fu sio n of coagulated egg yolk made up in a b u ffered
s a lt so lu tio n * This medium was subsequently m odified by re p la c in g
fre s h egg yolk w ith d rie d egg yolk in th e hope th a t a more standardized
p re p a ra tio n would r e s u lt (18)#
Amebae have been c u ltiv a te d in media co n tain in g alco h o lic
e x tra c ts of liv e r (1 9 )• E x tra c ts from th e liv e r of man, c a lf , b eef,
guinea p ig , and cat were a l l e f fe c tiv e in promoting growth of amebae*
E x tra c tin g substances from m a te ria ls which have been shown to be
e ffe c tiv e in c u ltiv a tin g amebae was designed to sim p lify th e medium
and a lso to co n cen trate th e substance re q u ired by amebae•
5 .
Defined or S y n th etic M edia. A fter replacem ent of d ip h asic media
w ith liq u id media, th e next major advance in c u ltu ra l techniques was
th e development of "defined" m edia. Hansen and Anderson (20) described
an e s s e n tia lly sy n th e tic medium capable of su p p o rtin g amebae asso ciated
w ith “organism t " , an u n id e n tifie d b acteriu m . This medium co n sisted of
b u ffered s a lin e c o n tain in g tra c e m in era ls, 12 amino a c id s, 10 sy n th e tic
B complex v itam in s, n u cleic a c id , c h o le s te ro l, and r ic e s ta r c h . These
workers found th a t th e liv e r e x tra c ts p rev io u sly used by o th ers could
be rep laced by th e sy n th e tic vitam ins (21)»
Another medium developed by Hallman e t a l. (22) re s u lte d in th e
replacem ent of r ic e s ta rc h by human serum albumin and ovalbumin, or
serum m ucoprotein i f adequate amounts of carbohydrate e ith e r as
glycogen or glucose were p resen t • Hansen (23) prepared a liq u id medium
which contained p ro teo se-p ep to n e, W ilson1s liv e r f r a c tio n L, c y ste in e ,
m ethionine, c h o le s te ro l, and a few organic s a l t s .
E lim in atio n of B a c te ria . With th e advent of th e se “le s s ”
complex media a new phase in n u tr itio n a l stu d ie s of amebae ev o lv ed .
I n te r e s t now centered about th e p o ssib le elim in a tio n of th e b a c te r ia l
f lo r a which is ap p aren tly mandatory fo r optim al amebic grow th.
i
i
Seemingly su ccessfu l c u ltu re of amebae fre e o f b a c te ria , or in th e ;
I
presence of v ery few b a c te r ia , was made in m odified liq u id media
(24-28) • However, th e re has never been any p o s itiv e proof th a t th e
b a c te ria were com pletely absent or rendered s t a t i c , and some of th e
in v e s tig a to rs concerned w ith t h i s aspect of th e n u tr itio n of amebae
have withdrawn t h e i r e a r lie r claim s th a t c u ltu re s th e y were u sing were
fre e of b a c te r ia .
P h illip s (2 9 ), and P h illip s and Pees (30) have elim in ated
b a o te ria from ameba c u ltu re s by re p la c in g them w ith th e protozoan
Trypanosoma c ru z i, but th e advantage of t h i s system over m onobacterial
systems in th e c u ltu re of amebae is not e v id e n t.
I t has a lso been found th a t b a c te ria may be rep laced by growing
c e lls of h ig h er an im als. S h affer e t^ a l. (31-32) have propagated
Endamoeba h is to ly tic a in s t e r i l e c u ltu re s of 5-14 day chick embryo
tis s u e w ith th e a d d itio n of s u ita b le s u b s tr a te . In a d d itio n th ey
showed th a t liv e r tis s u e was more a ctiv e in th is c a p a c ity th an o th er
tis s u e s te s te d and which included in te s tin e , h e a r t, m uscle, and b r a in .
Role of S ta rc h . As a lre ad y noted, D obell and Laidlaw (5-6)
f i r s t introduced s ta rc h in to ameba c u ltu re s . These w orkers stu d ied
12 n a tu ra l sta rc h e s and found r ic e s ta rc h to be b e s t. Rees (33) found
th a t amebae grew b e tte r in media c o n ta in in g s ta rc h , but m aintained
th a t i t was not re q u ire d , sin ce ameba grew w e ll w ithout i t . On th e
o th er hand, Hansen and Anderson (20) e s ta b lis h e d th a t r ic e s ta rc h
could not be e lim in ated from a c u ltu re of Endamoeba h is to ly tic a in
a s so c ia tio n w ith norganism t ” grown in a p a r tly s y n th e tic medium.
However, t h is appeared to be due to th e f a c t th a t when th ey rep laced
sta rc h w ith o th er carbo h y d rates, excess a c id ity re s u lte d from ferm enta-
7 .
tio n s by "organism t w w ith subsequent death of amebae •
To in v e stig a te the p o s s ib ility th a t growth of amebae was
d ir e e tly r e la te d to in g e stio n of p a r tic le s , Anderson and Hansen (21)
s u b s titu te d ch arco al, F u lle r^ s e a r th , f i l t e r c e l , c e llu f lo u r and
barium s u lfa te fo r s ta rc h , but w ith no success • They concluded th a t
"organism t " does not re q u ire s ta rc h and th a t th e d is in te g r a tio n of
s ta rc h p a r tic le s 20 miorons or more in diam eter to component granules
of 5-6 microns which took p lace in th e c u ltu re s , occurred only in th e
presence of amebae# Balamuth (34) showed th a t r ic e s ta rc h stim u lated
growth of amebae in la te stag es of c u ltiv a tio n , even though b a c te r ia l
growth was d e c lin in g , in d ic a tin g th a t th e e ffe c t of th e s ta rc h is
d ir e c tly concerned w ith amebic growth#
Hopkins and Warner (35) claim ed th a t amebae s e le c tiv e ly
in g ested some sta rc h g rain s but not o th ers# This supported th e view
of some in v e stig a to rs th a t th e ro le of r ic e sta rc h is to provide
p ro te in m a te ria ls to amebae (2 2 ), and i t is not ju s t a carbohydrate
source as p o stu lated by o th ers (28)«
S h affer a t a l# (26) p o stu la te d th a t ric e sta rc h co n tain s a
fa c to r which in h ib its amebic m u ltip lic a tio n and th a t b a c te ria acted
in some manner on r ic e flo u r which was b e n e fic ia l t o m u ltip lic a tio n
of amebae. Others have shown a minimum, optimum, and maximum amount
of r ic e powder fo r growth of amebae •
Growth F acto rs and Growth S tim ulants • B a c te ria being
o b lig a to ry f o r amebic growth, w ith th e exceptions noted above, many
in v e stig a to rs have attem pted to fin d th e resp o n sib le fa c to rs p resen t
in b a c te r ia . S everal attem pts were made to grow amebae in th e p re
sence of h e a t- k ille d b a c te ria w ith v ary in g degrees of success (27,
36-38) • K arlsson (39) has rep o rted a h e a t- , a l k a l i - , oxygen-labile
growth s tim u la tin g fa c to r in dead b a c te r ia l c e lls *
Ovahormone, hemoglobin, horse red blood c e l l s , and vitam ins of
th e B complex added to D obell and Laidlaw*s medium stim u lated amebic
growth (40) • DeLamater and Hallman (41) d e te c te d a h e a t- s ta b le ,
d ialy z a b le substance from the p ro te in -fre 'e f ra c tio n s of human serum
which th ey b eliev ed e s s e n tia l to th e growth of Endamoeba h i s t o l y t i c a .
Some w orkers thought th a t a reducing substance present in egg
w hite was necessary fo r amebic growth (42-43) • Storage of th is
m a te ria l re s u lte d in lo ss of a c tiv ity through o x id a tio n . Others
b eliev ed th e n ecessary fa c to rs fo r growth were p resen t in egg yolk (18)
Kelson (44-45) im plied th a t sm all amounts of agar were
n ecessary fo r growth of amebae in order to minimize th e presence of
le c ith in globules which were ingested by amebae, hence f o r e s ta llin g
s ta rc h in g e stio n and r e s u ltin g in decreased grow th. H ighly p u rifie d
agar was incapable of su p p o rtin g growth u n less magnesium in th e form
of s u lf a te or c h lo rid e , or a c e ta te was added.
The ro le of c h o le s te ro l was f i r s t p resented by Snyder and
Meleney (46) who s ta te d th a t i t was req u ire d by Endamoeba h is to ly tic a
fo r grow th. Subsequent to th is o b serv atio n Rees e t a l. (47) found th a t
9 .
c h o le s te ro l and 8 vitam ins of th e B complex stim u lated growth of
amebae in egg w hite media# The com bination of th e two f a c to rs was
necessary# G riffin and McCarten (48) u sin g Endamoeba h is to ly tic a
rep o rted th a t c h o le s te ro l in adequate amounts could be used to rep lac e
serum in c u ltu re media#
A stim u la to ry fa c to r fo r Endamoeba h is to ly tic a has been found in
fre s h y east cake but i t was shown to b© h ig h ly u n stab le (49)#
One group of w orkers observed stim u la tio n of amebic growth upon
th e a d d itio n of e x tra c ts of human feces (50)#
Blumenthal e t a l. (51) found th a t th e phosphate system of th e
S to n e’s B uffer commonly used in th e Boeck-Drbohlav medium could be
rep laced by D L -alanine, L-glutam ic a c id , or D L -aspartic acid b u f f e r s •
In a d d itio n , th e se w orkers a lso showed .th a t in th e absence of phosphate
in th e liq u id p o rtio n o f d ip h a sic m odified k e lso n ’s egg medium,phosphate
could d iffu s e out of th e s o lid p o rtio n and t h is d iff u s ib le phosphate
was n ecessary fo r growth (52) • They a lso rep o rted th e p o s s ib ility of
an amebic requirem ent fo r a low m olecular w eight organic in term ed iate#
Using a sim p lifie d medium c o n s is tin g of a b a sa l s a lt m ixture
f o r t i f i e d w ith tra c e m inerals and r ic e powder, Greenberg e t a l. (55)
re p o rt th e apparent s p e c if ic ity of glucosamine, d ir e c t or in d ir e c t,
f o r th e growth of amebae•
A sparagine as w e ll as s ta rc h has been added to c u ltu re media
w ith apparent stim u la tio n of amebic growth (54-55) •
The e ffe c ts of m etabolic in term ed iates on th e growth of amebae
was stu d ied by M ichaelson e t a l. (56) who found th a t th e D K B s tr a in of
amebae w ith i t s a sso ciated mixed f lo r a could be m aintained on a medium
c o n s is tin g of a b asal s a l t m ixture, tra c e m inerals and v ita m in s, r ic e
powder, and e ith e r sodium pyruvate, sodium la c ta te , d ex tro se,
a -k e to g lu ta ric a c id , or sodium a c e ta te provided a s u ita b le n itro g e n
compound, urea or ammonium c h lo rid e , was added to th e c u ltu re medium*
Sodium su c c in a te , sodium c i t r a t e , u rea, ammonium c h lo rid e , or th e base
medium w ithout any s u b stra te o th er than s ta rc h were not capable of
su p p o rtin g amebic growth*
Of s ix amino acids t r i e d , g ly cin e, DL-glutamic a c id , and L-
leu cin e su sta in e d growth* D L -alanine, D L -aspartic acid and DL-
iso le u c in e did not*
Using th e in h ib ito rs tra n s -a c o n itic acid and sodium m alonate
th e se in v e s tig a to rs observed e ffe c tiv e in h ib itio n of growth of amebae
in a p y ru v ate-u rea medium by tra n s -a c o n itic acid in co n cen tratio n of
0*005 M and 0*05 M , but by only th e g re a te r of the two co n cen tratio n s
of sodium m alonate.
