STUDIES ON THE GLUCUSE METABOLISM
OF TRETRECHUMONAS AUGUSTA

Thesis far the Degree of Ph. D.
MECHKQAN STATE UNEVERSIT‘I’
STUART H. WiNSTGPé
1.9!67

THEblS MP: 7
g! LIBRAR Y '
Micki-3:421 Sm:

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“‘2 4‘11 '2 ersi L;

This is to certify that the

thesis entitled

STUDIES ON THE GLUCOSE METABOLISM
OF TRITRICHOMONAS AUGUSTA

presented by
Stuart H. Winston

has been accepted towards fulfillment
of the requirements for

Ph. D. d . M.P.H.
_ egree in—

Major professor /

Date «5/1/17 //‘;Z/ /95, 9

0—169

 

ABSTRACT

STUDIES ON THE GLUCOSE HETABOLISH
0F TRITRICHOHONAS AUGUSTA

by Stuart H. Hinston

with glucose as an energy source, Tnltgighgggnas augusta produced

lactic acid, acetic acid, succlnlc acid, 602 and H2 frou growth in a
continuous culture systen or under anaerobic conditions in a Varhurg
flask. The acids were extracted with ether, separated on a ceiite
chreuategraphic coluun and titrated to deterulne the quantity of each
acid. The gases were deterulned using a fernentation train with the
continuous culture systen or standard uanouetric techniques with the '
Uarburg apparatus. Feruentation balances gave carbon recoveries of
89% free the continuous culture systen and 95% free uauouetric
studies. Oxidation-reduction balances were 0.8l and 0.85 respectively,
showing a slight excess of reduced end product. The inherent errors
of deterninlng fernentatlon balances fron organisus grown on un-
defined uediun and under non-nutrient conditions with a glucose sub-
strate were discussed.

Evidence was obtained for the occurrence of an M EDJJ.‘
type of phosphoroclastic split of pyruvate in'ILLL.‘gggggta as the
source of acetate, 602 and £2. The evidence consisted of the
detection of acetyi phosphate and foruate as internediates plus a
couplets fernic hydrogen-lyase enzyme systeu. The possibility of the
for-etion of ATP via acetyl phosphate was discussed.

The detection of the naiic enzyne, funerase, and succinic dehydro-

genase, the presence of nalate and funerate and the absence of citrate,

Stuart H. Winston
isocitrate, a-ketoglutarate and oxaloacetate in cell honogenates of
L11. gm; suggest the fornatlon of succinate by a partial reversal
of the Kreb's cycle. The naiic enzyue reseubled the enzyne systeu_
described for pig liver in its requireuent for IADP as a co-factor.
Funarase was denonstrated for the first tine in trichononads by a
couplex coupling of the funerase free cell extracts with added uallc
dehydrogenase and the use of two artificial co-factors for transfer
and acceptance of electrons. The succinic dehydrogenase detected in
L15. m also seens to resenble the uamallan enzyne in that it
is readily inhibited by nalonate.

1:15, assist; has been shown to possess at least two pathways for
the oxidation of NADH. One was via the lactic dehydrogenase systeu
and the second was through the succlnic acid-electron transport
systeu. The latter systeu couples FAD reduction with NADH oxidation.
Phosphoryiation was associated with the above reaction resulting in
nearly one nole of ATP fonned for every NAB” oxidized. The inportance
of this systen in the overall energy netabollsu of ILL!- W was

discussed.

STUDIES ON THE GLUCOSE METABOLISM
OF TRITRICHOHOHAS AUGUSTA

By

Stuart H. Winston

A THESIS

Submitted to
Hichigan State University
in partial fulfillment of the requirenents
fer the degree of

DOCTOR OF PHILOSOPHY

Departnent of Microbiology and Public ilealth

l967

6%9395
‘j-Qé-éfi

ACKNOWLEDGEMENTS
The author wishes to express his thanks to his major adviser,
Dr. D. H. Twohy, for his encouragement and helpful advise. He also
wishes to thank the many other faculty members and fellow graduate
students In the Department of Microbiology and Public Health who
contributed useful suggestions, help, or encouragement which con-

tributed to the completion of this thesis.

ll

TABLE OF CONTENTS

Chapter

'"TRODUCTI 0“ O O O O O O O O O O O O O O O O O O O O O O V .
L'TEMTURE REVI EH 0 O O O O O O O O O O O O O O O O O O 0

MATERIALS AND METHODS . . .
RESULTS . . . . . . . . . .
DISCUSSION AND CONCLUSIONS .
SUMMARY . . . . . . . . . .
LITERATURE CITED . . . . . .

Page

l9
hZ

5h
56

LIST OF TABLES

Fernantation balance of Trig. augusta in

continuous culture . . . . . . . . . . . . . . . .

Glucose fermentation balance of TEIS- gggggt;
in the Harburg apparatus . . . . . . . . . . . .

nononstration of acetyi phosphate production
bYMOMooooooooooooeoee

Evidence for the presence of riboflavin, FAD and
ATP in T:[§.,gggggtg as deternlned by paper
chrenatography . . . . . . . . . . . . . . . . .

Anount of ATP resulting from electron transport
associated with succinate production . . . . . .

Specific activity of enzymes associated with
the production of end products by T[[;.'ggggstg .

iv

Page

20

22

2h

38

38

hi

9.

IO.

LIST OF FIGURES

Formic dehydrogenase activity of a cell homo-
genate of T: I. augusta . . . . . . . . . . . .

Hydrogenase activity of an extract of T511.
augusta cells . . . . . . . . . . . . . . . . .

Formlc hydrogenlyase activity in cell extracts

Ole'IE.Iugg$§O.c.............

Malic enzyme activity in an extract of TE!!-
‘Egusia O I O O O O O O O O O O O O O O O O 0 0

Activity of fumarase in cell homogenates of

I;1t.,gggggt_ . . . . . . . . . . . . . . . . .

Succinic dehydrogenase in a crude extract of
lcwoooeooooooooooo...

Maionate inhibition of succinate formation in whole
cells of Tris. augugta . . . . . . . . . . . . . .

Lactic dehydrogenase activity of an extract of Tgig.

.ugugsa O O O O O O O O O O O O O O O O O O O O O 0

Activity of enzyme(s) involved in the terminal

respiration system of Tcit. augggg . . . . . . . .

Proposed scheme for succinate production, hydro-

gen and carbon dioxide evolution and terminal

respiration in Tr! . augusta . . . . . . . . . . .

Page

25

27

28

3]

33

3h

35

37

39

#8

iNTRODUCTION

Igltnlghgggggs aggusta (Alexeleff) is a highly plastic pyrifonm
protozoan ranging in size from is to 27 microns long by l3 to is
microns wide. In addition to three anterior flagella, a fourth forms
both the outer margin of the undulating membrane and the trailing
flagellum. A rod-like costa follows the base of the undulating mem-
brane, while a thick central axostyle extends the length of the body
and protrudes posterioriy. The organism possess other structures
commonly found in other animal cells such as the golgl apparatus
(parabasal body), vacuoles and endoplasmic reticulum. Abnonmal mito-
chondria-like structures lacking cristae have been reported IH.ILL1-

HILL! (l) and in W £LI_¢.!.§.L (7), but the actual presence of

mitochondria in trichomonads has been questioned in recent investi-
gations on 1111. Mil}. (A7) and I. xagInalis (#8).

1111, ggggggg is a common commonsai living in the colon of
tadpoles, frogs and toads. The normal environment of this organism
has a low oxygen tension and is rich in other microorganisms.
Bacteria, unadsorbed food and intestinal secretions present in the
lumen of the colon probably serve as food for this protozoan. Trich-
emonads can only be cultured in complex undefined media at low oxygen
tensions. The optimum temperature and pH for growth approximate that
of the host and the habitat in the host. Forms like 1:11. auggsta
from cold blooded hosts grow in a temperature range of from l6 to
32 C and a pH range of 5.5 to 9.0 (22). The optimum generation time
is from 6 to 8 hours.

The principle studies on the carbohydrate metabolism of trich-

I

2
omonads have been done on the pathogenic species. These organisms
have been shown to utilize glucose, fructose, maltose and galactose
and most of the common di- and polysaccaride sugars for growth or gas
production. Pentoses and sugar alcohols do not generally support
growth. There Is no evidence of amino acids, lipids or other organic
compounds, other than carbohydrates, serving as an energy source in
the trichomonads studies to date. The nutritional requirements of
trichomonads have been reviewed by Shorb (#6).

Numerous investigators have determined one or more of the end
products formed by these organisms as a result of anaerobic carbohydrate
fermentation. Pyruvate, lactate, acetate, succinate, 602 and H2 have ,
each been detected by more than one of these investigators (23, 30, 37,
#9 and 50). The schema of formation of pyruvate and lactate have
been well documented (2, l3, 23, 60), but the pathways for the pro-
ductlon of succinate and acetate and the significance of these path-
ways to an electron transport system and energy production in the
trichomonads has not been ascertained. Also, the type of mechanism
involved in the evolution of H2 and (:02 has not been settled.

it is obvious that an understanding of the carbohydrate metabolism
of the trichomonads lags far behind that of the bacteria. It was the
purpose of this investigation to try and fill some of the gaps in the
present knowledge of the glucose metabolism of Iglt, auggsta by:

(I) computing fermentation balances to provide a more accurate analysis
of end products formed in relation to glucose utilized; (2) demon-
strating the complete pathway by which succinate is formed; (3) deter-
mining the role and nature of the linkage of succinate production to the
electron transport system; and (h) determining if formate is an inter-

and H .

mediate in the production of 602 2

LITERATURE REVIEH

The successful detection of the intermediates and enzymes of the
glycolytic pathway fortified the theory of the existence of a con-
ventional Embden-Meyerhof pathway in the trichomonads. Seven inter-
mediates of the system were identified lnII, vaginalis (6i). They
were: glucose-I-phosphate, glucose-6-phosphate, fructose-G-phosphate,
fructose-l-6-phosphate, 2-phosphoglycerate, 3-phosphogiycerate and
phosphoenolpyruvate. All of the glycolytic enzymes have been demon-
strated ind. xaginalis and most have been detected in other species
of trichomonads (23, no, 60). The only evidence for the existence of
a hexose monophosphate pathway in these organisms is the detection of

glucose-6-phosphate dehydrogenase in I, gagigalls (S9) and in Tgit.

1221593.. 1111. at; and Pentatrichomonas galiinagum (23).
Egd chgucts of Carbohydgate Metabolism: The end products from

anaerobic growth on a carbohydrate substrate have been determined
mostly for the pathogenic species of trichomonads. When cultivated
for #8 hours in a complex medium containing glucose ILL; M was
found to produce mainly Hz with trace quantities of N2 and methane
gas. Of the total acid produced from glucose, 73 Percent was succinic
acid, and 22 percent was pyruvic and lactic acids (#9, 50). Ninomiya
and Suzuoki showed that I, vaginalis could produce 602 and other un-
identified gases from a medium in which either pyruvate or glucose
served as the substrate (30). Kupferberg ggugl. were able to demona
strate only the evolution of C02 and the formation of lactate from
carbohydrate medium, (20) but Read showed that glucose was metaboiized
to H2, CO2 and unidentified acids by this same species (36). in later
3

1,

work, Road was able to detect the anaerobic formation of CO and H2 by

2
I, galligae from glucose, fructose and galactose (37). Doran found
that glucose, lactate and pyruvate stimulated gas production:L£L_.
15g; and III... 393 (ll, l3). From 30 to 50 percent of the acid
produced under anerobic conditions by these two species was lactic
acid. He observed that nearly two thirds of the gas produced by these
trichomonads from glucose was C02, and that if no substrate was pro-

vided only one half of the gaseous and product was CO The addition

2.
of either lactate or pyruvate stimulated both gas production and oxygen
uptake by these organisms. Other gases not adsorbed by KOH increased
only slightly under these conditions. Tetratglchomonas buttreyi has
been shown to produce similar end products from glucose (l2). From

#0 to 50 percent of the total acid was lactate. Gas production was
also stimulated by glucose, pyruvate or lactate. ILLE. augusga
PTOdUCOS H2 and 602 in equal or nearly equal amounts. The individual
acids produced were not determined (53). Llndblom reported succinate
to be the major acid produced by I;1§,1£gg§g§,.fi. gallinarum and a
nasal strain of ILLE- gul__s_ in culture, but a focal strain of 1m. 3313,

produced more lactate than succinate (23). All of the organisms he

studied fonmed 602 and H2.