They concluded from th e se d a ta , which could not be reproduced
in m onobacterial c u ltu re s of amebae, th a t p o ssib ly some phases of th e
K reb's tric a rb o x y lic acid cycle are im portant in th e n u tritio n and
m etabolism of amebae; but th a t th ese are probably re la te d to th e
b a c te r ia l fu n c tio n s ra th e r th an th e amebae, p rim a rily because of
f a ilu r e of m onobacterial c u ltu re s to support amebae w ith th e
in term ed iates and because of th e need fo r some su b stra te o th er th an
X X »
r ic e powder and a n itro g e n compound*
GeneraX MetaboXism
E a riy experim ents on th e metaboXism of Endamoeba h is to iy tic a
were concerned w ith acid and gas production from added s u b stra te s •
Yon Brand e t aX. (57) found th a t amebae growing w ith norganism t tt
produced a m ixture of gases c o n s is tin g of hydrogen as th e c h ie f con-
I
s titu e n t w ith smaXXer amounts of carbon d io x id e, m ethane, and nitro g en *
However, when ’ ’organism t tt was grown aio n e, identicaX resuX ts were
o b ta in e d •
Rees e t al* (58) were abXe to dem onstrate g re a te r carbon dioxide .
production from c u itu re s of Endamoeba h isto X y tic a and A erobacter
aerogenes, or amebae and * * organism t tt th an from c u itu re s of e ith e r of
th e baoteriaX symbionts aione exeept when g iucose-enriched medium was
used* The in crease in th e b a c te r ia i carbon dioxide formed in th e
presence of giucose was a ttr ib u te d to a d d itio n a i s u b s tra te fu rn ish ed
by amebae* No d iffe re n c e s in carbon dioxide production in any c u itu re s
were observed when th e b a c te r ia i symbiont used was CXostridium
p erfrin g en s * This he a ttr ib u te d to th e u t i i i z a t i o n of r ic e s ta rc h by
t h i s anaerobic organism*
Manometric measurements of carbon dioxide p roduction by amebae
were recorded by Nakamura (59-60) • In h is experim ents, washed amebae
c e iis in bicarb o n ate b u ffe r were piaced in Warburg v e s se is in an
anaerobic atm osphere, and in th e presence of a n tib io tic s to suppress
1 2 .
i
i f lo r a a c t i v i t y . Amebic a c tiv ity in t h is system was measured as cubic i
' mm. of carbon dio x id e form ed. These stu d ie s dem onstrated m etabolic
a c t iv ity of Endamoeba h i s t o l y t i c a ,
j At approxim ately th e same tim e Bradin and Kun (61-62) a lso using
manometric tec h n iq u e s, re p o rte d in a s e rie s of no tes th a t n e g lig ib le
; amounts of pyruvie a c id , l a c t i c a c id , and carbon d io x id e were formed
from glucose under anaerobic c o n d itio n s . However, th e y claim ed th a t j
when c y ste in e was added to t h i s system th e y obtained a 10 fo ld
in crease in gas pro d u ctio n and an e i $ i t fo ld in crease in la c ta te
; fo rm atio n . Hydrogen s u lfid e gas as w e ll as carbon d ioxide was produced
in th is system . Of tw elve o th er s u lfu r compounds t r i e d none was as
e f fe c tiv e as c y ste in e in term s of hydrogen s u lfid e p ro d u c tio n .
Using p re p ara tio n s of amebae which had been starv e d to exhaust
endogenous s u b s tra te s , th e y suggested a r e la tio n s h ip between carbon
dioxide and hydrogen s u lfid e production w ith th e g r e a t e s t gas production
oecuring from glucose, mannose, or g alacto se in th e presence of added
c y s te in e . However, a l l of th e i r work was accom plished in th e presence
of a n tib io tic s n ecessary fo r su ppression of b a c te r ia l m etabolic a c tiv ity .
This in tro d u ces a new v a ria b le in th a t th e re are no d a ta a v a ila b le on
th e e f fe c t of a n tib io tic s on amebae m etabolism .
In th e fo llo w in g year B aernstein (63) e t al*, u sin g c u ltu ra l
tec h n iq u e s, found th a t l a c t i c acid was produced from glucose and
3-phosphoglyceric acid by amebae grown in ro u tin e c u ltu re s in th e
presence of e ith e r organism t or A erobacter aero g en es. The b a c te r ia l
15.
i
organisms grown alone showed e ith e r no la c tic acid form ation or g re a tly
:reduced acid p ro d u ctio n . No acid was produced by any of th e se '
j a e ro b ic a lly grown c u ltu re s when th e added s u b stra te s were pyruvate and
l a c t a t e . However, w ith glucose as s u b s tra te and w ith a hydrogen gas
atmosphere th e re was g re a tly in creased la c tic acid form ation in a l l
.c u ltu re s but w ith more in th e amebae c u ltu re s , th a n in th e f lo r a c o n tro l
c u ltu r e s . Gas production in th e se experim ents was g re a te r th a n could
be accounted fo r by th e u ti l i z a t i o n of glucose from th e medium.
E ntner and Anderson (64) dem onstrated th e form ation of la c tic
and su ccin ic acid s as ferm en tatio n products by r e s tin g amebae c e l l s .
They a ls o claim ed th a t th e amount of l a e t i c acid formed is influenced
I 1
by th e a d d itio n of carb o h y d rates, glucose or m altose, and was governed
by th e source of amebae• When amebae were obtained from c u ltu re s of
amebae a sso c ia ted w ith b a c te ria , glucose suppressed w hile m altose
s lig h tly enhanced la c tic acid fo rm a tio n . W ith amebae th a t had been
grown in th e presence of Trypanosoma c ru z i opposite r e s u lts were
o b ta in e d . In Trypanosoma c r u z i- amebae c u ltu re s , s ta rc h is not manda
to r y but th e trypanosomes are allowed to develop in th e presence of
glucose p rio r to in o c u la tio n w ith amebae. In baoterial-am ebae c u ltu re s ,
s ta rc h is re q u ire d .
Most in v e s tig a to rs stu d y in g th e m etabolism of Endamoeba
h is to ly b ie a have re p o rte d , as a g e n e ra lity , th a t Endamoeba h is to ly tic a
u t i l i z e s glucose e ith e r d ir e c tly or in d ir e c tly as s ta r c h . However,
Hallman e t a l . (65) f a ile d to fin d u t i l i z a t i o n of e x tr a c e llu la r glucose
14*
by amebae grown w ith A erobacter aero genes and in the presence of a n ti- ;
b io tie s • These workers m aintain th a t th is r e s u lt in d ic a te s th a t
u ti l i z a t i o n of e x tr a c e llu la r dextrose per se is not a mechanism fo r
d e riv a tio n of energy by amebae*
In a d d itio n to th e ir e a rly work on cy ste in e and i t s in flu en ce
on carbohydrate m etabolism Kun and Bradin (66) have suggested th a t
s u lfu r has a fu n c tio n in th e m etabolism of Endamoeba h i s t o l y t i c a ,
These in v e s tig a to rs p o stu la te d th a t Endamoeba h is to ly tic a contains a
tr io s e phosphate oxidase system whieh is capable of tra n s f e r r in g
hydrogen to th e s u lfu r atom of cy stein e* A m e ta llo -p ro te in enzyme and
a c o fa c to r (or c o fa c to rs) p resen t in r a t liv e r acetone powder used in
th e se experim ents are b eliev ed t o be c o n s titu e n ts of th e c a ta ly tic
system*
One in v e s tig a tio n on amebic m etabolism has o ffered evidence fo r
th e presence of su c cin ic dehydrogenase* By th e use of su ccin ate
an alo g s, Seaman (67) was able to dem onstrate a com petitive in h ib itio n
of amebic su ccin ic dehydrogenase a c tiv ity * No o th er p o s itiv e demon
s tr a tio n of t h i s enzyme has been made•
Other Enzymes of P o ssib le M etabolic S ig n ifican ce
Evidence fo r th e production of a g e la tin a se was given by Hallman
e t a l* (22)* Confirm ation of t h i s ob serv atio n th a t a g e la tin a se or a
p ro tease is produced by Endamoeba h is to ly tic a was given by Rees afc a l ,
(58) who suggest th a t th e fu n c tio n of th e se enzymes is t o d ig e st away
! 15*
p ro te in surrounding s ta rc h g rain s th u s making th e s ta re h a v a ila b le to
i i
, th e amebae •
i
A stro n g h isto ch em ical acid phosphatase re a c tio n was rep o rted in '
Bndamoeba h is to ly tic a obtained from c u ltu re s , th e i n te s tin a l t r a c t , and
from in fe c te d tis s u e (6 8-69)• This o b serv atio n was confirm ed by
Blumenthal e t al* (7 0 ) who dem onstrated th e presence of both acid and
'a lk a lin e phosphatase a c tiv ity in is o la te d amebic c e lls by chem ical
methods •
S ev eral w orkers have dem onstrated th e production of an e x tra
c e llu la r am ylolytic substance by Endamoeba h is to ly tic a (63, 71)*
C H A PTER I I
STA TEM EN T O F THE PROBLEM A N D PLAN O F EXPERIM ENT
i
i
The g en eral purpose of th i s re sea rc h was to stu d y some asp ects
- of carbohydrate m etabolism by Endamoeba h i s to ly tic a , and more
s p e c if ic a lly , to in v e s tig a te some of th e step s of th e Embden-Myerhof-»
. Parnas m etabolic pathway* The choice of an approach to amebic
m etabolism was governed by a c a re fu l e v a lu a tio n of th e h is t o r i c a l
background on th is phase of th e b io ch em istry of Endamoeba h is to ly tic a
as w ell as by a c o n sid e ra tio n of se v e ra l o b serv atio n s made in stu d ie s
w ith amebae•
Many in v e s tig a tio n s have shown th a t d ie t is im portant in
experim ental in fe c tio n s of anim als w ith Endamoeba h i s t o l y t i c a .
A pparently d ie ts high in carbohydrate r e la tiv e to p ro te in co n cen tratio n
in crease s u s c e p tib ility of th e h o st to Endamoeba h is to ly tic a (72-75) •
D iets high in p ro te in decrease th e r a te of in fe c tio n (7 6 -7 7 ). An
exception to t h i s concept has been dem onstrated by an experim ent in
which amebae in fe c te d dogs fed a d ie t of canned salmon showed an
in crease in th e number of th e se in te s tin a l p a ra s ite s and th e symptoms
produced by th e in fe c tio n (78-79)* The same g en eral observ atio n s have
been made in humans (80-83)•
Numerous p a r a s itic pro to zo a, in clu d in g Endamoeba h i s to ly tic a ,
a v id ly engulf s ta rc h p a r tic le s e ith e r in th e ir n a tu ra l h a b ita t and
17*
l
!in c u ltu re (84) • D extrose or m altose may rep lace s ta re h in c u ltu re s ,
i
iprovided th e re is an adequate source of n itro g en p resen t (2 2 ).