Studies on Gas and Lactate Fogmation: The metabolic pathways for

the production of most of the end products fommed by the trichomonads
have either to be discovered or to be verified. The presence of a
glycolytic pathway in the trichomonads accounts for the formation of
pyruvate. Lactate is produced from pyruvate by a conventional pathway
involving reduced MAO and lactic dehydrogenase (2, l3). The scheme

for the formation of H2 and 602 from pyruvate is disputed. Llndblom

5
reported an adaptive type of formic hydrogeniyase system in EL. 7
M, g. galligagum and in two strains of '_|'_r_i_t,. _§_u_i_§ (23). He‘demon-
strated an active formic dehydrogenase, but showed only a weak hydroé
genase activity. Ryloy on the other hand, thought it was likely that
ILLS.- m possessed a phosphoroclastic system similar to that of
the Clostridia (#0). He based this assumption on his observations
that ILLS. m did not produce formate and could not use formate as
a substrate.

Eyidegge f2: a erb's Cycle: A complete Kreb's cycle seems to be
lacking in the trichomonads. The only enzymes from this cycle which
have been demonstrated by direct methods are malic dehydrogenase in:[.
yogigaiis, 3. W and 1m. 33L; and succinic dehydrogenase in
1. ML}. (3. 23, #3). Read speculated on the presence of aconltase,
lsocitrate dehydrogenase and a-ketogiutaric dehydrogenase in l. gelling;
based on the finding that the protozoan could oxidize a number of the
intermediates of the citrc acid cycle (37). There is no direct evidence
for the presence of these enzymes. Neither the condensing enzyme nor
fumarase have been detected by indirect or direct methods as this time
(23. 5|).

Kunitake‘gtwgl. attempted to demonstrate the presence of the Kreb's
cycle in I, gaginalis using glucose-U-Clh or succinate-2,3-C'h as sub-
strates. They were unable to isolate labeled Kreb's cycle inter-
mediates from cells grown with either substrate (21). However, labeled
602 and amino acids were detected. These investigators stated that
the pattern of labeling in the amino acids suggested the presence of a
citric acid cycle in I. vaginali . These investigators were unable to

find any activity of these Kreb's cycle enzymes in their cell homogenates.

6

if some sort of di- or tricarboxyllc acid pathway does exist in
these organisms, there must be a mechanism for the formation of a four
carbon intermediate. Since the condensing enzyme has not been detected,
the trichomonads must use some other method for tri- and dicarboxylic
acid production. In their first investigation, Wellerson.gt.gl. found
labeled CO2 was incorporated into the carboxyl group of lactate by I,
:aglnal|§ (58). Since it was not incorporated into malate or succinate,
they believed CO2 fixation onto pyruvate by the malic enzyme did not
occur. Later, these some investigators demonstrated a very active NADP-
linked malic enzyme in this trichomonad, suggesting a means forjI.

vaginalls to form malate from C0 and pyruvate (60). This type of

2
enzyme was also found in I;lt,,£gg£gs (25). Llndblom was able to show

a weak reversal of the malic enzyme reaction in‘fl. galllnargm by
incubating trichomonad homogenates with malate and NAD and measuring

the C02 evolved (23).

Studies g5 ngglgal Rgspipatlgn: Investigations into the terminal
respiration of the trichomonads have led to many speculations. Several
authors have failed to demonstrate the presence of cytochromes in these
protozoan (#0, 58). Only Suzuoki and Suzuoki reported a cytochome b
resembling the mammalian type in ILLS.- M (50). Whether or not this
cytochrome really exists has been questioned. It is not unusual to find
anaerobic organisms lacking a cytochrome system.

The most common electron carriers in anaerobes are the flavoprotelns.
Riboflavin and flavin mononucleotide have been isolated fromjl. vagigalis
(58), as has flavin protein oxidase (3) and NADH oxidase (26, 58). The

premises for the presence of an electron transport system in the

7
trichomonads was summarized by Baernstein (#): "Trichomonads are
essentially anaerobic and therefore depend on coupled reactions
mediated by pyridine and flavoproteins resulting in the production
of reduced compounds. They depend on conventional glycolysis
supplemented with other systems linked to NADP and to succinate to
furnish electron donors. Electron transport in anaerobic metaboiism
is limited to dehydrogenase couplings and to the excretion of the
reduced compounds. in some strains anaerobic production of hydrogen

supplements the usual mechanism of electron transport."

 

Common inhibitors of the citric acid cycle have been tested both on
isolated enzyme systems and whole cells of trichomonads. Both beta-
phosphonopropionato and arsonoacetate were effective inhibitors of
succinic dehydrogenase activity in cell extracts or I, gagigaiig (#3).
Neither inhibitor has any effect on the succinic dehydrogenase from
mammalian cells. Halonate a potent inhibitor of mammalian succinic
dehydrogenase had no effect on oxygen uptake by the four species of
trichomonads studied by Llndblom (23). Halic dehydrogenase fromjl.
mm was inhibited completely by p-mecuribenzoate but not by any
of the common Sfl-inhibitors (30). Neither cyanide or carbon monoxide
had any appreciable effect on the acid production of I, zagigaiis (20)
or on the oxygen uptake of ILL!- m (i0) and L xegigaiig (38).
This suggests that terminal respiration in the trichomonads is not
through the usual cytochrome system.

Vpclability [n the Metabolism of‘Trighomgnads: it is apparent
that all of the species of mm; so far studied can anaerobically

metabolize the hexoses to 602, H and various organic acids. The

2

8
relative amounts of each end product depend on the species studied and
on cultural conditions. Unfortunately, the wide variation in cuiturai
conditions often makes it difficult to compare the work of different
investigators. Read and Rothman (38) have called attention to variations
in the Qacid and Q9.“ throughout the growth cycle of I, vaginaiig.
Qacid and 09., also varied with the strain of the species being investi=
gatod. The variations were shown to depend upon the quality of the
medium, age of the lnoculum and the cultural history of the cells.
Their conclusions were substantiated by Llndblom with‘ILLt..figgtgg,
Trit. suis and.fi. gallinarium (23). His work showed that oxygen

 

 

uptake, CO2 and H2 evolution and anaerobic acid formation varied with
the age of a culture. Gas production was highest at l2 hours of
growth but varied considerably thereafter. Acid formation was highest
at #8 hours.

Due to the above reported variations, the cells employed in this
study were grown under constant conditions in a simple external control
continuous culture apparatus (l6). With this system a constant pop-
ulation of trichomonads could be maintained over long periods of time.
Since this population would be in a steady state the variation in its
metabolism from day to day would be expected to be at a minimum,
giving a more reliable picture of the glucose metaboiism of ILLS:
331311;. it is this type of stability which appears to be lacking in

previous studies on the trichomonads.

METHODS AND MATERIALS

Iggl;g;g_!gthgg§: A clone (53) of the parent strain T-l2 of
W aggusta isolated from £333 catesbiana was used in this
study. Stock cultures were maintained by serial passage in l6 X l25
mm screw cap test tubes containing l0 ml of Diamond's medium (8) with-
out agar. The medium was adjusted to pH 6.6 to 6.8 with phosphate
buffer and the cultures incubated at room temperature or at 29 C.

The continuous culture apparatus used in this study (52) con-
sisted of: l) a l l. reservoir flask containing fresh medium, 2) a
modified 500 ml. tissue culture spinner flask (Belco Glass Inc.
Vinoland, N.J.) which served as a growth flask, 3) a magnetic stirrer
to rotate the impeller in the growth chamber, #) an overflow flask,
and 5) a multispeed transmission pump (Harvard Apparatus Co., Dover,
Mass.) model # 600-000 which was employed to force medium from the
reservoir flask into the growth chamber.

A stock culture in the log phase of growth (approximately #8
hours old) was used to inoculate the growth flask of the continuous
culture apparatus. When full, the growth flask contained 3#0 ml of
Diamond's medium modified by the addition of lOI O.liM NaZHPOh (pH 6.6)
and by the substitution of 2.0mM concentration of glucose for maltose.
All of the above procedures of preparation, assembly and inoculation of
the apparatus were carried out using sterile techniques.

To insure anaerobic conditions for growth the entire apparatus
was flushed with nitrogen. Oxygen was removed from the nitrogen by

passage through copper turnings at 500 C and carbon dioxide by

9

l0
bubbling through i I laOfl. Trichomonad growth was limited by the low
concentration of glucose used in the modified Diamond's medium. After
inoculation the organisms were allowed to grow undisturbed for three
days at 29 C. Then the impeller was started to give a homogeneous sus-
pension of organisms in the medium and the pump turned on to force
fresh medium into the system. in this study a flow rate (f) of 5.h
ml/hr. was used, providing a dilution rate (0) of 0.0]58. D was
determined from D-f/v where v- the flask volume (3&0 mi). It rep-
resents the number of complete volume changes per hour. The mean
residence time of an organism in the growth flask is equal to l/D or
63.3 hours (l6). The generation time (9) in hours calculated by the
formula g-in 2/0, was ‘03.9 hours (ill).

WW: Steady state conditions were assumed
to exist when the trichomonad population showed no consistent in-
crease or decrease (l6). Duplicate samples for population counts were
withdrawn from the growth flask daily in a sterile i ml syringe fitted
with a three inch 22 gauge needle which was inserted through a rubber
stopper in one sidearm. Each sample wes killed and preserved with
l ml of 21 formalin diluted with 80% (v/v) Kreb's Ringers phosphate
(KI?) and stored not more than 2h hours prior to counting. Samples
were then diluted to 20 ml with 861 KRP and the cell population counted
with a lodelqA Couitor Counter (Couiter Electronics, Chicago, Iii.)
equipped with a lOOu diameter aperture. The instrument was set at
an aperture current of 2 and a threshold value of i5. All counts
were contacted for coincidental passage of more than one organism at
a time through the aperture (28).

h P a I e 8 Approximately

ii
2&0 ml of medium were decanted from the growth chamber of the continuous
culture apparatus into the recently drained overflow flask. The .
medium was then flushed from this flask with nitrogen, collected in
conical glass screw cap centrifuge tubes and centrifuged for 20
minutes at 880 X g. The supernatant was decanted and the cells were
suspended in a small volume of 80% KRP with 0.i% cysteine, adjusted
to pH 6.8 with ll such. The organisms were pooled and the process
of suspension, contrifugation and decantation repeated until the cells
were washed three times in cold 80% REP-cysteine. The cells at this
stage of processing were employed in experiments requiring whole
cells, except for organisms used for the determination of cell nitrogen
and glycogen. Coils used for these studies were washed in cold 80%
IR? without cysteine.