Coupled w ith th is n u tr itio n a l inform ation are th e observ atio n s th a t
polysaccharides may be spread throughout th e amebae c e ll w ithout
d e f in ite lo c a liz a tio n (8 5 -8 6 ). One of th ese p o lysaccharides is pre
sumed to be glycogen, because of th e dem onstration of glycogen vacuoles
in both tro p h o z o ite forms and c y s tic forms of amebae (8 7 ). In a d d itio n ,,
1
most of th e a v a ila b le inform ation on amebic m etabolism is confined to
th a t of carb o h y d rates. Data on p ro te in m etabolism im p licates carbo
h y d ra te s, fo r i t has been shown th a t s ta rc h may be rep laced by
ovalbumin, whole serum p ro te in s , or m uooprotein, i f th e re i s an
adequate source of carbohydrate such as glycogen or m altose (2 2 )•
F in a lly , i t has been rep o rted th a t Endamoeba h is to ly tic a
produces la c tic a c id , from endogenous s u b s tra te s , from glucose, and
' 3-phosphoglyceric acid (6 1 -6 4 ). These o b servations were obtained from ,
both c u ltu ra l experim ents, and by manometrie d eterm in atio n s under
anaerobic c o n d itio n s; a r e s u lt eompatable w ith th e o b serv atio n th a t
Endamoeba h is to ly tic a grows b e st under anaerobic or semi anaerobic
9
co n d itio n s (46, 88-94) .
The fo reg o in g d a ta suggest th a t carbohydrates are of prim ary
im portance in th e whole problem of am ebiasis; although v ery l i t t l e is
known in d e ta il of th e processes involved in carbohydrate m etabolism
of amebae. In th e lig h t of th e se two conclusions and w ith th e
r e a liz a tio n th a t e ffe c tiv e chemotherapy may be accomplished through
g re a te r knowledge concerning th e m etabolism of th e p a r a s ite , stu d ie s
were c a rrie d out to determ ine i f amebae m etabolized carbohydrates v ia
g ly co ly sis •
The f i r s t s e rie s of in v e stig a tio n s of th e carbohydrate
m etabolism of Bndamoeba h is to ly tic a was made u sin g washed amebae c e ll
p rep a ratio n s which were allowed to incubate in th e absence of sub
s tr a te s a t 37 *5° C under normal atm ospheric co n d itio n s in order to
determ ine i f la c tic acid is produced endogenously* Then, under th e
same co n d itio n s of in cu b atio n , a s e rie s of s u b s tra te s in clu d in g l a c t i c
a c id , pyruvic a c id , cy ste in e h y d rochloride and a com bination of
c y ste in e hydr© chloride and pyruvic acid were added to washed amebae
c e lls to stu d y t h e i r e ffe c tiv e n e s s as s u b s tra te s fo r la c tic acid
form ation as w e ll as t h e ir in flu en c e on th e o v e ra ll process of
carbohydrate m etabolism*
These experim ents were next follow ed by a s e rie s of in v e s tig a
tio n s usin g whole c e lls incubated a n a e ro b ic a lly w ith sodium pyruvate
to determ ine i f th e f in a l ste p of g ly c o ly sis , namely, red u ctio n of
pyruvic acid t o la c tic acid was p resen t*
Exam ination of th e d a ta from th e se experim ents w ith whole c e lls
showed th e presence of some phases of g ly c o ly tic m etabolism . This
in fo rm atio n , when considered w ith th e c o n s is te n t o b serv atio n th a t
washed ameba c e lls would d is in te g r a te before th e te rm in a tio n of th e
in cu b atio n perio d employed, re s u lte d in a study designed to e s ta b lis h
w hether th e locus of ameba enzyme a c tiv ity is w ith in th e c e ll# This
19.
was done by f ra c tio n a tin g ameba c e ll p rep a ratio n s in to two p a r ts ,
in so lu b le (p a rtic u la te s ) and so lu b le ( l y s a t e ) . These were assayed fo r
g ly c o ly tic a c tiv ity u sin g as s u b stra te s p a r tic u la te r ic e s ta r c h ,
so lu b le r ic e s ta rc h , and glucose#
Other stu d ie s were conducted w ith th e s im ila r f ra c tio n s under
anaerobic co n d itio n s to determ ine the e f fe c t of th e m etabolic
in h ib ito rs sodium flu o rid e and io d o acetic acid on th e ferm en tatio n of
g lu co se•
F in a lly , th e presence of re s id u a l b a c te ria in ameba tis s u e
p rep aratio n s re q u ired e v a lu a tio n of th e i r in flu en c e on th e r e s u lts •
Experim ents were c a rrie d out u sin g b a c te r ia l f lo r a th a t had grown
w ith amebae as a source of enzyme a c t i v i t y . This was done in order
to determ ine th e in flu en ce of re s id u a l b a c te ria on ameba m etabolism ,
as w ell as to o b tain inform ation on carbohydrate m etabolism of th e
b a c te r ia l symbionts norm ally p resen t in c u ltu re s of th e s tr a in of
amebae used in t h i s work#
C H A PTER I I I
EXPERIM ENTAL
Method fo r L actio Aeid D eterm ination and P re p a ra tio n of a Standard Curve
D e p ro te in iz a tio n . The method used fo r a l l la c tic acid determ ina
tio n s was p atte rn ed a f te r th a t of Barker and Summerson(9 5 )• To a 2 m l.
a liq u o t of re a c tio n or sample m ixtures was added 0.5 ml* of 50 per cent
tr ic h lo r o a c e tic a c id . The p re c ip ita te d p ro te in was th en removed by
e e n trifu g in g fo r 10 m inutes a t 15,000 RPM. The c le a r supernate was
th en decanted in to a 16 x 150 mm. t e s t tu b e . To i t was added 20 m l. of
d i s t i l l e d w ater and 0.5 m l. of 20 per cent (w/v) hydrated copper s u l
f a te (CUSO4 • 5 HgO) s o lu tio n . Next 0.5 grams of s o lid calcium
hydroxide (Ca(QH)2 ) were added, d isp e rse d i n i t i a l l y by shaking 15
tim e s, and th en 10 tim es th e r e a f te r a t 10 m inute in te rv a ls fo r 50
m in u tes• At th e end of th e 30 minute in te r v a l, th e tube was c e n tr i
fuged a t 2,000 RPM fo r 10 m in u tes.
C olorim etric P ro ced u re. One m l• of th e c le a r supernate r e s u l t
ing from th e copper s u lfa te -c a lc iu m hydroxide treatm e n t was added to a
20 x 150 mm. t e s t tu b e . The tube was placed in an ic e b ath and allow ed
to e q u ilib ra te fo r 10 m in u tes. Next 3.0 m l. of reag en t grade,
co ncentrated s u lf u r ic acid was added dropw ise• The tube was shaken to
mix th e co n ten ts and an a d d itio n a l 3 .0 m l. of acid was added. Follow -
2.1 •
in g th e a d d itio n of acid th e tube was shaken 20 tim es in order to in
sure complete m ixing. The tube was next; placed in a b o ilin g w ater bath
fo r e x a c tly 5 m in u tes. A fter removal from th e w ater b a th , i t was
placed in an ic e bath and cooled to room te m p e ra tu re. To th e acid
s o lu tio n was added one drop (0.05 m l.) of 4 per cen t hydrated copper
s u lf a te s o lu tio n . The tube was shaken 10 tim e s . Next two drops
(0.1 m l.) of a 1.5 per cent so lu tio n of para-hydroxydiphenyl reag en t
in 0.5 per cent sodium hydroxide were added. A fter shaking 10 tim es
o
to d isp e rse th e reag en ts th e tube was placed in a w ater bath a t 50 C T
f o r 50 m inutes, and th e reag en t was re d isp e rse d every 10 m inutes by
shaking 10 tim e s . A fter 30 m inutes th e tube was placed in a b o ilin g
/
w ater bath fo r e x a c tly 90 seconds ♦ The tube was cooled to room tem pera
tu re and i t s contents were tra n s fe rre d to a c a lib ra te d co lo rim eter tube
and th e percentage of lig h t tra n sm issio n was recorded a t a w avelength
of 550 in a Bausch and Lomb Monochromatic C o lo rim eter.
The co lo rim eter was s e t a t 100 per cent tra n sm issio n w ith a
blank co n tain in g a l l th e reag en ts except th e unknown m a te r ia l. Two m l.
a liq u o ts of b u ffer were s u b s titu te d fo r th e 2 m l. a liq u o t of tis s u e
p re p ara tio n s in th e b la n k .
A standard curve was prepared from chem ieally pure zinc l a c ta te ,
(2n(C3Hg03)2 • 3 H2O ). S u ita b le d ilu tio n s of a 0.1 per cent sto ck
s o lu tio n of zinc la c ta te were c a rrie d through th e assay procedure*
Three d eterm in atio n s in d u p lic a te were made. The tra n sm issio n v alu es
obtained were averaged, and th e v alu es th en obtained were used in
2 2.
p lo ttin g a stan d ard c u rv e.
The curve was co n stru cted "by p lo ttin g th e O p tical Density-
c a lc u la te d from th e per cent tra n sm issio n a g a in st th e c a lc u la te d
micrograms of la c tic acid per sam ple. See F igure 1 .
Composition of C ulture Medium and Conditions of C ulture
The D K B - l/ s tr a in of amebae a sso c ia te d w ith mixed b a c te r ia l
f lo r a was th e only s tr a in used in th e work d escrib ed in th is d is s e r ta
t i o n . The amebae were m aintained and c u ltu re d fo r experim ental pur
poses on a m odified Cleveland and C o llie r medium, th e com position and
p re p a ra tio n of which are shown in Table I . This medium, h e re a fte r
re fe rre d to as W M C C W medium, was used f o r c u ltu re of th e amebae w ith
one e x c e p tio n . In a sin g le experim ent done w ith lysed amebae c e lls
th e ’ ’M C C ” medium was a lte re d by in c re a sin g th e amount of liv e r con
c e n tra te to 0.5 per c e n t.
T est tubes co n tain in g ”100” medium were in o cu lated w ith 1.0 m l.
of sediment from the bottom of sto ck c u ltu re s of th e D K B s tr a i n of
amebae • The tu b es were th en placed in an incubator a t 37.5° C and
growth was allow ed to proceed fo r 48 h o u rs. At th e end of th is tim e
a l l th e tubes were removed and th e sedim ent a t th e bottom mixed by
l l Obtained from D r. Fred Ryden, V an d erb ilt U n iv e rsity , School of
M edicine, N a sh v ille, Tenn. June 6, 1949. The D K B s tr a i n of
Endamoeba h is to ly tic a was is o la te d in England in November, 1924,
by C liffo rd D obell •
FIG. 1. Standard Lactic Acid Curve
0.9
0.7
> -
H
05
Z
bJ
O
<
o 0.5
I -
Q .
O
0.3
MICROGRAMS OF LACTIC ACID
t- o
0 3
T A B LE I
CO M PO SIT IO N O F C U L T U R E M E D IU M
O riginal Modified
Compound Cleveland-Gollier medium Cleveland-Collier medium
(gm .A lter) (gm. A lte r)
Infusion from Beef Liver 272.0 272.0
Proteose Peptone
5.5 5.5
Disodium Phosphate 3.0 3.0
Sodium Chloride 2.7 2.7
Liver Concentrate N. F . ^ 2.5
Agar 11.0 20.0
All of the constituents ire re dissolved by boiling in one l i t e r of
d is tille d w ater. The solution was cooled and while s t i l l liq u id was dispensed
in te s t tubes in 3 ml. amounts. The tubes were then plugged with cotton and
autoclaved fo r 20 minutes a t 120° C and 15 pounds of pressure. A fter s t e r i l i
satio n the contents were allowed to so lid ify with the tubes slanted a t a 15°
angle. The slan ts were covered with 3 ml* of heat inactivated s te rile human
serum-Ringer's solution ( li S te rile rice starch (ca. 30mg.) was added
p rio r to use.
1 / A powdered aqueous ex tract of fresh or frozen beef liv e r from which
heat-coagulable proteins have been removed. This meets National Formulary
standards and i s comnercially prepared by the Wilson Laboratories, Chicago,
I llin o is .
2 / The human serum-Ringer solution i s prepared by adding 10 ml. of
S e its -filte re d serum which has been exposed to a temperature of $6° C fo r
one hour to 90 ml. of Ringer's Solution.
2 5.
p ip e ttin g th e co n ten ts up and down 15 tim e s. This procedure was
follow ed in a l l subsequent p rep aratio n s of amebae c e lls and re su lte d
in approxim ately a th re e fo ld in crease in amebae p o p u latio n when com
pared w ith normal ro u tin e c u ltu re s . A fter m ixing, th e tu b es were again
placed in an incubator and growth allowed to continue fo r an a d d itio n a l
24 hours • At th e end of th i s 72 hour period of in cu b atio n th e amebae
were h arv ested by poo lin g th e co ntents of a l l tubes and th e n c e n tr i- '
fug in g a t 1,000 RPM f o r 5 m inutes • The su p ern ate, co n tain in g b a c te r ia ,
was decanted and used as needed fo r experim ents re q u irin g co ncentrated
b a c te r ia l flo ra #
The sediment co n tain in g a m ixture of amebae, b a o te ria , unused
r ic e powder, and extraneous m a te ria l was tre a te d in one o f two ways
depending upon w hether whole c e lls or c e llu la r f ra c tio n s were being
assayed fo r a c t i v i t y .
T issue P re p a ratio n s
B uffer # A modif ied S to n e 's B uffer (9 6 ), pH 7 .4 , was used in a l l
am eba-oell and am eba-tissue in cu b atio n s (see Table I I ) . The b u ffe r
was f re s h ly prepared p rio r t o each experim ent •
P re p a ra tio n of Hhole C ell S uspensions. Amebae, h arv ested as
p re v io u sly d escrib e d , we^e suspended in 5 ml# of m odified S to n e 's
B uffer and a g ita te d m echanically w ith a p ip e tte 15-20 tim es • The
suspension was th en c e n trifu g e d a t 500 RPM fo r 5 m inutes * The
T A B L E I I
C O M PO SIT IO N O F M ODIFIED ST O N E ’S B U FFER
Final Concentration
Com pound (gm. A lte r )
NaCl 6.500
Ns^HPOjj 1.100
X H jjPO,, 0.300
K C 1 0.200
NaHCO, o.Uoo
CaCl2 * 2H20 0.050
M g S O L ♦ 7H 20
0.005
2 7.
su p ern ates were d iscard ed except when used fo r p re p a ra tio n of concen
tr a te d b a c te ria l c e lls in some assays * This w ashing procedure was
rep eated fo u r tim es w ith th e supernate from th e l a s t wash being used
as a b a c te r ia l c o n tro l* I t is re fe rre d to in a l l ta b le s and th e texb
as "c o n tro l ( la s t w ash)1 1 .
The washed sedim ent which contained amebae c e l l s , re s id u a l
b a c te r ia , and unused r ic e powder, was th en suspended in b u ffer and
ad ju sted to th e volume needed fo r a ssa y . The l a s t wash, d esig n ated as
"c o n tro l ( la s t w ash)", was ad ju sted to th e same volume as th e sedim ent.
P rio r to th e a c tu a l d eterm in a tio n of enzyme a c tiv ity , amebae p resen t
in th e sediment were counted by th e standard method of Paulsen (9 7 ).
B a c te ria were counted by th e accepted p la tin g method used in determ ina
tio n of b a c te ria pop u latio n s in w ater and m ilk (98)•
In some experim ents a s p e c ia l b a c te r ia l co n tro l was u sed . This
was prepared by c e n trifu g in g th e pooled b a c te ria from th e supernate
rem aining a f te r h a rv est of th e amebae and th e b a c te ria obtained from
th e f i r s t two washes of th e amebae sediment a t 15,000 RPM fo r 5
m in u tes. The co n cen trated b a c te r ia l c e lls were th e n suspended in 4 m l.
of b u ffe r, a g ita te d by p ip e tte 15-20 tim es, and c en trifu g e d a t 15,000
RPM fo r 5 m inutes • The supernate was d iscard ed and th e w ashing pro
cedure rep eated fo u r tim e s . A fter th e f in a l wash, th e c e lls were re
suspended in b u ffe r and ad ju sted to th e same volume as th e ameba
sediment and " c o n tro l ( la s t w ash )". This suspension of b a c te r ia l c e lls
is re fe rre d to in th e te x t and ta b le s of t h is d is s e r ta tio n as
28 •
C o n c e n tra te d f lo r a ” .
P re p a ra tio n of C e llu la r F rac tio n s > Washed ameba sedim ent,
suspended in 5 ml# of m odified Stone*s B uffer and placed in a 10 m l•
beak er, was incubated in a w ater bath a t 40° C* I t was a g ita te d
i n i t i a l l y w ith a s t i r r i n g ro d , and th en again a t 10 m inute in te rv a ls
fo r a p erio d of one h o u r. At th e end of t h i s tim e th e co n ten ts of th e
beaker were c e n trifu g e d a t e ith e r 500 PPM fo r 5 m inutes, or 15,000 PPM
fo r 5 m inutes, depending upon th e n atu re of th e experim ent. The
supernates were removed and sav ed . The sedim ent was suspended in
another 5 m l. of b u ffe r and su b jected again to th e h e at ly s is
procedure fo r a 30 m inute p e rio d . The supernate of th i s f in a l ly s is
was pooled w ith th a t of the o rig in a l ly s i s , and th e combined c le a r
su p ern atan t fra c tio n s were c a lle d “ly s a te ” • The sediment was next
ad ju sted to volume w ith b u ffe r and assayed fo r a c t i v i t y . This
fra c tio n is re fe rre d t o as “ameba p a r tic u la te s ” . Aaebae and b a c te r ia l
counts were made on th e p a r tic u la te f r a c tio n s . No amebae c e lls of any
type could be dem onstrated in th e p a r tic u la te f r a c tio n a f te r th e ly s is
tre a tm e n t.
In some experim ents c e n trifu g a tio n of th e suspension of amebae
sediment a f te r ly s is was c a rrie d out a t 15,000 PPM; in o th ers a t
500 RPM. In th e l a t t e r th e supernate was used per s e , or i t was
cen trifu g ed a t 15,000 RPM to c l a r if y i t . The r e s u ltin g sediment was
resuspended and assayed fo r a c t i v i t y . The v a ria tio n s in speeds of
29.
c e n trifu g a tio n were used to determ ine i f enzymatic a c t iv ity of ameba
p a r tie u la te s eould be removed by c e n trifu g a tio n a t 500 RPM. B a c te ria l
c o n tro ls were sub jected to the same ly sin g procedure when th e y were
used in a ssa y s. B a c te ria l and ameba counts were made, when d e s ire d , by
th e standard methods p rev io u sly d e sc rib e d .
T issue In cu b atio n Procedures
Whole C ells Under Aerobic C o n d itio n s. For th e se experim ents
washed amebae c e lls , c o n tro l ( la s t w ash), and concentrated f lo r a p re
p a ra tio n s were each placed in 20 m l. b e a k e rs. To th e se were added
1 ml • of b u ffered s u b s tra te , or 1 m l• of S to n e’s Buffer i f only
endogenous a c t iv ity was being m easured, and th e volume of each was
ad ju sted to 10 m l. Then th e suspensions were incubated in a w ater bath
a t 37.5° under normal atm ospheric c o n d itio n s. Two m l. a liq u o ts of
each were removed a t 0, 2, and 4 hour in te rv a ls and la c tic acid
d eterm in atio n s were c a rrie d o u t. The s u b stra te s used in th is p a rt of
th e in v e stig a tio n s and th e f in a l co n cen tratio n s of each in th e re a c tio n
m ixtures were la c tic a c id , 9 -2 0 /< g ./m l•; sodium pyruvate, l l / f g . / m l . j
c y ste in e h y d ro ch lo rid e, 2 5 0 ^ g ./m l. and a com bination of cy ste in e
hydrochloride and sodium pyruvate in co n cen tratio n s of 2 5 0 /rg ./m l. and
110^ g . / m l . r e s p e c tiv e ly .
2_/ C ysteine hydrochloride used in a l l experim ents was d isso lv ed in
m odified S tone’s B uffer and th e pH ad ju sted to 7 .4 w ith sodium
hydroxide (2 M).
Whole C ells Under Anaerobic C o n d itio n s* Measurement of amebic
enzyme a c tiv ity under anaerobic co n d itio n s was done in th e Warburg
Constant Volume Re spirom eter • Washed ameba c e lls and th e c o n tro l
(l a s t wash) p re p a ratio n s were th e only systems te s te d fo r a c tiv ity *
Each p re p a ra tio n was suspended in 10 ml* of b u ffer* Five ml* of each
suspension was th en placed in a Warburg v e sse l* To one s e t of v e sse ls
used fo r m easuring endogenous a c tiv ity was added 1 .0 ml* of b u ffe r and
to th e o th er s e t 1 ml* of b u ffered sodium pyruvate so lu tio n to give a
f in a l co n cen tratio n of 110/«g*/m l. of sodium pyruvate (1*0 x 10~ M)•
A reag en t blank and a pyruvate c o n tro l were also run*
P rio r to in cu b atio n two ml* of each v e s se l were th en removed
and assayed fo r la c tic acid* The values obtained c o n s titu te d th e 0
tim e la c tic acid c o n c en tratio n per v e sse l* The v e s se ls were th en
a ttach ed to th e manometers and flu sh ed fo r 5 m inutes w ith a m ixture
of 99*7 per cent n itro g e n and 0*3 per cent hydrogen* Next th e v e sse ls
were allowed to e q u ilib ra te w ith shaking fo r 10 m inutes w hile being
flu sh ed w ith th e gas m ixture* At th e end of th e e q u ilib ra tio n period
th e v e sse ls were closed o ff and allowed to incubate w ith co n stan t
shaking* At 2- and 4-hour in te r v a ls , 2 ml * a liq u o ts were removed and
assayed fo r la c tic a c id .