For these experiments that required cell homogenates the follow-
ing additional procedure was used to rupture the trichomonads. Cells
from the final pellet were suspended in 3 mi of 0.02! phosphate buffer
containing 0.l% cysteine (ph 6.8), placed into a Thomas model Il0083
tissue grinder, immersed in an ice bath and homogenized at i000 rpm
for seven minutes. The homogenate was then dialyzed for 2h hours at
h C against the some phosphate buffer with cysteine. After removal
from the dialysis tubing, the homogenate was centrifuged at 880 X g
for five minutes and the supernatant decanted and stored at -i2 C
for use the same day.

Determination of Gas Production: The C02 and H2 produced by
11:11. m were measured for short intervals with non-growing cells
or over 2k hour periods of growth in the continuous culture system

incorporated to be fermentation train described by leish (29). In

12

the latter system 0 and Coi-free nitrogen was flushed continuousiy

2
through the apparatus. The metabolic CO2 in the gas was first
trapped in lscarite. The H2 was converted into water by passage
through copper oxide at 550 C and adsorbed in calcium chloride.

The flsccritc and calcium chloride traps were weighted before and

after each experiment to determine the amount of gas absorbed.

Hanometric experiments were conducted at 30 C in a Precision
Scientific 20 unit Warburg respirometer with a shaking rate of l20/
minute according to standard procedures (5h). All experiments were
conducted using an O2 and CO2 free nitrogen atmosphere.

Carbon dioxide retained in the Kreb's phosphate buffer system
employed for the manometricaiiy determined carbon balance was ex-
pelled by adding 3" H250“ from a sidearm at the end of the experio
ment (5“). The H230“ was added to the contents of a control flask
at the start of the experiment to determine the initial CO2 in the
buffer. Hydrogen was measured in flasks containing 20% KOH in the
center well to absorb the CO2 (5h).

Qgtgggination of Organic Acids: Organic acids were separated
from cells and medium using the method for ceiite partition chromato=
graphy described by Swim and Krampitz (5i) and modified by Seaman
(#4). Eighteen ml samples of either medium or cell homogenates were
first extracted with ether in a reflux condenser described by Neish
(29) according to the method outlined by Seaman (hh). The concenu
tratod organic acid sample to be chromatographed was combined with 1
gram of ceiite (Johns-Hansviiie Filter Aid) in a mortor with a pestle
and added to the top of a previously prepared ceiite coiumn (hfi). A

sequence of chloroform and chloroform-butanol mixtures described by

Swim and Krampitz (Si) were passed through the column to separate the

13

acids in the other extracted sample. A positive pressure of N2 was
applied to the column to achieve a flow rate of 3-h mi/min. Succesive
5 mi fractions, regulated by the drop count, were coilected with the
aid of a fraction collector. Each fraction collected was titrated
with 0.0i5 I alcoholic KOH until phenol red indicator gave a stable
red color. The quantity of acid present in a given sample was deter-
mined from the volume of alkali required to neutralize each fraction
in a given peak (Rh). The volumes of K0! were plotted for successive
samples and the sum of the volumes for each peak converted to equi-
valents of organic acid. Samples of fresh medium were analyzed prior
to their use in the growth chamber and the initial concentrations of
any of the measured organic acids subtracted from the final values.
lixtures of standard organic acids were passed through the column to
check on the positions of peaks and on the efficiency of the method.

Oggggginatigg of GlucoseI Gizggggn agd litgggen: The yeast
extract and serum in the medium contained natural sugars in addition
to the glucose component added. Prior to the determination of the
glucose content of the medium, the sample was passed through a
short anion-cation exchange resin column (hXS cm). The resin was
that normally used in a deminoraiizer (Deeminite L iO Resin, Crystal
Research Labs., Hanford, Conn.). The column was washed with sufficient
water to double the samples volume. The resin removed unknown none
carbohydrate reactants which caused a nonospecific color reaction
with anthrone reagent. Glucose was determined by Roe's (39) anthrone
method.

For glycogen and nitrogen determinations the cells were removed

from the growth chamber and freed of medium by the method previously

ih
described for the harvest of intact cells. Glycogen was extracted
from approximately 8 X lo6 washed cells by the method of Good gt 3;,
(l5) and assayed by Bee's anthrone method (39). Standards containing
a known amount of glycogen were included in each group of analyses.
Cell nitrogen was determined by the Nesslerization procedure described
by Umbreit using a Bausch and Lamb Spectronic 20 spectophotomenter
(5h) .

Fenmontation balances based on substrate utilized and gas and
acid end product analyses were calculated according to the method out-
lined by Hood (63). Analyses of fresh and used medium showed that
pyruvate was utilized by Ills..gggggtg from the Diamond's medium.

This pyruvate was considered as a substrate along with glucose in
calculations of the fermentation balance.

Egg!!! Assays: The general procedures employed in the spectro~
photometric assays of malic enzyme (3i), succinic dehydrogenase (5)
lactic dehydrogenase (l9), pyruvate carboxyiese and phosphoenol-
pyruvate carboxylase (56) activity have been described in detail in
Iothods in Enzymoiogy, vol I. l955. Other determinations of enzyme
activity required modifications of described techniques.

(The enzymes of the formic hydrogeniyase system were determined
manometricaliy using the method of Peck, Jr. and Cost (33, 3h). The
components of each assay accompany the appropriate figures depicting
the results.

The following coupled reactions were involved in the determination
of fumarase activity inylglt. agggsta:

cell extract
(" Fmr‘t. + “20 -- -------- nu--- H.1't‘;

l d d

l5

(3) "AD" + h+ + Phenazine Hethosulphate (PMS) ------ reduced PMS + MAD;
a.) reduced PMS + Triphenylterazoiium Chloride (TPT) ====== reduced
TPT + PHS.

The fumarase present in the cell homogenate converted the fumarate to
malate. Commercial malic dehydrogenase was added to catalyze the
fonmation of oxaloacetate and reduced NAD. Phenazine methosulphate
acted as an electron acceptor from NADH + H+. it in turn reduced the
indicator, triphenyltetrazoiium chloride, a reaction which was followed
spectrophotometricaily at a wavelength of 650 mu. To check the
specificity of the enzyme in the cell extract, fumarate was omitted
from the control assay.

The assay for a coupled electron-high energy phosphate transfer
(EEPT) system was carried out in the cuvetto of a model D. U. Beckman
Spectrophotometer. To cell homogenate, which was dialyzed against Nu
tris (hydroxymethyl) mothyi-Z-aminoethane sulfonlc acid (TES) buffer,
was added adenoslne diphosphate (ADP), flavin adenine dinucieotide
(FAD), phosphate buffer (pH 6.8) and reduced NAD as a source of elec-
trons. The criteria of activity for the system was the measurement,
at a wavelength of 3&0 mu of the oxidation of the reduced MAD. One
control was used for each component, ie. with each component deleted
from the system. TES buffer was used in place of phosphate buffer to
determine if inorganic phosphate was essential for the acitivity of
the transport system.

in spectrophotometric assays, rate measurements were conducted at
ambient temperatures between 23 C and 26 C. Substrate or enzyme was
added at zero time and the components mixed by inverting the covered

cuvetto a few times. Appropriate blanks were used to zero in the

l6

spectrophotometer prior to each experiment. 7 .

oggeruihetioh of the Effect of Halonate on Succinate Pgoductionz'
lnorder to determine if the enzyme responsible for succinate production
l".I£L£- augusta was susceptible to maionate inhibition the following
experiment was perfonmed. 'Two Thunburg tubes were prepared to contain
final concentrations of 0.002! glucose, 3 X l06 cells and 0.0lH
maionate in 0.lH phosphate buffer (pH 6.8) to give a total volume of
l0 ml. Control tubes contained the above materials minus maionate.
All of the tubes were alternately placed under vacuum and flushed with
02 free I2 three times. The reaction mixtures were then incubated in
a 30 C water bath for one hour. After removal from the bath the con=
tents of the tubes were centrifuged at 880 X g for i0 minutes to re-
move the cells. The supernatant was then other extracted (hh) and
placed on a ceiite chromatography column (5i). The amounts of fumatate
and succinate in both the control and experimental mixtures were deter=

mined as described earlier (uh).

 

To determine FAD and riboflavin, i0 lambda of a 0.002H solution of each
was spotted on Hhatman # h filter paper strips (38X3h mm). Eighty
lambda of cell extract was then spotted on a third strip of the same
dimensions. An end of each strip was immersed in a solvent composed

of l60 grams of phenol, 30 ml of butanoi and lOO ml of water for 5
hours at 20 C (9). After the strips were air dried they were checked
for the fluorescence of the fiavins using a model ReSl Hineralite
(Ultraviolet Products Inc., San Gabriel, Calif.). The Rf values were
determined for these spots.

Adenosine triphosphate (ATP) was chromatographed on paper according

. . l7 , .
to the method described by Pabst Laboratories (32). An 80 lambda
aliquot of sample, from the assay mixture used for determining the
EEPT system was spotted on a 2lXi6§ inch sheet of Hhatman # i filter
paper. The control on the ATP chromatograph consisted of the above
mixture minus reduced NAD. The filter paper was immersed in a
solvent composed of i00 ml of O.iH phosphate buffer at pH 6.8, 60
grams of ammonium sulphate and 2 ml n-propanol. After developing for
l6 hours in an ascending position, the sheet of paper was air dried,
the spots located by observing the visual quenching of ultraviolet
light by ATP under a Hineralite lamp and the if values calculated.
The spots were cut out of the chromatogram and placed in vials which
contained 3 mi of 0.il hCL. The vials were placed in a 37 C incubator
overnight. The eluted nucleotides were then pipetted into 3 ml
quartz cuvettes and their absorbency at a wave length of 257 mu
determined in a model 0. U. Deckman Spectrophotometer. The concena
tration of.ATP present in each sample was calculated using an
extinction coefficient of ih.7 x iO'3 for ATP at 257 mu and on 2.0.

The amount of NADH oxidized was determined for the EEPT reaction
by measuring the change in optical density at 3&0 mu for one minute.
The optical density value obtained was then incorporated into the
following formula: Ln, - Mg, where, in equals the molar absorbency
index (extinction cooficlent), A; equals the absorbency and 3 equals
concentration. Using theyAg,value of 6.22 X lo",3 the concentration
of reduced IAD oxidized was determined from the spectrophotometric
reading. The ratio of ATP fonmed per IADH oxidized was then cale
cuiated.

Acetyl phosphate was analyzed by the method of Lipmann and

Tuttle (2h). Samples for the assay were obtained from Harburg vessels

which contained the following: Mi hydroxylamine -i"lCl, 3.5ll sodium
hydroxide, 0.lli acetate buffer (pil 5.“),dialyzed cell homogenate,
and 0.05“ sodium pyruvate as a substrate. The atmosphere in the
flasks was lOOX I2 and the reaction was carried out for one hour at
30 C.

m3 The emu organic and inorganic compounds used were
of highest grade of purity available cemorcially. The other chem-
icals were obtained from the following sources: benzyl and methyl
viologen fr. liann Research Laboratories, llew York, I. Y.; malic
dehydrogenase, oxidized and reduced MD and ADP from Sigma Chemical
Co., St. Louis, lio.: reduced nicotinamide adenine dinucieotide phos=
photo (IADPII), FAD and riboflavin from Calbiochem, Les Angeles,
Calif.: and ATP fr. the Pabst Brewing Co., Chicago, ill. The TES
buffer was obtained through the kindness of Dr. ilormon E. Good,
Department of Botany, liichigan State University, East Lansing,
llichlgan.