C e llu la r F rac tio n s » A ll incubations w ith c e llu la r fra c tio n s
were c a rrie d out under anaerobic co n d itio n s in a Warburg apparatus*
To each v e sse l was added 2*0 ml* of a c e llu la r f ra c tio n , 0*3 ml* of
51.
m odified S to n e’s B uffer co n tain in g 2 micromoles each o f ATP and DPN,
0.12 m l. of b u ffer c o n tain in g 2 micromoles of glucose, or 500 miorograms
3/
of so lu b le S te in H all r ic e s ta rc h — , or 10 m illigram s of p a r tic u la te
S te in H all r ic e s ta r c h .
In experim ents in which in h ib itio n of glucose ferm en tatio n was
b ein g stu d ie d , sodium flu o rid e and io d o ac etic acid were d isso lv ed in
m odified S tone’s B uffer in a co n c en tratio n such th a t when 0*1 m l. of
so lu tio n was added t o th e re a c tio n v e s s e l th e f in a l co n cen tratio n of
■ •5
in h ib ito r was 5 x 10 M. The volumes of a l l v e sse ls were ad ju sted t o
2 .6 m l. w ith b u f fe r , th e co n ten ts mixed, and th e v e s se ls were flu sh ed
fo r 5 m inutes w ith a m ixture of 99.7 per cen t n itro g en and 0.5 per cent
hydrogen. A fter 10 m inutes of e q u ilib ra tio n w ith co n stan t shaking a t
72 RPM d u rin g which tim e th e gas m ixture was flu sh ed th ro u ^ i, th e
v e sse ls were closed and th e in cu b atio n allow ed to proceed in an
anaerobic atmosphere fo r 3 h o u rs • At th e end of th e th ree-h o u r
in te rv a l 2.0 m l. a liq u o ts were removed from each v e s se l and assayed fo r
th e presence of la c tic a c id . L actic acid determ in atio n s were c a rrie d
out on amounts of th e o e llu la r f ra c tio n eq u iv alen t to th e amount p resen t
in 2 .0 m l. of th e 2.6 m l. per re a c tio n v e s s e l. These served as c o n tro ls .
3/ A h ig h ly p u rifie d , f in e ly m illed r ic e s ta rc h obtained from
S te in , H a ll and C o., I n c ., Hew York, N. Y.
B a c te ria l F erm entation of G lucose» Washed co n cen trated f lo r a
were suspended in 10 m l. of m odified S to n efs B uffer* One ml* was
removed and the b a c te r ia p re sen t counted* A liquots of t h is suspension
were then added to a s e rie s of Warburg v e s se ls so th a t th e f in a l
co n cen tratio n of b a c te r ia per v e s se l per ml • of re a c tio n m ixture was
6 6 6 6 6 6
5 x 10 , 15 x 10 , 30 x 10 , 70 x 10 , 160 x 10 , and 210 x 10 ,
re sp e c tiv e ly * To each v e s s e l was added 0*3 ml* of b u ffe r co n tain in g
2 micromoles of each ATP and DP$ and 0*2 ml* of b u ffe r co n tain in g
2 micromoles of glucose* The volume in a l l of th e v e s se ls was
ad ju sted to 2*6 ml* w ith b u ffe r, thoroughly mixed, and c a rrie d througjh
th e in cu b atio n procedure d escrib ed fo r c e llu la r fra c tio n s *
C H A P T E R IV
RESULTS A N D DISCUSSION
Aerobic In cu b atio n of Whole C o lli
The d a ta found in Tables I I I - V II summarize th e r e s u lts of
aerobic in cu b atio n s of whole ameba e e lls • From Table I I I i t can be
seen th a t ameba c e lls which were incubated w ith no added s u b s tra te ,
except th e sm all amounts of re s id u a l s ta rc h which could not be washed
o u t, produce v ery l i t t l e l a c t i c acid* Ho acid was produced by e ith e r
of the b a c te r ia l co n tro ls used in th is assay* The low degree of
endogenous a c tiv ity observed in th is experim ent is s ig n if ic a n t when
compared to th e amount of endogenous a c t i v i t y obtained w ith whole c e lls
in an anaerobic atmosphere (see Table V III) • In Table V III i t can be
seen th a t 5 to 7 tim es more la c tic acid was produced a n a e ro b ic a lly
th an under aero b ic co n d itio n s w ith but o n e -th ird th e number of amebae •
This o b serv atio n o ffe rs evidence in support of th e view , to be d isc u s s
ed l a t e r , th a t amebae are organisms p o ssessin g a g ly c o ly tic m etabolism*
As can be noted in Table I I I , no d eterm in atio n s of b a c te r ia l
p opulations were made in experim ents 1-5, and no co ncentrated f lo r a
were assayed in th ese experim ents • I t was assumed th a t th e re s id u a l
b a c te ria in th e ameba c e ll p re p a ra tio n would not produce la c tic acid
in th is aero b ie system* This assum ption was s u b sta n tia te d by ex p eri
ments 4 and 5 in which b a c te r ia l c o n tro ls were assayed and were found
T A B L E I I I
M IC R O G R A M S O F LA CTIC ACID P R O D U C E D
PER MIILILITER O F M E D IU M BI RESTIN G A M ER AF. C E L L S
Amebae ce lls
Control
( la s t wash)
Concentrated flo ra
Experiment
Incubation -,/
Time in hours-'
Amebae ytal. Bacteria/ml.
Incubation
Time in hours Bacteria/ffll#
Incubation
Time in hours
Bacteria/m l.
0 2 U
0 2 h
C M
o
1 0.5 1.0 1.0 7.5xL05 0.1 0.1
0.0
2 0.0
0.5 0.5
U.lxlO* 0.0 0.0 0.0
3 0.5 1.5
0.8 7.5xl05
0.5 0.0 0.0
h 0.1 1.0 1.0 6.9x10'* 19xL06 0.0 0.0
6
0.0 7zL0 0.0 0.0 0.0 88xl06
5 0.1 0.8 0.8 7.1*10* 3 8 x 1 0 6 0.0 0.0 o .o 2k a o 6 0.0 0.0 0.0 U90xl06
\J A^l incubations n e n caxried out a t 37*5° C under aerobic conditions*
TABLE IV
AEROBIC FORMATION OF LACTIC k C H ^ FROM SODIUM PYRUVATE^
Experim ent In cu b atio n In cu b atio n In cu b atio n
Time in hours j^ b a e /m l. B acte ria/m l. Time in hours B acteria/fcL. Time hours B ac te ria /m l.
0 2 h 0 2 h 0 2 U
1 .6 x l0 6 0.2 0 .0 0 .0 2 .6 x l0 6 0 .0 0 .0 0 .0 200.0xl06
13*2xl06 0 .0 0 .0 0 .0 18.0 xL 06 0 .0 0 .0 0 .0 lOOO.QxlO6
2 .8 x l0 6 0 .0 0 .0 0 .0 3 .2 x l0 6 0 .0 0 .0 0 .0 220.0xl06
1 / A ll r e s u lts are expressed as micrograms o f la c tic ac i'4 produced/m l. of medium.
2 / The f i n a l co n c en tratio n o f sodium pyruvate in th ese experim ents was 1.0x10”^ M
1 0.0 0.0 0.0 l.b ao *
2 0 .1 0 .0 0 .0 li.lxlO ^
3 0 .0 0 .0 0 .0 2.UxlO^
TABLE V
EFFECT OF CYSTEINE^/ ON THE AEROBIC FORMATION QF LACTIC AC!
Experiment 1
6 6
Experiment 2
T est System
Incubation
Time in hours
0 2 1 *
Amebae/ml. Bacteria/mL.
Incubation
Time in hours
0 2 1 *
Amebae/ml. B acteria/oil.
Amebae
4*
Cysteine • H C 1
0.2 1.7 0 .5 3.2x10^ 1.8xl06 0.0 1.2 1 .0
•
C M
l .l a i o 6
C ontrol ( la s t trash)
C ysteine • H C 1
1.2 0.5 0.0 3.2xl06 1.2 1.0 0.5 3.2x10^
Concentrated F lo ra
4*
Cysteine * H C1
0.0 0.0 0.0 I5ii.0xl06 0.0 0.0 0.0 88.0x10^
1 / The fin a l concentration of cysteine • H C 1 in these experiments iras 1.6x10”^ M
2 / A ll re s u lts are expressed as micrograms of la c tic acid produced/ml, of medium
TA BLE V I
EFFEC T OF C Y ST E IN E O il T H E A ERO BIC FORMATION
OF LA C T IC A C I D - / FROM PYRU V IC A C ID
E xperim ent 1 E xperim ent 2
T e st System
In cu b atio n
Time in .hours
0 2 it
Amebae/ml. B a c te ria /m l.
In c u b a tio n
Time in hours
0 2 U
Amebae/ml. ' B a c te ria /m l.
Amebae
C y stein e • H Cl^/
Sodium P y ru v a te 3 /
o*o U .i o«6 13.0x10^ 1 .6 x l0 6 0 .0 2 .0 0 .7 5.0x10** l.O xlO 6
C o n tro l (L a st wash)
+
C y stein e • HC1
+
Sodium P yruvate
0 .0 0 .0 0 .0 2 .6 x l0 6 0 .0 0 .0 0 .0 it.5x10^
C oncentrated F lo ra
+
C y stein e • HC1 0 .0 0 .0 0 .0 130. 0x l06 0 .0 0 .0 0 .0 6it.0xl0^
Sodium P yruvate
1 / A ll r e s u lts a re expressed as micrograms o f l a c t i c a c id produced/m l. o f medium
2 / F in a l c o n c e n tra tio n o f c y ste in e • HC1 in a l l o f th ese experim ents was 1.6x10*”^ M
3 / F in a l c o n c e n tra tio n o f sodium pyruvate in a l l o f th ese experim ents was l.O xlC - ^ M
TABLE VII
UTILIZATION OF LACTIC ACIdI/ A D D E D
T O SUSPENSIONS OF A M EB A E CELLS A N D OF BACTERIAL CELLS
Amebae C e lls C o ntrol ( l a s t mash) ,-i C oncentrated f l o r a
L a c tic a c id
c o n tro l
Exp. In c u b a tio n ? /
Time in hours
Amebae/ml. B a c te ria /m l.
In cu b atio n
Time, in hours
B a c te ria /m l.
In cu b atio n
Time in hours
B a c te ria /m l.
In c u b a tio n
Time in hours
0 2 h 0 2 h
° r * : ' 2
r.:
h 0 2 h
1 8.3 3 .3 0.3 k.2xlC p 1U7x106 8 .8 1 .0 0 .2 8bd.’ 06 8 .5 ■ a%3
■
0 .0 Ii320x l06 9 .0 8 .8 9 .0
2 15.3 5 .0 0 .3 0.7x10$ 22*106 U .8 5 .0 1 .3 5 x l0 6 1U.8 3 i0 0 .0 :.96xl06 1 5 .0 U . 7 1 5 .0
3 20.5 1 6 .3 '6.0 o.Uxio5
7xl06 2 11.0 8 .0 6*106 2 0 .5 1 6 .8 ^ 3 .0 r ‘ # 6 x io 6 • 2 0 .3 2 0 .0 2 0 .0
h 9.0 7 .0 3 .0 O.UxlO$ 17xl06 9 .0 6.3
1 .3
12xl06 8.8 1 .8
*
' 0.0* 190xL06 8 .3 8 .8 8 .3
1 / R esu lts a re expressed as micrograms o f l a c t i c a c id rem aining/m l. o f medium
2 / A ll in cu b atio n s c a r r ie d o u t a t 3 7 .5° C under aero b ic c o n d itio n s
TA BLE VIII
A N A E R O B IC PR O D U C T IO N O F LA CTIC ACID^ F R O M S O D IU M PY RU V A TE^
Experiment 1 Experiment 2 Experiment 3
Test system
Incubation
Time in hours Bacteria/m l.