RESULTS
l. The Glucose Fermentation Balance of.ILL;. augugta.

A. ngtinuggs Cgituge System: A typical fermentation
balance from the use of the continuous culture apparatus was sum-
mmrized in table l (part I). The mean trichomonad population was
1.21» x 105 cells per ml (0.036 .9 ll/ml) at the time a steady state
was reached. The gases produced by the organisms were collected
over a 2k hour period. Organic acid, glucose, cell nitrogen and
glycogen determinations were made on duplicate samples from the
flask at the end of the 2“ hour period. Similar determinations
were made on fresh medium as a basis for detecting net changes due
to the activity of the organisms.

. The cells produced several products from glucose. Significant
quantities of both 602 and Hz were evolved, but H2 production wos
over twice that of the 002 production. Forty nine percent of the
total acid extracted from the growth medium was lactic acid. Acetic
and succinic acids were found in approximetely equimolar concen-
trations, accounting together for #3 percent of the acid end pro-
ducts. Formic acid accounted for the remainder of the organic acid.
On a molar basis, the production of either 602 or H2 exceeded that
of either acetate or succinate or a combination of the two. .For
each glucose equivalent of substrate utilized there was in excess
of one C02 and 3 ”2 evolved.

The above analyses showed that 67 percent of the initial
glucose in the medium was used by the cells. Determlnations of the

organic acids in the medium before and after trichomonad growth

19

Table l. Fermentation balance of Trig. augusta* in continuous culture.

l. Substrates and extracellular Products.

 

 

' Oxidation Oxidized Reduced

Compounds ggnéhr. unggg Value Product Product

Glucose used 9.2 55.2 0

Pyruvate used l3.0 O +l l3

Total used .2

002 23.0 23.0 +2 lr6.o

H2 55.0 -l 55.0

Formate l.5 l.5 +l l.5

Acetate 3.8 7.6 0

Lactate 9.8 26.7 0

Succinate 0.0 l6,0 +l h,O

Totals 7h.8 51.5 55.0
-13 0a
38.5

Carbon recovery - 79.9% O/R - 0.70

*) l.2 X lO6 cells were present per ml of growth medium. The flow
rate of medium into the growth flask was S.h ml/hr.

a) The oxidized substrate was subtracted from the end products since
it entered into the system already in an oxidized state.

ll. lnterceliular products.

Halate
Fumarate
Succlnate
a-Ketoglutarate
lso-Cltrate
Citrate
Oxaloacetate
Lactate
Acetate
Formate
Pyruvate
Glycogen
Subtotals

Totals (part I + ll)

0.227

:5}

0.9l0
l.360
0.390

W
\l

5'

8

N-fi-‘POO
mN-F'hb-Pil
g 2

m
N
o

N
0‘

+2
+2
+l

03-1900 I i I

Carbon recovery - 82.26/9#.20 - 87.3%

i.82
2.72
.39

O.h2]
l.200

goSSI
h5.05
0/8 - “5.05/55.0 I 0.

2]

showed a definite pyruvate utilization from the fresh medium.. Thus
pyruvate was considered as a substrate in addition to glucose.‘ Com-
putations from the continuous culture apparatus gave a carbon re-
covery of 79.9 percent and an OIR ratio of 0.70 (Table l, part i).
Table l (part Ii) shows the results of the carbon recovery when the
intercellular organic acids were added as end products to the total
balance. The intercellular products were: malate, fumarate,
succinate, lactate, acetate, formate, pyruvate and glycogen. The
limiting concentration of glucose used kept glycogen reserves low.
incorporating the intercellular organic acids into the overall
balance increased the carbon recovery to 87.3 percent and the O/R
ratio to O.8l9.

B. W: A fermentation balance was deter-
mined uslng non-growing cells in Hbrburg manometer flasks. A pop-
ulation of 2.h X lO7 trichomonads (0.62h mg l/ml) was suspended
in 3 ml of 80% KRP buffer (ph 6.6) containing ZOO ul of glucose.
Control flasks lacking glucose were employed to correct for endo-
genous gas and acid production. Acetic, lactic and succinic acids
were the only organic acid and products detected (Table 2). in
this carbon balance acetate and lactate were produced in approxi-
mately eqiimolar concentrations and constituted 9i percent of the
total acid. Succlnate accounted for only 9 percent of the acid.

On a molar basis CO2 and H2 production was high. TheCO2 ex-
ceeded the acetate produced and the "2 production was higher than
the total for all organic acids. Only 6.7 percent of the glucose
used was stored as glycogen. The calculated carbon recovery

accounted for 95.7 percent of the glucose used by the trichomonads.

22

Table 2. Glucose fermentation balance of 1:11. aggg§;a* in

the Harburg apparatus.

 

 

Oxidation Oxidized Reduced
Compound :fllmfl Glucose mH}C Value Product Product
Glucose used i.OO 300 0
co2 0.927 h6.35 +2 92.7
Hz 2.370 -l ll8.3
Acetate 0.783 78.30 0
Lactate 0.750 ll2.50 0
Succinato 0.l50 30.00 +l 7.5
Glycogen 0. 067 M 0 __ __
Totals 287.25 l00.2 ll8.3
Carbon recovery - 95.7% 0/R - 0.85

*) 2.“ X l07 cells per flask in phosphate buffer pH 6.6

23
The O/R value was 0.85 suggesting either the loss or anabolic
incorporation of an oxidized end product.
Ii. Acetate, Hydrogen and Carbon Dioxide Production.
Both a clostridial-type of phosphoroclastic split and an
Egghgclghia.ggll type split of pyruvate have been proposed to occur
in trichomonads (23, #0). The clostridial—type split would form

acetyi phosphate and CO from pyruvate and H would be evolved by

2 2
the action of hydrogenase on reduced MAO without formate as an
intermediate product. In the g. gall-type of split acetyi phos-
phate is also formed but formate acts as the intermediate in the
production of both C02 and H2.
A. Acetyl Phosphate Production: If acetate is formed

from acetyi phosphate a molecule of ATP could be formed for each
acetate molecule. An attempt to demonstrate acetyi phosphate forma-
tion by cell extracts ofylglt, auggsta showed lh times as much i
acetyi phosphate formed with pyruvate as a substrate as compared

to endogenous, pyruvate free controls (Table 3). These results

suggest that acetyi phosphate is an intermediate in the pathway for

CO2 and H2 production in T:!§..ggggggg.

0. Th; fixdrggenlyase Systgm: This system has been shown
to consist of two different enzymes (34) coupled together as an

electron transfer system. Formic dehydrogenase catalyzes the
formation of CO2 and a reduced electron acceptor from formate.

”Y‘rtgenase fonns H from the reduced electron acceptor.

2
The manometric assay for formic dehydrogenase showed a siight
"9 ‘0 t0. PO'OISO 0f C02 from the substrate, possibly due to 002

retention in the buffer (Figure l). Gas evolution ceased after 20

2‘}

Table 3. Demonstration of acetyl phosphate production by T: ;.

 

.3!2!£££~
Samle O. 0. at 500 mu ulis‘iAcetyl POu/mg cell ilk/hr.
Control (no pyruvate) 0.003 0.28h
caplet. System. 0.052 3,800
Total 3.555

*) homogenate added contained 0.6h8 mg cell N.

a) The complete system for acetyi phosphate analysis contained: 0.5 ml
of O.lH acetate buffer (pH S.h), 0.5 ml of hydroxylamine solution
(0.25 ll ll" hydroxlamlne-HCI + 0.25 In] 3.5“ NaOH), 0.5 all cell
extract and 0.5 ml 0.05H sodium pyruvate (sidearm). The endogenous
system contained no pyruvate.

25

Figure l. Formic dehydrogenase activity of a cell homogenate
of I;_;. augusga. Each Harburg flask contained: l.6 ml. 0.065H
phosphate buffer pH 6.8, 0.5 ml. cell homogenate, (0.636 mg cell
11) 0.5 .1. 0.01 sodium formate (sldearm 1), 0.0 .1. (32011) benzyl
vlologen (sidearm 2). Control: fonmate replaced with buffer.

Gas phase; nitrogen. Carbon dioxide evolution measured for 25

minutes at BO'C.

uL coz/mc. ecu. N.

l50

l20

 

 

ronmc DEHYDROGENASE

X\x

 

10 15
MINUTES

25

26
minutes. “A similar cessation of enzyme activity was noted by,
Llndblom after 30 minutes (23). A total of l20 ul of C02 were
liberated from sodium fonmate in 20 minutes at 30 C. The formate
free control showed no C02 evolution.

The next step after finding formic dehydrogenase activity
was to determine if there wes an active hydrogenase in these
trichomonads. Hydrogen evolution was followed for 25 minutes with
methyl vlologen (reduced immediately before use by sodium hydro-
sulfite) acting as the source of electrons, ie. substrate for the
hydrogenase. Nearly 55 ui of Hz were released from the reduced
methyl vlologen in the first 20 minutes of the reaction (Figure 2)
Gas evolution ceased after 20 minutes as observed for CO2 pro-
duction from formate by formic dehydrogenase. In the control
lacking reduced methyl vlologen no Hz was released from the system,
which showed that the cysteine present in the system was not the
source of “2 for hydrogenase.

The third step in this series of experiments was to demon-
strate the activity of the complete formic hydrogeniyase system.
An artificial electron acceptor was not necessary In the complete
reaction when non-dialyzed homogenate was used in the assay. in
figure 3 one can see that the complete system did indeed function.

There '08 '75 ul of 0 released from pyruvate for each mg of cello

2
ular nitrogen. Once again a drop in the gas evolution was ob-
served after 20 minutes. There did not appear to be any lag in

the formmtlon of "2 gas by the cell extract as reported by Linda
blom with huogenates of ILLS.- £29.!!!» 1115. £911 and l_’_. gallmagm

(23). The specific activities of the above mentioned enzymes were

27

figure 2. Hydrogenase activity of an extract of Iglt. augusta
cells. Each Harburg flask contained: i.0 ml. 0.0625H phosphate
buffer pH 6.05, 0.5 mi. cell extract (0.636 mg cell N), O.hmi. of
32ufl methyl vlologen and 0.4 ml. of 80uH sodium hydrosulphite
added from the sidearm Just prior to start of experiment. The
centerwell contained 0.2 ml. of 20% K0“. The control lacking methyl

vlologen showed no fl evolution. The Gas Phase was nitrogen..

2
hydrogen evolution was measured at 5 minute Intervals for 25

minutes.

uL l-Iz/MG CELL N.

75

45

30

 

HYDROGENASE

1'15
MINUTES

20

25

28

Figure 3. Formic hydrogeniyase activity in cell extracts of
‘IglgL augusta. Harburg flasks contained: l.8 ml. 0.065H phosphate
buffer at ph 6.h5, 0.5 ml. cell extract (0.636 mg. cell N), 0.5 ml.
of 0.0H sodium pyruvate in the sidearm and 0.2 ml. of 20% KOH in
the centerwell. Prior to the addition of pyruvate no gas was

liberated over a l5 minute period. This served as the control. The

gas phase was N2.

uL I-Iz/MG CELL w.

 

150

 

50

NYDROGENLYASE SYSTEM

1115

 

MINUTES

°\.