Incubation
Time in hours ^og^ae/ml. B acteria/m l.
Incubation
Time in hours
Amebae/ml. B acteria/m l.
0 2
k 0
2 h 0 2 h
Amebae 0*0
1.5
3.5 2.5x10* I5xl06 0.0
1.3
2.5 2.1*xl0* 17xl06 0.0
2.5 U.3 l.fcdO* 17xl06
Amebae
Sodium Pyruvate
0.0 2.0 lu3 2.5xl06 I5xl06 0.0 li.8 8.0 2.LxlO* 17xl06 0.0 U.8 7.0 1.9x10* 17xl06
Control
( la s t wash)
0.0 0.0 0.0 21x106 0.0 0.0 0.0 12xl06 0.0 0.0 0.0 lUxlO6
Control
( la s t wash)
0.0 0.0 O.U 21xl06 0.0 0.0 0.0 12xl06 0.0 0.0 0.0 t o o 6
Sodium Pyruvate
1 / All re su lts are expressed as micrograms of la c tic acid produced/ml. of medium.
2 / The fin a l concentration of sodium pyruvate in these experiments was 1.0x10 M
40
I
t
to produce no la c tic a c id , even w ith th e b a c te ria per ml* numbering as
high as 490 x 10 .
The d a ta from th e experim ents in which sodium pyruvate was th e
su b stra te employed in th e aerobic in cu b atio n s tu d ie s are given in
Table IV* A ll v alu es have been co rrec te d fo r th e pyruvate co n tro l*
This ta b le shows th a t l a c t i c acid is not accumulated from pyruvate
* a e ro b ic a lly by any of th e c e ll p re p a ra tio n s • These r e s u lts are in
. accord w ith th e g e n e ra lly accepted view th a t l a c t i c acid is only
produced from pyruvic acid d u rin g anaerobic ferm en tatio n * In animal
tis s u e s red u ctio n of pyruvic acid to la c tic acid occurs under condi
tio n s of low oxygen te n s io n , sin ce under such co n d itio n s D PN is
, a v a ila b le c h ie fly in th e reduced form . In th e se experim ents e a rrie d
out under aero b ic co n d itio n s DPU e x is ts p rim a rily in th e oxidized form
hence th e form ation of la o tic acid v ia pyruvic acid red u c tio n w ill not
o c c u r•
Table V p resen ts d a ta obtained when c y ste in e was used as a
su b stra te * A ll valu es have been co rrec te d fo r th e cy ste in e co n tro l*
This amino acid was used to determ ine i t s e f fe c t on th e aerobic
form ation of la o tic acid p rim a rily because o f i t s ro le in th e a c tiv a
tio n of m etab o lio a lly s ig n if ic a n t enzymes such as trio se p h o sp h a te
dehydrogenase (9 9 ), and se c o n d a rily because of i t s suggested ro le as
a reducing substance fo r m ain tain in g low oxygen te n sio n in amebae
c u ltu re s (2 0 )• The d a ta in Table V in d ic a te th a t c y ste in e does
stim u la te endogenous l a c t i c acid production by ameba o e lls , e s p e c ia lly
when th is inform ation is compared to th a t of Tahle I I I * The r e s u lts
obtained w ith e y s te in e , suggest th a t i t may be a su b stra te fo r la c tic
acid form ation in th is aerobic system . In a d d itio n i t may have a
p o ssib le ro le in aerobic g ly c o ly sis even thouggi In tn e r and Anderson
(64) have in d ic a te d th a t in anaerobie endogenous m etabolism , c y ste in e
is not a s u b s tra te f o r la c tic acid production by amebae. The exact
ro le of cy ste in e is not y et c le a r and f u rth e r stu d ie s should be made
to a s c e rta in i t s fu n c tio n in amebic g ly c o ly s is . However, one
a d d itio n a l s e rie s of experim ents was c a rrie d out in which th e e f f e c t
of c y ste in e on th e form ation of la c tic acid from pyruvic acid was
stu d ie d , and th e se r e s u lts are summarized in Table V I. A ll v alu es
have been co rre cted fo r th e pyruvate c o n tr o l. Prom t h i s d a ta , i t is
seen th a t la c tic acid form ation has again been stim u la te d , but th a t
stim u la tio n occurs only in th e f i r s t two hours of a s sa y . When th e
l a c t i c acid v alu es in Table VI are compared, on th e b a sis of acid
produced per 50,000 amebae to th o se of experim ents in which o y stein e
is th e only s u b s tra te (see Table V ), th e r e s u lts suggest th a t th e
f a te of c y stein e in both experim ents may be v ia some mechanism o th er
th an th a t of conversion of pyruvate to l a c t a t e . I f th is ex p lan atio n
is c o rre c t c y ste in e could be converted d i r e c t ly to la o tic a c id . For
t h is re a c tio n to occur a s e rie s of step s in v o lv in g d e s u lfu ra tio n w ith
sim ultaneous h y d ro ly tic deam ination would be re q u ire d . In support of
t h is concept i s th e re c e n t work of Saz and Brownell (100) who showed
th a t o y stein e was converted a e ro b ie a lly to pyruvic acid by e x tra c ts of
42*
; E sc h e ric h ia c o l i » Hydrogen s u lfid e gas and ammonia were produced dur
ing th e re a c tio n th u s su g g estin g th a t th e mechanism involved was a
d e s u lfu ra tio n coupled w ith an o x id ativ e deam ination. S im ilar re a c tio n s
could a lso occur in th e am ebae-cysteine systems te s te d w ith th e
a d d itio n a l s te p in v o lv in g th e red u c tio n of pyruvic acid to l a c t i c
acid v ia la o tic dehydrogenase•
Another p o s s ib ility is th a t th e fu n c tio n of c y ste in e is to
m aintain a low oxygen te n sio n th u s c re a tin g an e s s e n tia lly anaerobic
system which would fav o r la c tic acid production from endogenous
s u b s tr a te s •
C ysteine may a lso stim u la te la c tic acid form ation by a c tiv a tin g
l a c t i c dehydrogenase (101) and o th er enzymes im portant in g ly c o ly s is •
In both of th ese experim ents, g re a te s t la o tic acid form ation
occurred in the f i r s t two hours of incubation* In th e l a s t two hours
of fo u r-h o u r in c u b a tio n s, la c tic acid v alu es are g re a tly reduced* In
th e experim ents s e t up t o stu d y th e e f fe c t of la o tic acid as a sub
s tr a t e fo r amebic m etabolism (Table V II), i t is ev id en t th a t when
la c tic acid is added to ameba c e ll p re p a ra tio n s i t is r a p id ly u tiliz e d
in a l l of th e t e s t systems • This would in d ic a te th a t disappearance of
la c tic acid could be e ith e r an ameba fu n c tio n or b a c te ria l* I f due
only to th e b a e te ria th e n i t is p o ssib le th a t a sym biotic r e la tio n s h ip
may e x is t between b a c te ria and amebae a t the le v e l of pyruvic acid
conversion to la c tic acid* S ta te d in o th er term s, i t o ffe rs th e
p o s s ib ility th a t amebae are capable of b reaking down s u b s tra te but
only w ith th e h elp of some b a c te r ia l fu n c tio n , probably aerobic in
n atu re* This concept is p a r t ia ll y supported by th e work of an
a sso c ia te in t h i s la b o ra to ry who has dem onstrated th e presence of a
trio sep h o sp h a te dehydrogenase in amebae, b u t none of th e dehydro
genases p resen t in th e K re b ^ cycle (102)* A ll of th e dehydrogenases
te s te d fo r and found to be absent in amebae, however, are p resen t in
b a c te ria norm ally growing w ith amebae*
Anaerobic Incubations of Whole C ells
When stu d y in g anaerobic production of l a c t i c acid by Bndamoeba
h is to ly tic a , pyruvic a c id , th e only s u b stra te employed, was found to be
converted to la c tic acid* The d a ta from th e se experim ents are to be
found in Table V III* The conversion of pyruvic acid to la c tic a c id ,
o ccu rrin g in th e anaerobic system but not under aero b ic c o n d itio n s,
is regarded as evidence fo r th e presence of la c tic dehydrogenase in
amebae •
C e llu la r F rac tio n s
The a b i l i t y of v ario u s c e llu la r fra c tio n s t o form la c tic acid
a n a e ro b ic a lly from se v e ra l s u b s tra te s im portant in g ly c o ly sis was
determ ined in a s e rie s of 6 ex p erim en ts• The d a ta from th e se
in v e stig a tio n s are summarized in Table IX*
One of th e most im portant observations made in th e d a ta in
Table IX is th a t no la c ta te form ation occurs fo r any ly s a te (so lu b le
component) f r a c tio n f o r any of th e c e ll systems involved • A ll
n a u n
u u m c m m a im a t tm v t u sx A t w a n s c a u u s m em o s
44
dalbas Control (la s t sash)
j Caestntrstad flora
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farU eolstsa I/sa'M parUcalrtas IjSftte to tM a M
- 1 s 3 1 * 5 6 ? /
k p a rla a t
1 8 3-6 1 8
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3 ^ 5 ^
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& 9 erlaaat
3V # $ ^
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0 0
0.0 0 4
M M M
stank
(5*/U .>
0.0 0.0
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( S o M .) ,
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6
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f f ] S m ^ " ,,h ,d ^ > ) r ** d ssOUsht «* » ■ so th* see ■ * ! « ta M s d a lth S .S of B U m r'e llw r n c w t n t t ,
J1 ' “ ^Wiwf'iiSS&JSSSnSiSu'**' ■ " “■ « « “ * • * > * •* ) » < -
B fci
45
a c tiv ity re sid e s in e ith e r th e amebae c e ll re sid u e obtained a f te r
l y s i s , or in th e co ncentrated f lo r a obtained e ith e r a f te r ly s is or
used as whole u n tre a ted c e lls • This d a ta o ffe rs evidence th a t
r
g ly c o ly tic a c tiv ity in End amoeba h is to ly tic a is a sso c ia te d w ith unknown
p a r tic u la te s tru c tu re s of th e ameba c e l l . This view i s supported from
th e d a ta in experim ent 5 obtained w ith th e c e ll fragm ents of th e c o n tro l
( la s t wash) • The fragm ents in th is experim ent r e fe r to th e m a te ria l
p resen t in th e supernate a f te r low speed c e n trifu g a tio n (500 RPM) of
th e re sid u e a f te r ly s is • fo rm ally th e re sid u e was sep arated by
c e n trifu g in g a t 15,000 RPM which re s u lte d in a w ater e le a r su p e rn a te .