20 25

29
listed in table 6.

The above detection of a complete formic hydrogeniyase system
in cell extracts of 1:15. gm coupled with the determination of
acetyl phosphate (Table 3) and formate (Table 1, part I and il)
favors the presence of an 5,.22LL-type of pyruvate spilt In this
protozoan.

Iii. Demonstration of a Pathway for Succlnate Formation.

Succinic acid is a known end product of glucose fermentation
for most of the trichomonads studied, but previous investigations
have failed to show the pathway for its formation. it seemed that
a determination of organic acids in the cells of ELLE. augusta
might give some indication of the enzymes present in the pathway
leading to the production of succinate. A separation of ether
soluble material from cell extracts by ceiite chromatography re-
vealed peaks for malate, fumatate, succinate, acetate, formate,
lactate and pyruvate (table l, part ii). Only small amounts of
succinate, lactate and malate were recovered with acetate, formate
and pyruvate making up the bulk of the acids. The presence of
acetate and formate was accounted for in the phosphoroclastic
split of pyruvate. Lactate probably was formed by the reduction
of pyruvate by lactic dehydrogenase. Since succinate Is the end
product of the pathway and malate and fumarate were found in the
cells all the intermediates were present for a reversal of part of
the Kreb's cycle. Previous Investigations had demonstrated malic
enzyme (59) and succinic dehydrogenase (‘53) In trichomonads.

The quantity of pyruvate in the cells and the demonstration

of malic enzyme suggested that pyruvate acted as the acceptor for

30
602 In the formation of malate. Both malic enzyme and pyruvate
carboxylase are capable of fixing CO2 to pyruvate._ A third enzyme,

phosphoenolpyruvate carboxylase, fixes CO to phosphoenolpyruvate.

2
Neither pYruvate carboxylase or phosphoenolpyruvate carboxylase
both of which catalyze the formation of oxaloacetate, were de-
tected In cell extracts ofjlglt. agggsga. The fact that oxalo-
acetate was not detected In cell homogenates of ILLE- m
(Table 1, part ii) supports the probable Importance of malic enzyme
in CO2 fixation.

The assay for malic enzyme by the method described by Ochoa
proved successful (3i). The increase of optical density (0.0.) at
a wave length of 300 mu showed the formation of reduced NADP
(Figure h) Indicative of malic enzyme activity. Controls lacking
malate, NAOP or cell homogenate were negative.

In order to form succinate by a reversal of the trlcarboxylic
acid cycle, malic acid must be converted to fumarate by fumarase
through the removal of one water molecule. Llndblom was unable to
demonstrate have» In Lit. M, ILLS.- ggLs and fi. Mm
(23). Attempts to detect fumarase in cell homogenates of._LL§.
Igggggig failed when the method described by Nassey (27) was used
with either fumarate or malate as substrates. Another assay was
devised which utilized the coupling of two reactions that normally
occur In the TCA cycle. The complete procedure is outlined in the
materials and methods and the reactants listed in figure 5. in
brief, this entailed the conversion of fumarate to malate and then
the oxidation of malate to oxaloacetate by malic dehydrogenase

which was added to the reaction mixture. Phenazlne methosulphate,

31

Figure A. Nailc enzyme activity in an extract of I;1_, augusta.
The cuvetto contained: 0.3 m1. 0.025N glycylgiyclne buffer pH 7.h,
0.06 ml. 0.05H HnCl2, 0.2 m1. 0.000675H NADP, 0.05 ml. 0.03M L-
malate pH 7.0, 1 ml. dialyzed cell homogenate, and water to adjust
final volume to 3 ml. The reaction was followed spectrophotometric-
ally by determining the optical density at 300 mu every 15 seconds.
Three controls (one lacking malate, one lacking NAOP and a third

without cell homogenate) showed no Increase in optical density.

.05

0. D. at 340 mu
5
no

.02

.01

  

MALIC ENZYME

x—X EXPER.

0—0 CONT.

   

30
SECONDS

 

32
substitutedfor NAO, acts as a ii+ acceptor in the conversion of
malate to oxaloacetate. The two electrons Involved were passed to
a tetrazolium salt with a resulting change In its 0.0. at 650 mu
I(Flguro 5). in the control which lacked~sodium fumarate In the
reaction mixture, there was no change in absorption. Unfortunately,
the spectrophotometer was not adjusted to give a zero reading for
the Inactive reaction mixture.

The final step in demonstrating the pathway leading to the
formation of succinic acid was the reduction of fumarate by
succinic dehydrogenase. The assay for this enzyme system was
carried out spectrophotometrically at #00 mu by the method described
by Bonner (5). The procedure and results are outlined with Figure
6. The fumarate free control showed no measurable activity.

The obligate anaerobe, Nlcgggoccus lactilytlgus, contains an
enzyme, fumarate reductase, which catalyzes the reduction of
fumarate to succinate far faster than the reverse reaction (35).
It is relatively insensitive to maionate inhibition (57). An
attempt was made to determine if the enzyme in‘11_t. augusta was
similar to that noted for‘!. lactllyticug by testing the inhibio
tion of the reaction by maionate. There was a definite decrease
in succinate production from glucose In the presence of maionate
(Figure 7). Only a trace of succinate could be measured in the
sample eluted from a ceiite chromatography column. It was also
obvious there was an accumulation of fumarate in the trichomonad
cells treated with maionate (Figure 7) and that the succinic den
hydrogenase in 11.1.1- _a_gg_g_s_t_g was susceptible to maionate at the

concentration employed. The specific activities of the enzymes

33

Figure 5. Activity of fumarase In cell homogenates 'T,ILL£-
augusta. Reaction mixture: 0.5 ml. 0.0l7N sodium fumarate, 0.5
m1. 0.033N phosphate buffer pH 6.8, 0.1 m1. 0.0015N NAO, 3 ml. l1
solution of Trlphenyltetrazollum chloride, 0.5 ml. phenazine metho-
sulphate (lmg./10 ml.), and 1.0 ml. dialyzed cell homogenate. A
three ml.aliquot was placed into a cuvetto and 20 units of malic
dehydrogenase was added to It. The reaction was then measured at
one minute Intervals at a wavelength of 650 mu. The control
system was composed of the above reaction mixture omitting the

sodium fumarate.

O. D. at 650 mu

.39

.38

I31

.36

 

 

FUMARASE

/0—0
0

'/'/'/4 c o N T.

3
MINUTES

 

3h

Figure 6. Succinic dehydrogenase in a crude extract 9f.ILL£-
aggugt . The cuvetto contained: 0.3 ml. 0.I N KCN, 0.3 ml. 0.01H
K3Fe(CN)6,O.2 ml. 0.2M sodium succinate 2 ml. O.l5N phosphate
buffer pH 7.2. The reaction was Initiated by the addition of 0.2
ml. of dialyzed cell homogenate. The change in optical density
was followed spectrophotometrically at 000 mu at 15 second Inter-
vals. The control system contained the all above components

except sodium succinate.

SUCCINIC DEHYDRDGENASE

  
    

.09
0_0
o“‘—’

3
E
O
3
g .06 o
N
9:
O
.5
i‘,‘ _ exam.
2
<
a .0 0 VIII/II. C 0 N T.

0 15 0 45 o 0 s

  

SECONDS

35

Figure 7. Naionate Inhibition of succinate fonmation in whole
cells of 1115,. aggusta. Each Thunburg tube contained: 1 m1 0.02N
glucose, I ml of a cell suspension containing 3 X 106 cells per ml,
0.I ml maionate and 7.9 ml 0.IN phosphate buffer pH 6.8. The
control consisted of the above mixture minus maionate. The Thun-
burg tubes were alternately placed under vacuum and flushed with
oxygen-free nitrogen three times. They were then Incubated one
hour at 30 C. The cells were then removed from the reaction mix-
ture by centrifugation and the supernatant extracted with ether
and separated on a ceiite column. The concentration of fumarate
and succinate are given with and without maionate In the reaction
system.

Key: F - fumarate, S - succinate.

Ml. 0.015M ALC. KOH

INHIBITION OF SUCCINATE FORMATION
BY MALONATE

NO MALONATE

 

 

TUBE NUMBER

36
involved In the succinate pathway are given in table 6.

IV. Oxidation-Reduction Systems Associated with Lactic and
Succinic Acid Production and Adenosine Trlphosphate
Formation. ’ _

Lactic dehydrogenase has been reported in I, vaginalls (2, 60).
Using the method of Kornberg (19) an attempt was made to demon-
strate lactic dehydrogenase in 111;. augusta by following the
oxidation of NAON+N to NAO with sodium pyruvate as substrate. The
results showed the enzyme to be very active in this organism (Figure
8). When either pyruvate or NADN was omitted from the reaction mix-
ture no enzyme activity occurred.

Flavln adenine dinucieotide is usually the flavin associated
with succinic dehydrogenase. To determine if this coenzyme was
present, homogenates of l X 107‘I;1;. aggggta organisms and standard
solutions of FAD and riboflavin were chromatographed on paper.

Spots were located by their fiourescence and the calculated Rf
values of the unknowns were found to be identical to those obtained
for standard solutions of FAO and riboflavin (Table h).

It next remained to determine If FAD participated in the re-
actions associated with the formation of succinate. An assay was
devised whereby reduced NAD and oxidized FAD were added to a cell
extract of 1.1213.- W to detect any Interaction of these twoco-
enzymes. The complete assay contained added FAD, ADP, NADH, phos-
phate buffer (pH 6.8) and cell homogenate. The oxidation of NADH
was followed spectrophotometrically at 300 no at 15 second intervals
for one minute (Figure 9). The oxidation of NADH appears to be

dependent on the presence of ADP and inorganic phosphate. For the

37

.Flgure 8. Lactic dehydrogenase activity of an extract of
111_,maugusta. The cuvetto contained: 0.1 ml 0.01M sodium pyruvate,
0.1 m1 0.002H NADN, 1.0 ml of 0.1" phosphate buffer pH 7.# and 0.2
mi dialyzed cell extract. Two controls were prepared: one lacking
pyruvate and a second lacking NADH. The oxidation of the re-
duced NAD was followed spectrophotometrically at 3h0 mu with the
change In 0.0. recorded every 15 seconds. Neither contrei showed

a detectable change in 0.0.

.20

L
as

CHANGE in 0.0. at 340 mu

.04

b
9° .

 

 

15

LACTIC DEHYDBOGENASE

I ' i I l."/‘ l '1',

30 45
SECONDS

_ EXPER.

 

60

75

38

Table h. Evidence for the presence of riboflavin, FAD and ATP in
Trit. augusta as determined by paper chromatography.

 

Controls Rf Values* Cell Nomogenate Rf Values*
10 lambda 0.002N Rb 0.81 80 lambda 0.81
10 lambda 0.002N FAD 0.15 80 lambda . 0.15
10 lambda 0.002N ATP 0.08 80 lambda O.h9

*) Rf values were determined by observing the fluorescence of the
flavins and the visual UV. quenching of ATP using a Ninerallte
lamp.

Table 5. Amount of.ATP resulting from electron transport assoc-
Iated with succinate production.