From th e d a ta of experim ent 5 i t is seen th a t th e a c t i v i t y in th e
c o n tro l ( la s t wash) fo r la c ta te production from glucose is higher th an
th a t of th e resid u e co n tain in g th e amebae fragm ents , The la c tic
acid is not produced by th e r e s id u a l f lo r a sin ce th e number of b a c te r ia
£
p resen t (27 x 10 /m l,) would account fo r only 3 micrograms of la c tic
acid as shown in F igure 2 , Hence i t appears th a t th e production of
la c tic acid from glucose is an enzyme fu n c tio n a sso c ia te d w ith amebae
c e ll fragm ents of such d e n sity th a t th ey could not be cen trifu g ed
down a t 500 RPM,
The d a ta fo r th e la c ta te production by amebae fragm ents are
rep resen ted by th e v alu es recorded fo r endogenous a c t i v i t y . Since i t
was not p o ssib le to wash th e resid u e c o n tain in g amebae fragm ents
fre e of th e r ic e s ta rc h , th e re s id u a l endogenous la c tic acid formed
was assumed to r e s u lt from ameba a c tiv ity on th e re s id u a l s ta r c h .
4 6.
C M
_ O
CD 0 0
aiov oiiovi jo swvbooaoiw
PIG* 2* P roduction of la c tic acid from glucose by "bacteria
obtained from amebae c u ltu r e s • J
NUMBERS O F BACTERIA ( X 10
4 7.
A ll of th e d a ta on th e ferm en tatio n of glucose by ameba c e ll p a rtic u
la te s are summarized and recorded in Table X . Exam ination of t h i s
ta b le re v e a ls c le a r ly th a t l a c t i c acid is produced from g lu c o se . The
low value obtained in experim ent 6 oannot be explained except on th e
b a sis th a t th e amebae were c u ltu re d on M C C medium enrich ed w ith
a d d itio n a l liv e r c o n c e n tra te . The e x tra c o n c en tra tio n of th e liv e r
f ra c tio n may have a ffe c te d th e m etabolic mechanisms involved in
, glucose fe rm e n ta tio n .
; In th e study of th e e f fe c ts of sodium flu o rid e and io d o acetic
acid on th e ferm en tatio n of glucose, th e d a ta show th a t th e r e la tiv e
in h ib itio n of glucose m etabolism by c e ll p a r tic u la te s in th e presence
— 3
of 5 x 10 M sodium flu o rid e is g re a te r th an th a t observed fo r th e
concen trated f l o r a . This d iffe re n c e is emphasized in Table XI where
I
th e in h ib itio n d a ta of Table IX have been summarized and presen ted as
th e per cent in h ib itio n of glucose ferm en tatio n by amebae c e l l
p a r tic u la te s compared w ith th e in h ib itio n obtained w ith concentrated
f lo r a as a source of enzyme a c t i v i t y . From Table XI i t is seen th a t
th e average in h ib itio n of amebae p a r tic u la te s is 31 per cent and th a t
of f lo r a about 10 per c e n t. These v alu es are not com patible w ith th e
r e s u lts obtained fo r th e in h ib itio n of animal tis s u e g ly c o ly sis in
w hich, depending on th e n atu re of th e tis s u e being stu d ie d , from 75
per cent to 95 per cent in h ib itio n is obtained w ith 5 x 10“ ® M
flu o rid e (103-104). Two ex p lan atio n s fo r th ese d a ta are p o s s ib le .
F i r s t , th e v alu e o b tain ed , although in d ic a tin g some in h ib itio n of
TABLE X
LACTIC ACID FORMATION^ FR O M G LU CO SE
B Y A M E B A E CELL PARTICULATES
Experiment
A B C D E
Endogenous Glucose (B-A)
Lactate formed
by residual
flora
Corrected
value
(C-D)
1 8.3 19.5
11.2 6.1* 1*.8
2
1.5
7.8
6.3 2.0
li.3
3
0.8 12.0 11.2 2.8 8.U
h 0.8
7.3 6.5 2.0
U.5
5 13.5 23.3
9.8 2.0 7.8
& 1.3 3.5 2.2 v 1.2 1.0
1 / All values expressed as micrograms of lactic acid/ml. of incubation
m edium
2 / In this experiment the am ebae were grown on m cc m edium enriched
with 0.5$ liver concentrate N. F.
TABLE X I
PER CENT INHIBITION O F LACTATE FORM ATION FR O M GLUCOSE
BY 0,005m SODIUM FLUORIDE A N D 0.005m IODOACETIC ACID
5*0 x 10~^ M Sodium Fluoride 5.0 x 10”3 M Iodoacetic Acid
Test System
1 2
Experiments
3 U 5 A V . 1 2
Experiments
3 h 5 A V .
A m ebae Particulates
38*5 18.3 52.0 36.5 10.0 31.0 92* 0 ^ 93.7 97.0 99.5 100.0 97.6
Concentrated Flora 0.0 16.7 0.0 22.7 9.9 96.9 92.3 97.0 81.0 91.8
1/ This experiment done using 0.001M iodoacetate and is not used in calculating averages*
r t*
c o
5 0.
en o lase, could be low, due e ith e r to an in s e n s itiv ity of th e enzyme of
th ese c e llu la r p re p a ra tio n s to flu o rid e in h ib itio n or to th e presence
of substances in te r f e r in g w ith th e a c tio n of flu o rid e ions • Secondly,
th e observed in h ib itio n may be due to b lo ck in g of the a c tiv ity of o th er
enzymes im portant in g ly co ly sis ra th e r th a n ju s t th e a c tiv ity of
enolase • C oncentrations of flu o rid e of from 5 x 10“ ^ M to 5 x 10“ ^ m
in h ib it e f fe c tiv e ly a v a r ie ty of m e ta b o lic a lly im portant enzymes such
as gluco-6-phosphatase (105), adenylic k in ase (106), su ccin ic
dehydrogenase (107), and pyrophosphatase (1 0 8 ).
The exact n atu re of th e observed in h ib itio n could be determ ined
by experim ents in which an accum ulation of D -2-phosphoglyceric acid
could be dem onstrated in a system of amebae c e ll p a r tic u la te s , glucose,
and f lu o r id e . A p ilin g up of D-2-phosphog ly ce ric acid would be evidence
fo r th e presence of e n o la s e •
C e llu la r p e rm e ab ility must a lso be considered (Table IX ), and
e s p e c ia lly in th e experim ents w ith concen trated f lo r a , sin ce th e
co ncentrated f lo r a used in th e flu o rid e in h ib itio n stu d ie s were w hole,
u n tre ated c e lls • The low percentage of in h ib itio n obtained w ith
flu o rid e in experim ents w ith concentrated f lo r a may be due to f a ilu r e
of th e in h ib ito r to p e n e tra te th e in ta c t c e l l . However, th e d a ta show
92 per cent in h ib itio n of b a c te r ia l fe rm e n ta tio n of glucose by iodo
a c e tic acid and 98 per cent w ith amebae c e ll p a r tic u la te s , implying
th a t th i s in h ib ito r gets in to th e c e ll* These observ atio n s suggest
th a t c e llu la r p e rm e ab ility may not be th e only ex p lan atio n fo r th e
| 5 1 .'
I I
: r e s u lts obtained w ith f lu o r id e . The sig n ific a n c e of th ese o b se rv atio n sj
'w i l l be discu ssed l a t e r .
B a c te ria l Ferm entation of Glucose
i
The d a ta obtained on th e anaerobic ferm en tatio n of glucose by
th e b a c te ria norm ally p resen t in c u ltu re s of th e D K B s tr a i n of amebae
are given in F igure 2 . The amount o f la c tic acid formed from glucose
appears to be a fu n c tio n of th e number of b a c te ria p r e s e n t. These
r e s u lts are im portant because in a l l th e assays conducted on ameba
p re p a ra tio n s, th e re s id u a l numbers of b a c te ria per m l. of re a c tio n
medium were in th e range of 10-40 m illio n . From F ig u re 2 i t is
. apparent th a t la c ta te v alu es obtained fo r am ebae-free b a c te r ia l system s,
compared to th e b a c te r ia l ranges in ameba p re p a ra tio n s, are of such
minimal magnitude th a t re s id u a l b a c te r ia l a c tiv ity cannot be s ig n i f i
can t in th e assays c a rrie d out on amebae p re p a ra tio n s . F u rth e r, in
a l l experim ents inv o lv in g th e c o n tro l ( la s t wash) no la c tic acid was
produced endogenously or from added s u b s tr a te s , whereas w ith th e
co n cen trated f lo r a , la c ta te was formed even by r e la tiv e ly few b a c te r ia .