 

uN ATP
Reaction Change In As uN NADN' uN NADH
Nixture As*257mu 3h0mu/min/ml. 0n ATP*/ml. Oxid./ml. Oxidized
Exogenous' 0.105 0.0u2 0.071 0.071
Endogenous” 0.015 ----- 0.010 -----
Exo. - Endo. 0.090 0.0112 0.061 0.071 0,061 .
0o07'
0.847

a) Exogenous Reaction Nixture: 1.7 mi. 0.IN phosphate buffer pH 6.8,
0.2 ml. of 0.002“ ADP, 0.002" FAD, and 0.002" NADH and 0.7 II.
dialyzed cell extract.

b) Endogenous Nixture: Above minus the reduced NAD.

*) ATP was separated from the reaction mixtures by paper chromato-
graphy as described In the methods section. Hilllmoles of1ATP
were determined using the following equation: Am-As/C; where
Am - molar absorbancy Index; As - absorbancy and C - concentra-
tion in mH/liter.

') The amount of NADN oxidized per min. per ml. was determined from
the change in absorbancy at 3h0 mu using the same equation given
above.

39

t Figure 9. Activity of enzyme(s) involved in the terminal.
respiration oijLLt. augugta. In cuvetto: 0.1 ml. 0.002N NADN,
0.1 ml. 0.00211 FAD, 0.1 ml 0.00211 ADP, 2.2 ml 0.111 phosphate
buffer pH 6.8 and 0.5 m1. cell homogenate dialyzed in TES buffer.
Controls: 1) no NAON, 2) no FAD, 3) no ADP and A) no inorganic
phosphate. The oxidation of NADN was determined spectrophoto-

metrically at 3&0 mu at 15 second Intervals for one minute.

CHANGE in 0.0. at 340mu

 
 

09 NAD-FADi-Iz-ATP nscsnznnmc SYSTEM
' COUPLED wmc SUCCINATE FORMATION

o‘————0

TS

omnwmno

1’”,

   
     
 
   
 
   

 

”III/4 - FAD

ummmu-ADP

-NADN

on
-Pi

  

."

L’. «00“"fl

sou-nun O lino-nuns" Ouuuuuuue 0
‘oeou

B .2 I

    
 
  

   

~th0

o 45
SECONDS

      

  

    

O

. p 00
first 30 seconds the addition of oxidized FAD had no effect on,

the rate of NADN oxidation, but after 30 seconds FAD is required
for optimum activity. Because the FAD-NADN oxidation reduction
system became inoperative in the absence of ADP and Inorganic phos-
phate it appeared asthough a reaction similar to the first step

of an oxidative phosphorylyation scheme might be present in this
anaerobic protozoan.

'To determine if indeed there was a production of ATP in the
above reaction paper chromatographs of the complete reaction mix-
ture were compared to mixtures containing no NADN and to ATP
standards. The Rf values of the spots from both reaction mixtures
were identical to the Rf values of the standard ATP (Table h).

A 7-fold greater yield of ATP was found in the complete mixture
than in the endogenous system (Table 5). A quantitative comparison
showed that for every NADH molecule oxidized 0.8h7 molecules of

ATP were fonmed. The specific activity of the enzymes involved is
listed in table 6.

In comparing the specific activities of all the enzymes
studied In this Investigation lactic dehydrogenase Is by far the
most active. Succinic dehydrogenase, the second In activity Is
only half as active as lactic dehydrogenase. The activities of

the other enzymes were much lower than succinic dehydrogenase.

#1

Table 6. Specific activity of enzymes asSociated with the pro-
duction of end products by T:|§.ygggg§;g.

 

 

Em
W
Formic Dehydrogenase
Hydrogenase

Formic Nydrogenlyase

Succinic Acid nggucglon
Nailc Enzyme

Fumarase

Succi ni c Dehydrogenase

Systggs f0; Oxidatlgg g: NADN
Lactic Dehydrogenase

Terminal Respiration-Type System
(redn. of FAD, oxid. of NADN and
fonmatlon of ATP)

Sggciflc Activitxt

3.5“r
2.5

7.0

6.98“
2.0
69.8

107.5‘I
17.1

*) Specific activity based on the conversion of l uN of substrate/

min./mg. cell N.

a) Specific activity expressed as units/mg. cell N, where one unit
of enzyme is defined as that amount required to cause a change

in 0.0. of 0.01/min.

DISCUSSION AND CONCLUSIONS
This Investigation showed that T:I§.,ggggs§g was capable of
metabolizing glucose to a mixture of end products which included

acetate, lactate, formate, succinate, CO2 and N Similar end

2.
products have been reported for other species of trichomonads

(12. 18. 23. 37, #9. 58, 60). However, all the metabolic path-
ways involved in the formation of these and products are incom-
pletely known except for that of lactic acid formation. Demon-
strations of lactic dehydrogenase in I, yaglnallg (2, 60) and in
1:11, gaggggg In this study provide conclusive evidence that
lactic acid can be formed by the reduction of pyruvate. This
mechanism illustrates one means by which ILLE- m; can oxidize

NADN so that this co-factor can recycle through the glycolytic

pathway.
Gas egg Aggtate Fgcggtlgg.

Suzuoki and Suzuoki were the first Investigators to detect
formic dehydrogenase in a trichomonad (50). However, they were
unable to show a complete hydrogeniyase system. Two different
pathways for the formation of acetate, CO2 and N2 have been
postulated, both similar to those found in certain faculative and
obligate anaerobic bacteria (23, #0). Because Ryley could not
obtain iIz evolution from formate in homogenates of ILL- 123.11.!
under anaerobic conditions, he proposed that I;1§..figgtgg,must
produce “2 by a pathway similar to the phosphoroclastic-type
split of pyruvate found In the Clostridia (#0). Llndblom de-
tected strong formic dehydrogenase activity, but only weak

#2

#3 y .

hydrogenase activity in' members of the 1m. LESLIE. complex and fl.
gallinarum (23). Ne reasoned that the trichomonads produced N2
like the‘ggtggggggtggiggggg,because of their strong formic de-
hydrosenase activity and his Inability to demonstrate the presence
of a clostridiaI-type of reaction.

in this Investigation both acetyi phosphate and formate plus
the two enzymes of the fonmic hydrogeniyase system were detected
In 1.13.110 mm. The theory of an Egtgpgagtgflacgae - like
phosphoroclastic system being Involved In trichomonad gas evolu-
tion seems to apply to 111;. m and probably to other tricho-
monads as well. The acetyi phosphate is most likely the precursor
of the acetate produced as an end product by this protozoan.
Bacteria such as thelggtggggggtgglggggg can hydrolyze acetyi phos-
phate to fanniocetate and high energy phosphate which reacts with
ADP to form ATP. The presence of acetyl phosphate is presumptive
evidence that ILLI- £019.23. and other trichomonads may form ATP
by this method. however, It is possible that other species of
trichomonads have different or added mechanisms for N2 + 002
evolution. An NADlI oxidase system has been demonstrated In I.
.zgglggllg,whlch may serve to oxidize NADN with the release of
molecular I2 (26, 58).

ShflfiflllflflLlflllhflflflJUb

None of the previous Investigations on trichomonads have
shown a complete pathway for succinic acid production. Hhen
Kunltake‘gthgl, used labeled glucose as a substrate for the growth
of I. vaginalis they found a significant amount of the labeled

carbon appearing In amino acids (21). The labeled carbon In a

.##

succinate substrate appeared in CO They considered the

2‘
labeling pattern as evidence for a Kreb's cycle even though they
were unable to demonstrate the enzymes of the pathway in cell
free systems. Two possible pathways have been suggested for the
production of succinic acid (23). One possibility was the con-
denstatlon of two acetate molecules (as proposed by Seaman (#5)
for‘Tetrahymena) derived from either a phosphoroclastic reaction
or from the hexose monophosphate pathway. The second was the
formation of malic acid from the fixation of CO2 to lactate or
pyruvate. Llndblom did not speculate on how succinate would be
formed from malate other than to state that fuaarate, though
undetected, might exist in these protozoans(23).

After an earlier failure (58), Hellerson and Kupferberg
found a mamnalian type of malic enzyme In '_I'_. will; (60).
Ryley provided Indirect evidence for CO2 fixation In 151;,
.123533 by showing succinate formation was dependent on the presence
of C02 (#0). A "malic type enzyme"was also detected in.£.
galliggrum which removed CO2 from malate and left lactic acid
as the end product (23). The Inability of Investigators to
demonstrate fumarase In cell extracts of several trichomonads
left the pathway for succinate production open to conjecture (#,
23). Seaman reported that cell extracts of I, gaginalls possessed
a succinic dehydrogenase (#3). This enzyme was not detected in
whole cells of‘I, vagigalis, but Read admitted that the inability
of succinate to permeate Into Intact cells could explain his neg-

ative results (37. 62).

A. Nailc eggzgg: The results of this investigation onllzlt.

us
gggggtg supports previous observations that the malic enzyme is
essential for malate fonmatlon. \The enzyme found In this organism
resembles the enzyme described for I, vaginal]; In that It required
reduced NADPII (60). The NADPI'I required by this system j_n yj_v_c_1
might come from the conversion of glucose-6-phosphate to 6-phos-
phegluconate by glucose-6-phesphate dehydrogenase, an enzyme which
has been reported In the Idl- £223.22 complex and fl. gallinagg
(23). it is also possible that a mechanism for the Intercenverslon
of NADP by reduced NAD to IADPN may be present In these cells al-
though no attempts have been made to demonstrate such a system.

The malic enzyme demonstrated in Trit. augusta fixes CO to pyru-

2
vate and not lactate. it is possible that the lactate detected by
Llndblom, as the result of malate decarboxylation (23), actually
resulted from the rapid reduction of pyruvate by a very active
lactic dehydrogenase. ills undialyzed preparations may have con-
tained a sufficient concentration of NADN to facilitate lactate
formation.

B. M: For succinate formation to occur by reversal of
the Kreb's cycle the conversion of malate to fumarate by fumarase
ls essential. The demonstration of fumarase activity in 111;.
auggstg proved to be not only the most difficult but also the most
essential contribution to the determination of the pathway for
succinate formation. A coupled reaction was necessary to demon-
strate fuarase activity because of the apparent need for strong
electron acceptor such as phenazine methosulphate to make the re-

action proceed to completion. The In 111.! association of the

enzyme with the succinic dehydrogenase system and its flavin

#6
aceptor may serve to control Its activity.

C. Sucginlg dehydgggenas : The demonstration of succinic
dehydrogenase in 1.12.1.5.- augusta supported the report of this enzyme
in L yogigailg (#3) and completed the list of enzymes essential
for succinate formation by ILLI- aggusga. The strong Inhibition of
succinic dehydrogenase In this organism by maionate shows that the
trichomonad enzyme resembles the mammalian enzyme more than the
fumarate reductase reported for £1. lactllyticus (35. 57). IIalonate
inhibition of succinate dehydrogenase will not stop the grovlth of
trichomonads (#6). Since maionate will Inhibit trichomonad motility
(#0), perhaps the additional energy supplied by the terminal respir-
ation system Is In part responsible for active locomotion.

Electggn Tgansporg Sygga Associatgd um Sgcgimte Pathway.

Flavlns have been detected in I. vaglgaIIs (58) and indirect
evidence has been reported for the presence of a flavoproteln
terminal oxidase In this organism (#, I9, 37).. An NADII oxidase
has alsolbeen described for 1. vaginal]; that operates In the
presence of oxygen (58). The significance of an NADII oxidase sys-
tem that Incorporates oxygen is difficult to evaluate since these
trichomonads fail to grow In an aerobic environment.

Studies on 111;. augusta suggest that the oxidation of FADI-I2
and the reduction of NAD occur jointly with the operation of the
succinic dehydrogenase system. The coupling of these electron
transport systems result In the anaerobic reduction of fumarate
by NADII. Phosphorylation appears to be concurrent with succinate
formation or the accompanying electron transfer. In this way the

formation of succinate not only provides a means of reoxidizlng

1+7
NADII, but also generates energy in the form of ATP with nearly one
ATP produced for every MON oxidized. It is possible that the NADH
oxidase system proposed by Nellerson _e_t_ _a_l_. for '_i'_. xaglnalis (58)
mightrequlre flavins for activity and be part of the electron
transport system observed in ILLS: aggugta. Unfortunately,
Hellerson and his co-workers did not look for ATP production in
association with the oxidation of NADII. The proposed pathways for
the formation of all end products except lactate from pyruvate and
the accompanying electron transport systems are Illustrated In
figure 10.

Systems for succinate production similar to the one described
for ILL- augusta have been observed In other parasites such as
W m and Agcaglg 1m. Uhen grown anaerobic-
ally I. 5.031. produces acetate, lactate and succinate from glucose
by an identical pathway to that seen In ILLS- aggusta (39).
Succinate Is also produced by this organism under aerobic conditions
(6). 1. mg; differs from 1,115,. m In that a complete Kreb's
e761. has been shown to be functional as ".11 as c02 fixation to
form succinate. This organism has also been shown to possess a
terminal respiration system with cytochromes (39).

liuscle strips of A. lumbricoides were observed to fix C02 to
pyruvate to yield malate by means of the malic enzyme system. This
system differs from that In ILLE- 3.9.9.2259. inthat It can utilize
either oxidized IAD or NADP as a co-factor,,but shows higher
activity with NAD (#1). Both fumarase and succinic dehydrogenase
are demonstrable in the worm (#l). Fumarate was reported to be

reduced by NADll to form succinate In conjunction with several flavo-

#8

Figure 10. Proposed scheme for succinate production, hydrogen

and carbon dioxide evolution and terminal respiration In.TrIt.

IQQUSSO.»

PROPOSED SCHEME FOR SUCCINATE

PRODUCTION - HYDROGEN EVOLUTION

AND TERMINAL RESPIRATION IN TRI-
TRICI'IOMONAS AUGUSTA

Pyruvate ——) Acetate
e
COZ nadph Formate
malic d fhogmic nase
r
enzyme e I! 08 nad
Maiate
Cozs-NADH
fumarase
Fuma rate '
EAEHfiZ
succinic "AD. "2
NADI'I dehydrogenase
HADP-I-Pi

Succinate

#9

protein carriers. These carriers were reported to be succinic
dehydrogenase and NADN dehydrogenase (l8). Kmetec speculated that
the coupling of these systems In the nematode would provide not
only a means for oxidizing NADN, but also would generate ATP (18).
It Is this type of system which seems to be functioning in m.
a s a. Nowever,,5, umbgicojdgg decorbexylotes succinate to
form propienate rather than excreting It as an end product (#1).

The bacterium, ngglongbacteg‘gggglggggg, also has the ability
to form succinate and propienate by a similar pathway (6#). .fi.
aggbiggsgg uses the enzyme phosphoenolpyruvate carboxylase to fix
602 to phosphoenolpyruvate to form oxaloacetate. Oxaloacetate Is
then converted to succinate by a reversal of the Kreb's cycle.
However, preliminary work has shown that this pathway is not the

major source of energy In this organism. In fact the fixation of

CO2 by 2. $221322! requires energy (6#).

Fgcgggtatiog Balagcgs.
The end products produced by an organism gives an Insight to

the metabolic pathways that may be present and the probable elec-
tron acceptors and donators Involved. It Is for this reason the
stoichlometry of the glucose metabolism of 11:1;- gm was _
studied. There were some differences between the fermentation
balances obtained from the continuous culture system and the Har-
burg appartus, even though cells for both systems were grown in
continuous culture. A trace of formate was detected In the ex-
tracts from the continuous culture medium but not in the Narburg
sample. The fonmate probably come from cells damaged by the Im-

peller of the continuous culture apparatus.

50

A large difference was observed between the quantity of
succinate formed In continuous culture and that measured In the

Harburg balance. Nanometric experiments using T; g. foetus with-

 

out C02 In the gas phase showed low yields of succinate (#0)
similar to the results obtained with ILLL. augusta. Since the
trichomonads produce an excess of C02, a low partial pressure of
this gas would still exist in the fluid phase without adding CO2
to the gas phase. In the continuous culture system the greater
volume of fluid and a presumed reduced rate of exchange between
the liquid and gas phase may have kept higher 002 tensions in the
medium. This may have promoted succinate production In spite of
the constantly changing N2 atmosphere In the growth flask.
Lactate production was high in all fermentation balances.
The reason ILLI- m produces such large amounts of lactate,
when the fonmation of succinate appears to be more profitable, is
not known. Lactate production by means of the glycolytic pathway
is however associated with rapid growing organisms. For example,
homofermentatlve Iactobacllll In a complex medium develop so
rapidly that In 16 hours or less growth Is limited by the amount
of lactate accumulated (63). Possibly lactate Is produced
Initially 57.1LL1- agggsta to promote rapid growth, and after
there Is a certain accumulation of lactate or other limiting con-
ditions exist in the medium, metabolism Is shifted to produce
acetate and succinate to gain more energy (ATP) for cellular pro-
cesses. Llndblom stated that, "the trichomonads he studied pro-

duced more acid during the early stages of their growth and since

acid production points to an anaerobic metabolism, perhaps In

Illlil‘lllll‘lilllllllll II]! All Illl ll ilt‘l llllinll Ill-III. Ill-I'll (III .11 III II (1.1. (I1 .
I‘ll-

51
young cultures the giycoliytic pathway ls preferred" (23). Read
and Rothman reported that I,_vaginalis after being cultured for.
long periods of time tended to produce greater amounts of lactate
(38). Perhaps the growth of trichomonads under artificial cone
ditions ls selective for organisms which grow most rapidly, Ie.
those which produce the most lactic acid.

The quantities of CO and N2 reported in both carbon balances

2
were too high on a molar basis for the amount of acetate formed
If all CO2 and N2 is formed via the breakdown of pyruvate to

acetate, 00 and N . it Is possible that these gases might arise

2 2

from other pathways. A breakdown of amino acids present In the
growth medium might be one such mechanism. There are large
quantities of glutamic and aspartic acids present in the serum

(17) and In the trypticase of Diamond's medium (Baltimore Bloc

logical Lab. assay, unpublished data). Perhaps these amino acids

are decarboxylated by Trit. augusta and in the process CO2 released.
ILLi-.£2§£ ; has been shown capable of adsorbing amino acids from

medium and maintaining them as a pool of free acids (17). Some

of the 602 and N2 produced In the Harburg balance may come from
this amino acid pool. Peck Jr. and Cast have speculated from
their data on hydrogenase that more than one type may exist, or
that perhaps the enzyme Is double-headed with two prosthetic

groups (33). They proposed that one prosthetic group might
mediate electron transport to physiological acceptors of relatively
high redox potential, while the other might be concerned with re-
duction of electron acceptors of low potential and/or with the

formation of molecular Hz. The hydrogenase In TL! . augusta may

52
not be specific for the removal of N2 from formate. Itmight also
formrli2 from amino acids such as the cysteine added to the medium
to aid anaerobiosis and from reduced co-factors such as NAOH,
FADI-I2 and NADPN. The NADN or a flavin oxidase reported In I, .
vaginalis may be Identical to the hydrogenase ofjlgit. augusta (3,
58, 62). There Is also the possibility that other unidentified

pathways exist for CO and N2 evolution which are responsible for

2
the high gas production.

Ryley followed the anaerobic fermentation of Intercellular
glycogen by Igit.‘ngtgg,manometrlcally (#0). He obtained a
fermentation balance which contained acetate, lactate, succinate,
N2 and negative C02. His carbon recovery was 83% and the 0/R ratio
was 1.0. The amount of succinate produced was three times greater

than the acetate formed in a gas phase of 95% N2 and 5% CD It

2.
appears that cht. fggtus can use stored glycogen to form succinate
and re-cycle NAD, and that It is not dependent on lactate pro-
duction. Since the balances on T:]§.ygggg§tg,were done using an

external glucose substrate It Is difficult to compare these re-
sults with those of Ryley on Trig..figgtgs.
W:

The present work Illustrates some of the pit falls In studying
the fermentation characteristics of an organism using a complex
undefined medium. however, the presence of lactic dehydrogenase,
acetyi phosphate, the formic hydrogeniyase system and the Inter-
mediates and enzymes necessary for succinate do Indicate the path-

ways in which the major end products of T51 . augusta are formed.

53
The reduction of fumarate and the concurrent oxidation of NADH
appear to serve as a terminal respiration system for this tri- .
chomonad. The system is capable of carrying out the production of
ATP by an oxidative type of phosphorylation. However, the system
is not essential for the growth of trichomonads at least under

laboratory conditions.

SUMMARY

1. Glucose fermentation balances were performed on‘ILLLJ
'agggsta during growth In a continuous culture system and during
maintenance In Narburg vessels using standard manometric tech-
niques. Analysis of the medium in both cases showed the some
major and products were produced. These were lactate, acetate,
succinate, 002 and N2. The carbon recovery with a glucose sub-
strate In the manometric study was 95% and In the continuous
culture system 89%. however, the gas production In both bal-
ances was too high In consideration of the proposed metabolic
pathway for gas production and the quantity of acetate formed.
Speculations on the reasons for this high C02 and N2 evolution
are given.

2. A complete formic hydrogeniyase system has been demon-
strated in cell homogenates of 111;. m. Aise formic acid
and acetyl phosphate have been found In other extracted homogenates.
The system resembles the phosphoroclastic-type observed In
Escheclghla‘ggll.

3. The enzymes essential to the conversion of pyruvate to
succinate (malic enzyme, fumarase and succinic dehydrogenase) and
the Intermediates Involved (malate and fumarate) have been de-
tected In cell homogenates of ILL auggstg.

To demonstrate fumarase It was necessary to utilize a
coupled reaction Incorporating an external source of malic den

hydrogenase. Electrons were transfered from phenazine methen

5#

55
sulphate to a tetrazolium salt. The succinic dehydrogenase
of 1m. augugta seems to resemble the maionalianenzyme and not
the fumarate reductase described In the anaerobic micrococcus
.5. Iagtllytlcgg.

#. Besides the lactic dehydrogenase system a second type of
oxidation mechanism was detected In 1:15. agggsta which may rep-
resent a means for terminal respiration In the trichomonads as
well as a mechanism for.ATP formation. The Importance of this

pathway Is discussed.

9.

10.

ll.

12.

LITERATURE 01150

Anderson, E. and Ii. ii. Beams. 1959. The cytology of T: It-
;Lchm'msi, as revealed by the electron microscope. J.
NOI'PI'l. L—g 205-236o

Baernstein, N. 0. ‘1959. Lactic dehydrogenase In Tpichg-
m vagig 11;. J. Parasit. 35: #91-#98.

Baernstein, N. 0. I961. Nolic dehydrogenase in Trichg-
_m_g_g_a_s_ yogiggig. J. Paroslt. Lil: 279-28#.

Baernstein, N. D. 1963. A review of electron transport
mechanisms In parasitic protozoa. J. Parasit. #2: 12-21.

Bonner, N. D. 1955. Succinic dehydr enase, p. 722-729.
In S. P. Colowick and N. O. Kaplano?ed.), Nethods In
Enzymology, vol. I. Academic Press, Inc., New York.

Bovnaan, I. B. 8., E. J. Tobie, and T. Von Brand. 1963.
C02 fixation studies with the culture form of Tgpago-
gag ggu; . Comp. Biochem. Physiol., 2: 105-11 .

Chakraborty, J., N. N. Das Gupta, and N. N. Ray. 1961.

An electron microscope study of ngghomongs gzlcetl.
Cytologica .26: 320-326.

Diamond, L. S. 1957. The establishment of various tri-
chomonads of animals and man in axenic cultures. J.
Paraslt. £11: #88-#90.

Dimant, E., D. R. Sanadi, and F. N. Nuennekens. 1952.
Isolation of flavin nucelotldes. J. Amer. Chem. Soc.
1‘1: 5##0-5###.

Doran, D. J. 1956. Aerobic metabolism of Tgltzichomonag
f and chghgggas £22.- from swine. J. Protozool.
1: Suppl.) 13.

Doran, 0. J. 1957. Studies on Trichomonads. I. The
metabolism of Tzitrlchgoggs Mg; and trichomonads from
tlaie nasal cavity and cocoa of swine. J. Protozool. 5:

I 2"90o

Ooran, D. J. 1958. Studies on trichomonads. Ii. The

metabolism of a Tglchomonas batrachogum-type f lagellate
from the cocoa of swine. J. Protozool. 5: 89-93.

56

1‘ ‘- II I ill I'll I II I III I.

13.

l#.

15.

16.

17.

18.

19.

20.

21.

' 22.

23.

2#.

25.

57

Doran,'0. J. 1959. Studies on trichomonads. III. Inhibi-
tors, acld production and substrate utilization by #

strains of Tcitgichggonag feet g. J. Protozool. 6: I77-
I82.

Fencl, Z. ’1966. Theoretical analysis of continuous culture
systems., p. 69-153. In I. Nolek and Z. Fencl (ed.),
Theoretical and Methodological Basis of the Continuous
Culture of Microorganisms, Academic Press, Inc., New York.

Good, C. A,, N. Kramer and M. Somegyi. I933. The deter-
mination of glycogen. J. Biol. Chem. 1993 #85-#9l.

Norbert, D. 1959. Some principles of continuous culture.
Rec. Prog. MIcrobIol., Symp.'!LL. Intern. Congr. for
Microbiol., Stockholm. p. 381-396.

Johnson, A. E. 1962. The free amino acids In Tzitgl-
mm. Exp. Parasit. _l_z: 168-175.

Kmetec, E. and E. Buedlng. I961. Succinic and reduced
0PM oxidase systems of Ascagjg muscle. J. Biol. Chem.
2.3—3 5811-593.

Kornberg, A. 1955. Lactic dehydrogenase of muscle, p. ##1-
##3. In S. P. Colowick and N. 0. Kaplan (ed.), Methods
in Enzymolegy, vol. 1. Academic Press, Inc., New York.

Kupferberg, A. B., N. O. Singher, G. Lompson, L. Levy and
A. A. Romano. 1953. Studies on the metabolism of T -

.ghggggg; vagjnallg. Ann. M. Y. Acad. Sci. 56: 1006-
1015.

Kunitako, 0., C. Stltt and P. Saltmen. 1962. Terminal

respiration In Trlghggonas gaglgglj . J. Protozool..2:
371-373.

Leo, J. J., 5. Pierce, 8. H. Nutner, B. J. Smith and D. R.
Gurski. I962. Trichomonads from poikiletherms: nutri-
tional and physiological notes. J. Pretozool.l2: ##5-

59.

Llndblom, G. P. 1961. Carbohydrate metabolism of tricho-
monads: growth, respiration, and enzyme activity In four
species. J. Protozool..§: 139-150.

Llpmann, F. and L. C. Tuttle. l9#5. A specific micro-
technique for the determination of acyl phosphates.
J. Biol. Chem. 152: 21-28.

Magllocca, R. 1963. Incorporation of 'uC-labeled bicar-
bonate In Trjtrjchomonas foetus In vitro. Studl Sassaresi
SOZo lo a: 231+-239o

 

26.

27.

28.
29.

30.

31.
32.

33.
3‘1.
35.

36.
37.

33.

58

Magllocca, R. 1963. Respiratory enzymes of Tritrlchomonas
foetu . Studl Sassaresi Sez. 1. #l:'53#-5#0. _

Massey, V. '1955.' Fumarase. Po 729°735. In S. P. Colowlck
and N. 0. Kaplan (ed.), Methods In Enzymology, vol. 1.
Academic Press, Inc., New York.

Nattern, C. F., F. S. Brackett and B. J. Olsen. 1957.
Determination of number and size of particles by
eéectricai gating: blood cells. J. Appl. Physiol. 10:
5 '70-

Nelsh, A. C. 1952. Analytical Methods for Bacterial Ferm-
entations, National Research Council of Canada, Prairie

gogional Laboratory, Saskatoon, Canada. 2nd revision,
2 p.

Ninomiya, N. and Suzuoki, Z. 1952. The metabolism of 1;;-
ghomgga; a nail , with comparative aspects of trichomonads.
J. Biochem. Japan) 32: 321-331.

Ochoa, S. 1955. "Mallc" enzyme from pig liver and wheat
erm, p. 739-7#8. In S. P. Colowick and N. O. Koplan
(ed.), Methods In Enzymology, vol. 1. Academic Press, Inc.,
New York.

Pabst Laboratories. I956. Paperchromatography, p. 20. in
Pabst Laboratories, Circular OR-IO, Ultraviolet Absorption
Spectra of 5' Nucleotides, Pabst Brewing Co., Milwaukee,
Hisconsln.

Peck, Jr., N. D. and N. Gest. 1956. A new procedure for
assay of bacterial hydrogenases. J. Bacteriol.'11: 70-

Peck, Jr., a. 0. and N. Gest. 1957. Formic dehydrogenase
and the hydrogeniyase enzyme complex In coll-aerogenes
bacteria. J. Bacteriol.'13: 706-721.

Peck, Jr., N. 0., O. N. Smith and N. Gest. 1957. Compare-
tive biochemistry of the biological reduction of fumaric
acid. Biochem. at Biophys. Acts. 25: l#2-I#7.

Read, C. P. 1955. Comparative studies on the physiology of
trichomonad flagellates. J. Parasit. 51: (Suppl.), 16.

Read, C. P. 1957. Comparative studies on the physiology of
trichomonad protozoa. J. Parasit. 51; 385-39#.

Read, C. P. and A. N. Rothman. 1955. Metabolism of Trich-
omonas vaginall . Am. J. Nyg..§L: 2#6-260.

.l .151 la! All" 1. .5 It (I

39.

#l.

#2.

#3.

#9.

50.

51.

52.

59

Roe, J. N. 1955. The determination of sugar In blood and
spinal fluid with anthrone reagent. J. Biol. Chem. 21;:
335-3#3.

Ryley, J. F. 1955. Studies on the metabolism of the protozoa.
5. Metabolism of the parasitic flagellate Trichomonas
foetus. Biochem. J. 53: 361-369.

Ryley, J. F. 1956. Studies on the metabolism of the protozoa.
7. Comparative metabolism of eleven species of trypano-
somes. Biochem. J.,ég: 215-222.

Saz, N. J. and J. A. Nubbard. I957. The oxidative decorb-

oxylation of malate by Ascacis lgmbgicolde . J. Biol.
Chem.‘ggfiz 921-928.

Seaman, C. R. 1953. Inhibition of succinic dehydrogenase
of parasitic protozoans by an orseno and phosphono analog
of succinic acid. Exp. Parasit.'g: 366-373.

Seaman, G. R. 1959. Experiments In Microbial Physiology and
Biochemistry, p. #2-#6, Burgess Publishing Co., Minn-
eapolis, Minnesota.

Seaman, G. R. and M. D. Naschke. I955. Reversable cleavage
of succinate by extracts of Tgtrahyggna. J. Biol. Chem.

311: 1-12.

Shorb, M. S. l96#. The physiology of the trichomonads,
p. 38#-#59. In S. N. Nutner (ed.), Biochemistry and
Physiology of Protozoa, vol. III. Academic Press, Inc.,
New York.

Simpson, C. F. and F. N. White. l96#. Structure of Igjghg-
moggs fggtus as revealed by electron microscopy. Amer. J.
Vet. Res. 25: 815-82#.

Smith, B. F. and Stewart, B. T. 1966. Fine structure of
Mamm- Exp. Par-sit. .12: 52-63.

Suzuoki, Z. and T. Suzuoki. 1951. Carbohydrate metabolism
of ngchomonas foetus. J. Biochem. (Japan) 38: 237-25#.

Suzuoki, Z. and T. Suzuoki. I951. Hydrogen evolution of
TEIchomonas foetus. Nature 168: 610.

Swim, N. E. and L. O. Krampitz. l95#. Acetic acid oxida-
tion by Escherichia coll: Evidence for the occurrence of
a tricorboxylic acid cycle. J. Bacteriol. 61: #19-#25.

Twohy, D. H. 1961. A continuous flow culture system for
WM- 4- Prat-noi- §= (51112191.). 5.

53.

5#.

55.

55.

57.

58.

59.

61.

62.

63.

60

Twohy, D. V. and P. A. Tucker. 1961. Characteristics of
Tgitrichomonas au usta from a continuous flow culture.
J. Protozool. 8: ‘Suppl.), 5.

Umbrelt, H. R., R. H. Burris, and J. F. Stauffer. 1959.
Manometrlc Techniques, Burgess Pub., Minneapolis,
Minnesota, #th. ed.. 338 pp.

Umbreit, H. U. 1960. Metabolic Maps, vol. II. p. 29-#9,
Burgess Publishing Co., Minneapolis, Minnesota.

Utter, M. F. and K. Kurahashi. l95#. Purification of
oxalocetlc carboxylase from chicken liver. J. Biol.

warringa, M. F. P. J., 0. N. Smith, A. Guidetta and T. P.
Singer. 1958. Studies on succinic dehydrogenase. VIII.
Isolation of a succinic dehydrogenase-fumaric reductase
from an obligate anaerobe. J. Biol. Chem. ;1_: 97-103.

Hellerson, R., G. Doscher, and A. B. Kupferberg. 1959.

Metabolic studies on Tgichomonas xaglnalls. Ann. N. Y.

Hellerson, R., G. E. Doscher, and A. B. Kupferberg. 1960.
Carbon dioxide fixation in Tgichomonas vaginal! .

Hellerson, Jr., R. and A. B. Kupferberg. 1962. On
zigczlzsis In Trichomonas yaglnall . J. Protozool. 2:
l - 2 .

Hirtschofter, S. and T. L. John. 1956. The metabolism of

Tglchomonas vaginaljs: the glycolytic pathway. J. Proto-
zool . 3: 83-85.

letschafter, S., P. Saltman, and T. L. John. 1956. The
metabolism of ngchomonas yaglnall : the oxidative path-
way. J. Protozool. 3; 86-88.

Hood, N. A. 1961. Fermentation of carbohydrates and re-
lated compounds, p. 59-150. In I. C. Gunsalus and R. Y.
Stanier (ed.), The Bacteria V01. 11: Metabolism, Academic
Press Inc., New York.

Heed, N. G., and C. N. Workman. 1936. Mechanism of glucose
dissimulation by the preplonlc acid bacteria. Biochem.
J. 39: 618-623.