This d iscrep an cy in a c t i v i t y in glucose ferm en tatio n between c o n c e n tra t
ed f lo r a and c o n tro l ( la s t wash) is b eliev ed due to a d iffe re n c e in th e
type of b a c te ria in each of th e c o n tr o ls . The b a c te ria growing in
in tim ate co n tact w ith th e amebae rep resen ted by th e c o n tro l ( la s t wash)
p re p a ra tio n s , may possess a d if f e r e n t ty p e of m etabolism th a n those
growing near the su rface of a c u ltu re tu b e (co n cen trated f lo r a ) •
52 •
Experiments u sin g m onobaoterial c u ltu re s of amebae would h elp to
c la r if y th is problem*
F in a lly , in tie w of th e tendency of amebae to ly se i t is
p o ssib le th a t th e c o n tro l ( la s t wash) would co n tain amebic resid u e
in a d d itio n to b a c te ria * This view might th u s account fo r some of
th e d iffe re n c e s observed in th e d a ta on glucose ferm en tatio n between
th e co n tro l ( la s t wash) and th e concentrated f lo r a as w ell as r e s u lt
in an o v erco rrec tio n of th e d a ta obtained fo r amebae ferm en tatio n of
g lu co se•
C H A P T E R V
INTERPRET ATI O H O F RESULTS
G lycolysis today g e n e ra lly im plies th e breakdown of carbo
hy d rates by ferm en tativ e f is s io n w ithout th e in te rv e n tio n of
atm ospheric oxygen* As a r u le , a fu rth e r r e s t r i c t io n is th a t such a
ferm en tatio n r e s u lts in a c id ic products of which la c tic acid is th e
predominant one in animal tis s u e s * The d a ta p resen ted in th i s
d is s e r ta tio n o ffe r evidence fo r th e presence in End amoeba h is to ly tic a
of some of th e step s of animal tis s u e g ly c o ly sis • The f i r s t phase of
t h i s d isc u ssio n w ill cen ter about th e g ly c o ly tic m etabolism of th e
ameba* The second phase w ill deal w ith some asp ects of th e carbo
hy d rate m etabolism of th e b a c te r ia l symbionts norm ally growing w ith
th e D K B s tr a i n of amebae, and th e p o ssib le ro le of th e se f lo r a in th e
m etabolism of End amoeba h i s t o l y t i c a *
The o b serv atio n th a t s ta rc h , so lu b le or p a r tic u la te , can be
converted t o l a c t i c acid a n a e ro b ic a lly by Endamoeba h is to ly tic a c e ll
components suggest th e presence of th e enzyme, phosphorylase•
Credence f o r th e presence of phosphorylase is given by th e f a c t th a t
d is s o lu tio n of in g ested s ta rc h granules oocurs wi-thin th e c e l l (35)*
This disappearance of s ta rc h w ith in th e ameba c e ll could be due e ith e r
to amylase a c tiv ity or to phosphorylase a c tiv ity *
Endamoeba h is to ly tic a probably possesses a glucokinase as seen
in th e d a ta in which la c tic acid is produced from glucose • “ Whether
a lin k between glucose and s ta rc h is p re se n t, could be a sc e rta in e d
through th e dem onstration of th e re v e rs ib le conversion by phospho-
glueomutase of glucose-6-phosphate to g lu c o se -l-p h o sp h a te * However,
a t t h i s tim e i t is g e n e ra lly acoepted th a t th e common r e s u l t of
a c tiv ity of both glucokinase and phosphorylase in g ly c o ly tic m etabolism
is th e production of th e common in term ed iate of th e main stream of
g ly c o ly sis , glueose-6-phosphate; th u s r e la tin g both apparent ty p es of
enzyme a c tiv ity to th e concept th a t amebae possess g ly c o ly tic m etabol
ism* The d a ta on s ta rc h and glucose ferm en tatio n suggest th en th a t
th e e a rly stag es of anaerobic catabolism of carbohydrates in amebae
appear to be s im ila r to th a t of o ther animal tis s u e s •
Evidence fo r th e presence in Endamoeba h is to ly tic a of enzymes
o th er th an glucokinase and phorphorylase involved in th e i n i t i a l stages
of carbohydrate ferm en tatio n cannot be obtained from th e p resen t data*
However, th e experim ents perform ed in which glucose ferm en tatio n was
— 3
in h ib ite d by 5 x 10 M io d o a c e ta te , as evidenced by dim inished la c tic
acid form ation, in d ic a te th e presence of th e enzyme trio sep h o sp h a te
dehydrogenase* I t is known th a t th e in h ib ito ry a c tio n of io d o acetic
acid on th is enzyme is q u ite s p e c ific w ith e f fe c tiv e in h ib itio n
o ccu rrin g a t a c o n ce n tra tio n of 1 x 10"^ M (109)* At hig h er concentra
tio n s io d o acetic acid w ill in h ib it many su lfh y d ry l co n tain in g enzymes
(110), but i t s a c tio n is s t i l l more s p e c ific fo r trio sep h o sp h a te
dehydrogenase th an fo r th e o th er enzymes* The e f fe c ts of io d o acetic
aoid on glucose ferm en tatio n by amebae c e ll p a r tic u la te s in d ic a te th a t
, th e tr io s e stag© of g ly c o ly sis in amebae, invo lv in g th e conversion of
' 3-phosphoglyceraldehyde to 1 ,3-diphosphoglyceric a c id , is th e same as
th a t in animal t i s s u e s . The rem aining, f in a l stag es of th e g ly co ly sis
of carbohydrates in amebae are only p a r t ia ll y resolved* The in h ib itio n
stu d ie s w ith sodium flu o rid e suggest enolase a c tiv ity * The conversion
of a 2-phosphoglyceric acid to phosphoenolpyruvic acid cataly zed by
1 enolase is an im portant re a c tio n because i t is a step in which u t i l i z -
able energy is made a v a ila b le fo r work* The d a ta on t h i s phase of th e
in v e stig a tio n s suggests th e p o ssib le presence of e ith e r a flu o rid e
in s e n s itiv e en o lase , extrem ely high c o n c e n tra tio n of en o lase , or none
at a ll* I f th e enolase of Endamoeba h is to ly tic a is in s e n s itiv e to
flu o rid e io n , th e production of la c tic acid from ferm en tatio n of
glucose or s ta rc h could be explained on th e b a s is of a need fo r a more
s p e c ific in h ib ito r* I f th e co n ce n tra tio n of enolase is v ery h i$ i, th en
' th e u su a lly excepted in h ib ito ry co n c en tratio n of flu o rid e would be
only p a r t ia ll y e ffe c tiv e in blocking enolase a c tiv ity and some la c tic
acid could a ris e v ia th e f in a l stag e s of g ly co ly sis* F in a lly , i f th e re
is no enolase p resen t in amebae th e la c tic acid found may be o r ig in a t
ing from sources o th er th an th e normal sequence of ste p s in anaerobic
g ly co ly sis in v o lv in g pyruvie acid conversion to la c tic acid* Some of
th e se p o ssib le pathways are th e phosphoglyceraldehyde-m ethyl glyoxal
s e rie s of re a c tio n s suggested by Myerhof (1 1 1 ), th e anaerobic dism uta-
tio n of pyruvic acid cataly zed by e ith e r a sin g le enzyme (112) or as a
coupled oxidoreduction inv o lv in g th e la c tic dehydrogenase and th e
5 6.
|pyruvic oxidase system of Lippman (113), th e d ecarb o x y latio n of both
1-m alie and o x alo acetic acid s to la c tic and pyruvic acids re s p e c tiv e ly
(114), or through th e form ation of aceto in from pyruvic acid in which
re a c tio n s a -a o e ty lla c tie acid is a p o ssib le in term ed iate (1 1 2 ).
The o b serv atio n th a t pyruvic acid is converted to l a c t i c acid
a n a e ro b ic a lly by Endamoeba h i s t o l y t i c a , considered w ith th e o b serv atio n
th a t some enolase a c tiv ity is p re se n t, f u rth e r suggest th a t th e f in a l
stag es of anaerobic ferm en tatio n of carbohydrates by Endamoeba
; h is to ly tic a are id e n tic a l to th o se of animal t i s s u e s .
Aerobic stu d ie s made w ith in ta c t ameba c e lls showed th a t la c tic
acid was ra p id ly u tiliz e d , and a p p aren tly by th e b a c te r ia l f l o r a . This
o b serv atio n ex p lain s in p a rt th e re d u c tio n in l a c t i c acid produced in
experim ents in which endogenous amebic g ly c o ly tic a c t iv ity was measured
The low v alu es obtained can a lso be explained on th e b a sis of th e need
of anaerobic co n d itio n s befo re g ly co ly sis may p ro ceed . The la c k of
in creased la c tic acid form ation from pyruvate in aerobic stu d ie s is
c o n s is te n t w ith th e view th a t amebae possess g ly c o ly tic m etabolism .
The e f fe c t of cy stein e on la c tic acid production in aerobic stu d ie s
may be due to i t s use as a s u b s tra te , as alread y in d ic a te d , or more
probably to i t s e f fe c t on th e a c tiv a tio n of trio sep h o sp h a te dehydro
genase and o th er s u lfh y d ry l-c o n ta in in g enzymes of m etabolic im portance.
When considered in to to a l l of th e se observations obtained in th e
aerobic phases of th is stu d y support th e view th a t g ly co ly sis is
p resen t in amebae, but a lso suggest th a t amebae may co n tain some
aerobic mechanisms f o r the form ation of la c tic acid#
B a c te ria l Data
Exam ination of th e d a ta c o lle c te d on b a c te r ia l f lo r a re v e a ls
th a t th e b a c te r ia a sso c ia te d w ith th e amebae have a m etabolic p a tte rn
fo r th e anaerobic ferm en tatio n of carbohydrates q u ite sim ila r to th a t
assumed fo r th e amebae# The only in c o n siste n c y in th is s im ila r ity was
observed in th e stu d ie s on flu o rid e in h ib itio n in which th e b a c te r ia l
ferm en tatio n of glucose was in h ib ite d only 10 per cent w hile th a t of
amebae was in h ib ite d 31 per cent# One p o ssib le ex p lan atio n fo r th e
low value in b a c te r ia may be due to c e llu la r p erm ea b ility - th e
flu o rid e ion not being ab le to p e n e tra te in to th e c e ll# Another
p o s s ib ility is th a t th e value obtained is due to th e in h ib itio n of
enzymes o th er th a n enolase as p rev io u sly suggested fo r amebae fragm ents
F u rth er in v e s tig a tio n would determ ine i f t h i s d iffe re n c e in observed
flu o rid e in h ib itio n between b a c te r ia l p re p a ra tio n and amebae p rep ara
tio n s is s ig n if ic a n t, e s p e c ia lly in term s of a sym biotic re la tio n s h ip
between f lo r a and amebae a t th e enolase stag e of g ly c o ly tic metabolism#
The d a ta fo r a c t iv ity of th e f lo r a c o lle c te d in the aerobic stu d ie s
o ffe r th e p o s s ib ility th a t th e sym biosis of b a c te r ia and amebae e x is ts
a t another le v e l of m etabolism . Since b a c te r ia u tiliz e d la c tic acid
q u ite ra p id ly and amebae produce t h i s compound as an end product of
carbohydrate m etabolism , i t is conceivable th a t th e b a c te ria become
in tim a te ly involved in amebae m etabolism a t t h is le v e l of g ly c o ly s is .
5 8.
This could he through some mechanism in v o lv in g pyruvic oxidase or
enzymes of a sim ila r n a tu re .
The ex p lan atio n of th e e f fe c t of b a c te ria on la c tic acid
form ation in experim ents in which cy ste in e was a s u b s tra te is not
known. I t must be emphasized, a ls o , th a t th e dependence of amebae
on b a c te ria or o th er liv in g c e lls fo r su rv iv a l is s t i l l not u n d ersto o d .
C H A P T E R VI
S U M M A R Y
When w hole, washed amebae c e lls of th e D K B s tr a in were incubated
a e ro b ic a lly in a m odified S tone’s B u ffer, no l a c t i c acid was produced
from e ith e r endogenous s u b s tra te s or sodium p y ru v ate . The a d d itio n of
c y ste in e to th e aerobic systems e ffe c te d a s tim u la tio n of la c tic acid
from th e endogenous s u b s tra te but not from sodium pyruvate •
A naerobically la c tic acid was accumulated from sodium pyruvate
by whole amebae c e lls •
A stu d y of fra c tio n a te d amebae c e l l components showed th a t the
in so lu b le fra c tio n s ( p a rtic u la te s ) were capable of ferm enting r ic e
s ta rc h , so lu b le and in s o lu b le , and glucose to l a c t i c a c id . Glucose
••3
ferm en tatio n by t h is system was in h ib ite d 98 per cen t by 5 .0 x 10 ^ 1
io d o acetic acid and 30 per cent by 5 .0 x 10 g M sodium f lu o r id e . Ho
ferm en tativ e a c tiv ity could be dem onstrated in th e so lu b le p o rtio n of
th e fra c tio n a te d amebae c e l l s .
The b a c te ria growing w ith th e amebae were also-show n to convert
glucose in to l a c t i c a c id . Io d o acetic acid (5 .0 x 10“ * M ) in h ib ite d
95 per cent of t h i s a c t i v i t y . Sodium flu o rid e in th e same concentraticn
in h ib ite d only 10 per cent •
The r e s u lts of th e in v e stig a tio n s have been in te rp re te d in terms
of th e Embden-Myerhof-Parner m etabolic pathway fo r carbohydrate
catabolism * The presence in th e amebae c e ll of th e enzymes phosphory
la s e , glucokinase, trio se p h o sp h a te dehydrogenase, en o lase, and la c tic
dehydrogenase is discussed*
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University of S o u th ern Cafftornfa Lmrery

Asset Metadata

Creator Michaelson, Joseph Bernard (author)

Core Title Some aspects of the carbohydrate metabolism of Endamoeba histolytica

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Degree Program Biochemistry and Nutrition

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

Tag health sciences, nutrition,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-501

Unique identifier UC11347927

Identifier DP21563.pdf (filename),usctheses-c17-501 (legacy record id)

Legacy Identifier DP21563.pdf

Dmrecord 501

Document Type Dissertation

Rights Michaelson, Joseph B.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA