Vol. 52

Studies on the Metabolism of the Protozoa3. METABOLISM OF THE CILIATE TETRAHYMENA PYRIFORMIS

(GLAUCOMA PIRIFORMIS)

BY J. F. RYLEY*Molteno Institute, University of Cambridge

(Received 21 February 1952)

During the past 20 years, numerous studies havebeen carried out on the nutritional requirements andmetabolic activities of certain small, holotrichousciliates, known by a variety of names, which can begrown in bacteria-free pure cultures. As a result ofmorphological studies carried out with twenty-ninedifferent cultures of these ciliates, Corliss (1952) hasconcluded that they are nearly all different strainsof one and the same organism, which should in hisopinion be called Tetrahymena pyriformi8. In thepresent paper, the 'Glaucoma piriformis' ofA. Lwoff(1932), M. Lwoff (1934) and Lawrie (1935) and the'Tetrahymenageleii' of Chaix, Chauvet& Fromageot(1947) will be referred to as Tetrahymena pyriformi8(GL),the 'Colipidiumcampylum' ofWilber& Seaman(1948) and Seaman (1949), and the 'Tetrahymenageleii' of Seaman (1950, 1951) will be referred toas Tetrahymena pyriformi8 (S), the 'Colipidiumcampylum' of Hall (1941) will be referred to as

Tetrahymna pyriformi8 (H), the 'Tetrahymenageleii' of Pace & Ireland (1945) and Pace & Lyman(1947) will be referred to as Tetrahymena pyriformi8(W), while the 'Tetrahymena geleii' of Baker &Baumberger (1941) and Niel, Thomas, Ruben &Kamen (1942) will be referred to as Tetrahymenapyriformi8 (T); Kidder & Dewey (1945) give theorigins of some of these strains.A striking chemical characteristic ofT. pyriformi8

(GL) is the unusually high content of intracellularglycogen (Ryley, 1951; Manners & Ryley, 1952). Inthe present study, the anaerobic metabolism of T.pyriformi8 is shown to depend on a phosphorylativebreakdown ofthis reserve ofglycogen, with succinic,lactic and acetic acids as end products, while theaerobic metabolism is shown to depend on processesother than glycogen breakdown.

MATERIAL AND METHODS

Organism. The culture of Tetrahymena pyriformi8 (GL)was kindly supplied by Dr E. G. Pringsheim of the BotanySchool, Cambridge; it was the strain described by Lwoff

* Present address: Imperial Chemical (Pharmaceuticals)Ltd., Biological Laboratories, Morley, Wilmslow, Man-chester.

(1932) under the name Glaucoma piriformis. The organismwas cultivated in a sterile medium (pH 7 3) containing 1%(w/v) 'Oxoid' brand bacteriological peptone and 0.4%(w/v) NaCl. Cultures were maintained in tubes at roomtemperature, sub-inoculations being made every 2 weeks.For experimental studies, cultures were grown at 30°, inflasks containing 2-4 1. medium, with an air space of 1 1. Theorganismswere harvested after 6 days' growth, using a smallangle centrifuge; it was found necessary to use a centrifugewhich came to rest quickly, and to remove the supernatantimmediately by suction. With most centrifuges tried,although the ciliates were sedimented readily, by the timethe centrifuge had stopped the organisms had swum backinto the supernatant. The centrifuged cells were washedand suspended in either 0-45% (w/v) NaCl, a Ringer-phosphate solution containing 0{047M-NaCl, 0-002M-KCI,0-001m-MgSO4 and 0-012M-phosphate buffer (pH 7-3), ora Ringer-bicarbonate solution containing 0 05M-NaCl,0 003M-KCI, 0001 M-MgSO4 and 0016M-NaHCO3, whichwhen used in conjunction with a gas mixture of 5% C02-95% N. (v/v), had a pH of 7-3. Homogenates were made bywashing the organisms in water, and then shaking a thickaqueous suspension with glass beads in the Mickle disin-tegrator for 5 min. Cell-free preparations were obtained bycentrifuging this homogenate for 15 min. at 11 000 rev./min.in the Servall angle centrifuge. The supernatant was a clear,light-brown fluid, free from microscopically visible particles.

Materials. Glucose oxidase and catalase were kindlysupplied by Dr E. F. Hartree. The 'glycogen used was apurified specimen prepared from Tetrahymena pyriformis(GL), described by Manners & Ryley (1952). Adenosinetri-phosphate (ATP) was prepared from the barium salt, kindlysupplied by Dr E. C. Slater. Potassium glucose-l-phosphateand calcium fructose-1:6-diphosphate were gifts fromDr T. Mann.

Spectroscopic observations. These were made by means ofa microscope fitted with a Zeiss microspectroscope ocular.

Purpurogallin number (P.Z.). This was determined by themethod of Sumner & Gjessing (1943), slightly modified.A mixture of 2 ml. 5% (w/v) pyrogallol solution, 2 ml.0 5m-phosphate buffer (pH 6 0), 1 ml. 1% (v/v) H202 and15 ml. water were warmed to 200 in a beaker. An aqueoussuspension (1 ml.) of the ciliate, which had been twicefrozen and thawed to disrupt the cells, was added, and themix:ture incubated at 200 for 5 min.; the reaction wasstoppedbytheadditionofl ml.2 N-H2SO4. As much proteinwas present, and interfered with the extraction of purpuro-gallin, 1 ml. 100% (w/v) trichloroacetic acid was added, andthe mixture centrifuged. The supernatant was extractedtwice with ether, and the extract diluted to 25 ml, withether. Purpurogallin was estimated photoelectrically in the

31-2

483

J. F. RYLEYextract. The P.Z. is defined as mg. purpurogallin formed permg. material under the standard conditions used.

Metabolic experiments. These were carried out in Warburgmanometers, using cups (vol. 20-25 ml.) fitted with two sidearms. Gas phases of air or 5% C02-95% N2 (v/v) were used;the experiments were mostly carried out at 37°. The effectof CO on respiration was studied in Barcroft differentialmanometers at 190 and at 300. Metabolic rates were basedon the N content of the suspensions, being expressed as q.2and qN9,, i.e. as p1. gas/mg. N/hr. It was found that 1 mg.ciliate N was equivalent to 8-9 mg. dry weight, or about80 mg. wet weight of cells.

Estimation of metabolite8. Glycogen was estimated by themethod of Good, Kramer & Somogyi (1933). Reducingsugar was estimated iodometrically by the method ofSomogyi (1945), in Ba(OH)2-ZnSO4 filtrates; glucose wasdetermined from the loss in reducing value of such a filtrateafter treatment with glucose oxidase and catalase (Mann,1946), while fructose was estimated colorimetrically (Roe,1934). Fructose estimations were also carried out in tri-chloroacetic acid filtrates to determine the total of free andphosphorylated sugar; values for phosphofructose werecalculated from the difference in fructose content of thesetwo filtrates. Phosphate fractions were estimated by themethod of Fiske & Subbarow (1925); total phosphorus wasestimatedafter digestion with 60% HC104anda littleHNO3.Using trichloroacetic acid filtrates, pyruvic acid wasestimated by the colorimetric method of Friedemann &Haugen (1943), and lactic acid by the method of Barker &Summerson (1941). Acetic acid was estimated by distilla-tion of a tungstic acid filtrate (Buchanan, Sakami, Gurin &Wilson, 1947), while ethanol was determined by thebichromate method of Nicloux, le Breton & Doutcheff(1934). Succinic acid was estimated manometrically bymeans of a washed horse-heart preparation of succinicoxidase.

RESULTS

Aerobic metabolism

The free-living protozoan P. pyriformis (GL), although itoccurs naturally at a much lower temperature, is able tomaintain constant motility and metabolism at 370 for aperiod ofseveral hours at least. It was thought preferable towork at this higher temperature, rather than 220 or similartemperatures utilized by previous workers, as this reducedthe duration of the experiments. The organisms werecultivated at 300, which was found to be the optimumtemperature for growth; at 370, although the ciliatesflourished, growth was not quite as good as at 300.

Respiratory activity. In a non-nutrient medium at 370, theciliate respired with a qO2 of 480 (mean of seven values,ranging from 380 to 565). From Fig. l it can be seen that therespiration continued at an almost linear rate for 60 min.;other experiments have shown no marked decrease inrespiration after 5 hr. in a similar system. Fig. 1 also showsthat respiration proceeds at an almost linear, but reducedrate (qO2 =225) at 250. Although the ciliate maintains a highendogenous respiration in the absence of extracellularnutrients, several added compounds were able to increasethe 02 uptake still further. Thus, as shown in Table 1,acetate, propionate, butyrate, and to a lesser extent pyru.vate, increased the respiration, while glucose, ethanol, anda number of acids associated with the tricarboxylic acid

300 -

00~~~~~~~~~~~~~~~~~~~~~0

200 _ .., --

100 -

0 20 40 60Time (min.)

Fig. 1. Aerobic and anaerobic metabolism of T. pyriformis(GL). Each manometer cup contained 1 14 mg. ciliateN in a volume of 1-5 ml.; gas phase air or 5% CO-95%N2; 0-0,0* uptake (pl.) at 370; *--0 02 uptake(PI.) at 250; 0-0, anaerobic C02 production (p1.) at370 in bicarbonate buffer; 0-----0, anaerobic CO2production (1u.) at 250.

Table 1. Effect of extracellular substrateson re8piration

(Respiration of ciliates in Ringer-phosphate solution(pH 7-3) followed for 30 min.; 0-01M substrate then addedfrom side bulb, and respiration followed for a further60 min. Table gives 02 uptakes observed during this latterperiod. Total vol. 1-5 ml.; temp. 370; gas phase air.)

02 uptake StimulationSubstrate added (p1./hr.) (%)

Exp. 1 (0-5 mg. ciliate N)None 206 0Glucose 226 10Ethanol 213 3Sodium citrate 223 8Sodium succinate 219 6Sodium formate 218 6Sodium acetate 385 87Sodium propionate 400 94Sodium butyrate 406 97

Exp. 2 (0 57 mg.NoneSodium succinateSodium malateSodium fumarateSodium malonateLithium lactateSodium pyruvatePyruvate + malonatePyruvate + malonate + fumarateSodium acetate

ciliate N)265300309275276308368385385454

01316441639454571

484 I952

METABOLISM OF TETRAHYMENA PYRIFORMIScycle had little effect. The dicarboxylic acids had even lesseffect on the respiration when tested atpH 6-0, or ifadded toorganisms which had been starved prior to use by keeping inRinger-phosphate solution for 20 hr. at room temperature.With one particular ciliate preparation, which during aperiod of 60 min. consumed 23 /emoles 02, it was found that0-01 M-glucose increased the 02 uptake by 1-96 tzmoles or8%, while 3-87 1moles glucose had disappeared. The oxida-tion of 3-87,umoles glucose would require a much greaterquantityof O2than the extrauptake of 1-96 ,u moles observed.This may indicate a sparing of the unknown substrateresponsible for endogenous respiration, or may indicate anassimilation of the sugar.

18-5 ,umoles glucose; had the oxidation been incomplete, theglycogen utilization would have been even greater.

Effect of inhibitors on respiration and motility. Thesensitivity of endogenous respiration to a number of in-hibitors was studied; the results are summarized in Table 2.The presence of cyanide (0-46 x 10-3M), using the balancedKOH-KCN mixtures in the centre well as recommended byRobbie (1946), reduced respiration by only 18%; thisinhibition was maintained over a period of5 hr. On anotheroccasion, in the presence of glucose, the same concentrationof cyanide gave a 34% inhibition of 02 uptake. CO(CO/02 = 19), had no inhibitory effect on respiration in thedark, at either 19 or 300, but rather gave rise to a small

Table 2. Effect of inhibitors on respiration

(Each manometer cup contained about 0-6 mg. ciliate N in a total vol. of 1-5 ml.; gas phase air; temp. 37°. Inhibitoradded from side bulb, and 02 uptake over a period of 1 hr. compared with that of control.)

InhibitorNonePotassium cyanideCarbon monoxide (300)Sodium azideSodium azideSodium azide2:4-Dinitrophenol2:4-Dinitrophenol2:4-Dinitrophenol2:4-DinitrophenolHydroxylamineSodium iodoacetateSodium arseniteSodium fluoridePhlorrhizin2:2'-Dipyridyl4:4'-Dipyridyl8-HydroxyquinolinePhenylthiourea

Concn.(M)

0-00046(CO/02 = 19)

0-010-0010-00010-0010-00020-00010 000020-0020-00010-010020-010 00030-00030-00030-0003

Nature of endogenous respiration. A determination of therespiratory quotient was made, using the direct method ofWarburg on organisms in a Ringer-phosphate medium;corrections were applied both for the initial and the finalbound C02 in the medium. This gave an R.Q. value of 0-85,which suggests that endogenous respiration is probably notdependent on carbohydrate metabolism. A similar con-clusion can be derived from experiments in which intra-cellular glycogen was estimated before and after a period ofactive respiration. Six manometers were set up, each con-taining 1 mg. cellular N in Ringer-phosphate solution.After manometric equilibration, 2N-H2SO4 was tipped fromthe side bulb into three of the flasks, while the ciliates in theother three flasks were allowed to respire for a period of150 min. Respiration was stopped by the addition ofH2SO4, and glycogen was estimated in the contents of eachof the flasks. The initial glycogen present was equivalent to45-2,umoles glucose. During the incubation, a total of111 ,umoles 02 was consumed by the ciliates in the threeflasks, while the decrease in glycogen content amounted toonly 1-79 ,umoles glucose. Had the 02 uptake observed beendue to the complete oxidation of carbohydrate, there wouldhave been a glycogen disappearance corresponding to

Respiration(as % that of control)

pH 7-3 pH 6-0 pH 5-5100 100 10082105

70693810088969583100

171431192865144143

40272157

stimulation. Azide, another potent inhibitor of the cyto-chrome oxidase system, was used at pH 6-0 and 5.5, butacted as an inhibitor only at high concentrations. Indeed, atconcentrations of 0-001M and less, azide caused a pro-nounced stimulation of the 02 uptake. Motility studies,however, showed that azide inhibited motility to a smallextent at a concentration of 2 x 10-5M, and that higherconcentrations had a larger effect; the influence of azide onmotility increased with the time of incubation. 2:4-Dini-trophenol had similar effects to azide on respiration; con-centrations greater than 0-0002M inhibited respiration atpH 6-0, while concentrations less than 0-0001 M stimulatedrespiration. In all cases, there was an adverse effect onmotility; in the presence of 0-001 M-dinitrophenol, motilitywas abolished within 30 min. at room temperature, while in0-0002M-dinitrophenol, motility was gradually reduced,until the organisms were completely immotile after 230 min.The controls to these experiments showed that respiration atpH 6-0 was as good as that observed at pH 7-3, but that atpH 5-5, 02 uptake was reduced by 30%.

Respiration and motility were insensitive to high con-centrations of fluoride, while arsenite had little effect at aconcentration less than 0-01 M; this concentration of

VoI. 52 485

J. F. RYLEYarsenite was sufficient to render the ciliates almost stationaryafter 100 min. at room temperature, while a concentrationof0-003M had a smaller, but noticeable, effect after this time.Iodoacetate at a concentration of 3 X 10-5M had no effect onrespiration and motility, but 0 0001 M-iodoacetate reducedboth considerably over a period of 60 min.; 0-0003M-iodoacetate began to produce inhibitory effects after 15 min.incubation, and within 1 hr. the organisms were completelyimmotile. Phlorrhizin and the metal-binding substancestested were without noteworthy effect, save that 8-hydroxy-quinoline caused a slight reduction in motility. In all cases,loss of motility was an irreversible process, being accom-panied by damage to the cells.

Haem compoundsCytochrome system. Direct spectroscopic examination of

a suspension of T. pyriformis (GL) shows the presence of anumber of haem pigments. Under conditions of good aera-tion, the most prominent feature of the spectrum is thea-band of oxyhaemoglobin (Keilin & Ryley, 1951). Whenreduced with a small amount ofsodium dithionite (Na2S204),a cell suspension examined at room temperature showed:(a) A narrow, weak cytochrome c band at 550 m,u. (b) Astrong cytochrome e band at 551-553 m1A. (c) A weak cyto-chrome b band at 563 m,u. (d) An exceedingly weak band at605 m,u., probably due to cytochrome a. (e) Double ,-bandsat 521 and 530 m,. (f) A very weak and diffuse band at580-590 mit., possibly due to cytochrome a,, or to incom-pletely reduced haemoglobin. This band moved to 568-578 m1A., as a weak and very diffuse band, in the presence ofCO; the other bands were not affected by CO. (q) A veryfaint band at 635 mdi., probably due to methaemoglobin,derived by oxidation of some of the haemoglobin.Manometric experiments showed that a homogenate of

the ciliate was unable to catalyse the oxidation ofp-pheny-lenediamine, either in the presence or in the absence ofaddedcytochrome c. In connexion with this lack of cytochromeoxidase activity, the results of Table 2 concerning theeffects of azide, cyanide and CO on respiration, should benoted.

Peroxidase. Lawrie (1935) reported that T. pyriformis(GL) contained a peroxidase. The peroxidase activity of theprotozoan has been confirmed, and the purpurogallinnumber determined. Saturated ethanolic benzidine (1 ml.)was mixed with 1 ml. water, 6 drops glacial acetic acid, and1 ml. of a suspension ofthe ciliate. A few drops ofH202 wereadded; the mixture rapidly became blue in colour. Micro-scopic examination immediately after adding H202 showedthat the cells became uniformly blue throughout, but thatthis coloursoon diffused out ofthe cell. (Similarexperimentswith Paramecium caudatum showed a concentration of theblue colour in and around unidentified granules in the cell.)Boiled organisms gave only a slight production of colour,indicating that the benzidine reaction was mainly due toa thermolabile enzyme, rather than to the intracellularhaematin as such. Similar colour-producing reactions wereobtained with other phenolic compounds, such as pyrogallol,p-phenylenediamine, guaiacum or pyrocatechol, whentested with H202 in 0- M-phosphate buffer (pH 7-3). Ineach case, colour production was dependent on the additionto the system of H202 and cell suspension. During theseexperiments, a considerable production ofgas was observed,due to decomposition ofthe peroxide by catalase. Using themodified method of Sumner & Gjessing (1943), it was found

that 14-4 mg. dry wt. of cells formed 0-475 mg. purpuro-gallin, giving a P.Z. of 0-033.

Cell homogenates were able to oxidize reduced cytochromec peroxidatically. A small amount of dilute cytochrome csolution was reduced by adding a trace of p-phenylene-diamine. Cell homogenate was then added, and the mixtureexamined spectroscopically. The intense band of cyto-chrome c at 550 m,u. did not fade in spite of aeration for atleast 1 hr., indicating that no active cytochrome oxidase waspresent. When H202 was added to the system, the cyto-chrome c band quicklydisappeared, reappearingwhen all theperoxide had been utilized; further addition of H202 causedthe band to disappear once more.

Anaerobic metabolismUnder anaerobic conditions, Tetrahymenapyriformi (GL)

is ableto survive and maintain motility forprolonged periodsof time. A suspension of the ciliate in Ringer-bicarbonatesolution was placed in a Thunberg tube, which was evacu-ated and filled four times with an O2-free CO2/N2 mixture.Normal motility was maintained by a large proportion ofthe cells (examined microscopically with a i in. objectivewhile in the tube) for at least 72 hr., although a number ofthe organisms died during this time; a similar picture wasobtained with a control suspension exposed to the air.Initially, all the cells stained a dark-brown colour withdilute iodine, but many of those which had been kept underanaerobic conditions for 72 hr. gave very little colorationwith iodine; the cells kept for the same period of time underaerobic conditions still gave a good iodine stain. When sus-pended in a Ringer-bicarbonate medium under anaerobicconditions, the ciliate produced acid (Fig. 1). From sixexperiments carried out at 370, a mean qco, of 180 wasobserved (range 123-238), while at 25°, a qco value of 70was obtained. This liberation of CO2 was increased by 33%in the presence of 0-01 M-glucose, 35% of the added glucosebeing utilized.

Nature of anaerobic endogenous metabolism. Underanaerobic conditions the ciliate fermented its intracellularglycogen reserves and maintained a high degree of motility.Six manometers were set up, each with 1 mg. ciliate N in1-5 ml. Ringer-bicarbonate solution, and a C02-N2 gasphase. After equilibration, acid was tipped into three of theflasks in order to estimate the initial bicarbonate and after110 min. acid was tipped into the other flasks to estimateresidual bicarbonate. Estimations showed that the glycogenreserves had been depleted by 32% during the fermentation,while 55-0 ,equiv. acid had been produced, and 7-05 /&molesCO2 had been assimilated, i.e. for each glucose unit of glyco-gen metabolized, 2-75 equiv. of acid were formed, with theassimilation of 0-35 molecules of CO2.A similar experiment was carried out on a somewhat

larger scale, the flask contents being analysed for glycogen,succinic, lactic, pyruvic and acetic acids and ethanol. FromTable 3 it can be seen that the glycogen was mainly con-verted to succinic acid, with smaller amounts of lactic andacetic acid. The metabolites estimated accounted for 91%ofthe carbon supplied, and for 97-5% ofthe CO2liberated byacid formation.

Effect of inhibitors on anaerobic metabolism. From Table 4it can be seen that ofthe seven compounds tested as possibleinhibitors of the fermentation, only iodoacetate had amarked effect. In the presence of 0-001 M-iodoacetate, CO2evolution was reduced by 75% over the period of 60 min.

486 I952

METABOLISM OF TETRAHYMENA PYRIFORMISstudied; the metabolism stopped within 20 min. of addingthe inhibitor. In contrast to the anaerobic metabolism ofglucose by Trypano8oma lewi8i (Ryley, 1951), which ishighly sensitive to the metal-binding substances 2:2'-dipyridyl and 8-hydroxyquinoline, these same compoundswerefoundto stimulatethe rate ofan aerobic acidproductionby Tetrahymena pyriformis (GL).

Table 3. Anaerobic glycogen fermentation byTetrahymena pyriformis (GL)

(Ciliate N 8-8 mg. was incubated for 120 min. in a totalvol. of 18 ml. Ringer-bicarbonate solution, using sixmanometers; gas phase 5% CO-95% N.; temp. 37°.Reaction stopped, and residual bicarbonate estimated byadding 0-4 ml. 2N-H2SO4 to each flask. Contents of flasksneutralized, pooled, and deproteinized for estimation ofmetabolites. Yields expressed in terms of moles metabolitefound per glucose unit of glycogen disappearing, fromwhich values for carbon and acid recoveries have beencalculated. During the fermentation, a total of 71-1 ,emolesglucose (as glycogen) were used, and 28-9 ,Amoles CO2 werefixed.)

MetaboliteSuccinic acidLactic acidPyruvic acidAcetic acidEthanolSumC g. atoms supplied (asglycogen and C02)Acid yield (frombicarbonate destroyed)

Moles0-940-470-000-320-01

Uarbon(g. atoms)

3-761-410-000-640-025-836-41

ACld(g. equiv.)

1-880-470-000-320-002-67

Intermediary carbohydrate metaboli8m

Amyla8e and malta8e. Cell-free preparations of T. pyri-formi8 (GL) were incubated with starch, glycogen or maltoseat pH 7-3 in the presence of NaCl, and the reaction followedby estimating reducing sugar and glucose at suitable timeintervals. From Table 5, it can be seen that starch andglycogen were hydrolysed at approximately the same rate toreducing sugar, 80% of which was found to be glucose.During the 90 min. incubation, 23-4 mg. maltose werebroken down, and 24-8 mg. glucose were recovered. At thebeginning of the experiment, the samples already containeda small amount ofreducing sugar, due no doubt to hydrolysisofprotozoal glycogen in the course ofpreparation of the cell-free extract.

Table 6. Hexokina8e activity ofcell-free Tetrahymenapyriformis (GL) preparationm

(Each manometer cup contained 0-7 mg. N in the form ofcell-free preparation, 0-026m-NaHCO,, 0-005M-MgSO4,0-06M-NaF, 0-05M-KCI, 0-0045M-glucose and 0-0031 M-ATP in a total volume of 2-5 ml. Mixture incubated40 min. at 370 with gas phase of 5% C02-95% N2.)

InitialFinal

Inorganic P[PO]

(Q.moles)1-70

11-81

Acid-labile P[P7-PO](,umoles)15-224-01

Reducingsugar

(,umoles)14-114-5

P esterified= 1-lOmoles. CO2 liberated by acid pro-2-74 duction = 145 pl. (6-48 ,umoles).

Table 4. Effect of inhibitor8 on anaerobic metaboliwm

(Each manometer cup contained about 0-9 mg. ciliate Nin a total vol. of 1-5 ml.; gas phase 5% C0g-95% N2;temp. 37°. Inhibitor added from side bulb, and CO.evolution over a period of 1 hr. compared with that incontrol.)

InhibitorSodium fluorideSodium arseniteSodium iodoacetate2:2'-Dipyridyl4:4'-Dipyridyl8-HydroxyquinolinePhenylthiourea

Conen.(M)

0-020-010-0010-00030-00030-00030-0003

UV2 evomlon(as % thatof control)

866625

127100121100

Hexokina8e. An attempt was made to demonstratehexokinase activity in cell-free preparations. Warburgmanometers were set up as indicated in Table 6, the reactionbeingfollowed bythe liberation ofCO2due toacidformation.During the experiment, 145 pl. (6-48 ,umoles) CO2 wereevolved; there was a large breakdown of ATP to inorganicphosphate, but about 10% was esterified. This was observedon two occasions. It was not possible to follow the hexo-kinase reaction by the disappearance ofglucose since, duringthe experiment, there was a very slight increase in glucose,due no doubt to hydrolysis of polysaccharide present in theenzyme preparation. An identical system containing asimilar homogenate of Trypano8oma leWi8i showed markedhexokinase activity: 60% ofthe added glucose was utilized,and ATP was metabolized, equal amounts of inorganic andesterified P accumulating.

Table 5. Amylase and maltase activity in cell-free Tetrahymena pyriformis (GL) preparationw

(Each tube contained 3-9 mg. N in the form of cell-free preparation, 0-027M-acetate-veronal buffer (pH 7-3), 0-017M-NaCl, and starch, glycogen and maltose respectively, in a total volume of 9 ml. Tubes incubated at 37°, 2 ml. samplesbeing removed at intervals. Reducing sugar expressed in terms of mg. hexose. All figures given in mg.)

Incubation time(min.)

0

3090

I Substrate addedReducing sugar found

I Glucose foundReducing sugar foundReducing sugar found

l Glucose found

Starch362-021-746-8515-4712-35

Glycogen362-031-866-4116-7513-26

Maltose60-235-64-4

40-048-129-2

VoI. 52 487

J. F. RYLEYPho8phoryksme. Although Tetrahymena pyriformis (GL)

contains hydrolytic enzymes capable of degrading glycogenand starch, in a system containing protozoal glycogen andphosphate buffer cell-free preparations of the ciliate causeda breakdown of glycogen, accompanied by an uptake ofinorganic phosphate. During the first 15 min. ofincubation,over 50% of the inorganic phosphate disappearing wasconverted to an easily hydrolysable P compound (Fig. 2).On further incubation this fraction decreased, while the lesseasily hydrolysable phosphate fraction increased. Fructoseestimations in trichloroacetic acid extracts and in Ba(OH)2-ZnSO4 extracts showed that the process was accompanied by

25 r

- 15

E0

3 100.

5

incubating Cori ester with a cell-free preparation of theciliate. From Table 8 it can be seen that the easily hydro-lysable phosphate fraction was reduced on incubation, 50%of the phosphate being recovered as inorganic phosphate,the other 50% being converted into less easily hydrolysablephosphate, part of it in combination with fructose, pre-sumably as fructose-6-phosphate.

Table 7. Pho8phorylase activity of cell-free Tetra-hymena pyriformis (GL) preparation

(Each tube contained 0-5 mg./ml. N in the form of cell-free preparation, 4 mg./ml. starch or glycogen and 0-025M-phosphate buffer (pH 7-3) in a total volume of 9 ml.Samples (2 ml.) deproteinized before and after 2 hr. incu-bation at 370, with Ba(OH)2-ZnSO4 or with 5% trichloro-acetic acid (TCA). Reducing sugar, glucose and fructoseestimated in Ba-Zn filtrates, and fructose, P0 and P7 inTCA filtrates. Results expressed as ,umoles/ml. of enzymesystem.)

Inorganic P esterifiedGlucose-i-phosphate formedGlucose-6-phosphate formedFructose-6-phosphate formedReducing sugar liberatedFree fructose found

0 30 60 90 120Time (min.)

Fig. 2. Phosphorylase activity of cell-free T. pyriformis(GL) preparation. Each tube contained 0-85 mg./ml. Nin the form of cell-free preparation, 4 mg./ml. Tetra-hymena glycogen and 0-025M-phosphate buffer (pH 7-3)in a total volume of 6 ml.; temp. 370; *-*, inorganicphosphate; 0-0, [Pt-P7] phosphate fraction; A-A,[P7-PO] phosphate fraction.

the formation of fructose phosphate. After 120 min. incu-bation, there had been an esterification of 7-6 tg. atoms/ml.P, and an accumulation of 2-71iemoles/ml. fructose phos-phate. In a similar experiment (Table 7), it was found thatcell-free preparations of the ciliate were able to phosphoro-lyse starch as well as glycogen.

Phosphoglucomutase and oXoisomerase. The ability of T.pyriformis (GL) to transform glucose-l-phosphate into amixture of hexose-6-phosphates has been shown directly by

Glycogen5-361-291-852-228-100-16

Starch3-550-421-811-428-240-22

Table 8. Pho8phoglucomuta8e and oxoisomeraweactivity in cell-free Tetrahymena pyriformis (GL)preparation8

(Each tube contained 1 mg. N in the form of cell-freepreparation, 0-002M-MgCl2, 0-023m-acetate-veronal buffer(pH 7-3) and 3 mg. potassium glucose-l-phosphate in atotal volume of 4-5 ml. Incubation carried out at 370, andsystems deproteinized with 1-5 ml. 10% (w/v) TCA.)

Time [P0](min.) (pg. atoms)

0 1-4510 4-9160 5-39

[P7-PO](pg. atoms)

8-643-260-74

[P180-P7](ug. atoms)

0-100-771-87

[Pt-P180](jug. atoms)

0-291-552-48

Glucose-I-phosphate used = 7-90 ,umoles. Inorganic phos-phate liberated=3-94,emoles. Less easily hydrolysablephosphate ester formed = 3-96 ,umoles. Fructose-6-phos-phate found (as fructose)=2= 77,umoles.

Enzymic degradation offructose diphosphate. Table 9 showsthat a cell-free enzyme preparation from T. pyriformis (GL),incubated anaerobically with potassium fructose-1:6-diphosphate (FDP), gave rise to triosephosphate (alkali-labile phosphate); the conversion was greater in the presenceof cyanide, which acts as a keto fixative. Using a similar

Table 9. Aldolase activity in cell-free Tetrahymena pyriformis (GL) preparations

(Each manometer cup contained 0-4 mg. N in the form of cell-free preparation, 0-025M-NaHCO3, 0-06M-NaF and 0-01 M-fructose-1:6-diphosphate (FDP) in a total volume of 2-5 ml.; gas phase 5% C2-95% N2; temp. 37°. Mixtures incubatedfor 1 hr., and deproteinized with 1-5 ml. 10% TCA.)

SystemEnzyme +FDPEnzyme +FDP +0-06M-KCN

Alkali-labile Pformed(1&moles)

8-9521-10

Inorganic PFDP used liberated(/Lmoles) (1umoles)

6-35 1-313-20 1-0

488 I952

I I I

201

METABOLISM OF TETRAHYMENA PYRIFORMISsystem, it was found that 0.001 M-2:2'dipyridyl had no effect metrically. Even in the absence of FDP, appreciableon aldolase activity atpH 7-3. However, atpH 6-0, aldolase amounts of acid were formed, owing to the fermentation ofactivity was inhibited by 5% in the presence of 0-001 M- some substrate present in the enzyme preparation itself;2:2'-dipyridyl, and by 34% in the presence of 0-005M- however, when FDP was added, CO2 evolution was doubled.dipyridyl.

150

100

0)

050

0 30 60 90Time (min.)

Fig. 3. Fructose diphosphate metabolism by cell-free T.pyriformi8 (GL) preparation. Each manometer cup con-tained 0-73 mg. N in the form of cell-free preparation,0-025M-NaHCO3,0-008M-fructose-1:6-diphosphate(FDP),0-008M-pyruvate, 0-OOOlM-cozymase, 0-0018M-phos-phate and 0-06M-NaF in a total vol. of 2-5 ml.; gas phase5% CO0-95% N2; temp. 370; *-*, C02 evolution (/l.)in presence of FDP; 0-0, CO2 evolution (pI.) inpresence of FDP and 0-004M-arsenate; A-A, CO2evolution (pd.) in absence of FDP.

Speck & Evans (1945), using malaria parasite prepara-tions, attempted to demonstrate the oxidation of triose-phosphate, coupled with the simultaneous reduction ofpyruvate, fluoride being added to the system in order toprevent the breakdown of phosphoglyceric acid. A similarsystem has been used in the present case of T. pyriformi8(GL). Fig. 3 illustrates an experiment in which a cell-freepreparation ofthe ciliate was allowed to act uponFDP underanaerobic conditions, acid production being followed mano-

4)

0~e

0 30 60Time (min.)

90 120

Fig. 4. Lactic oxidase activity of cell-free T. pyriformi8(GL) preparation. Eachmanometer cup contained 1-0mg.N in the form of cell-free preparation, and 0-04M-phos-phate buffer (pH 7-3), in a total volume of 2-5 ml.; temp.370; gas phase air; *-*, 02 uptake (ul.) in presence of0-03M-lactate; 0-0, 03 uptake (pl.) in presence of0-03M-lactate and 0-00005M-methylene blue; 0-0-,--,03 uptake (1l.) in presence of 0-00005m-methylene blue.

In the presence ofFDP, fermentation proceeded at the samerate in the presence as in the absence of added pyruvate orcozymase. Chemical analysis showed that at the end of thisexperiment about 40% ofthe added FDP had disappeared;triosephosphate formation accounted for 60% of the FDPdisappearing, while the remainder was converted to lacticacid. The production oflactic acid was not influenced by theaddition of extra pyruvate although, whenpresent, pyruvatewas utilized. Arsenate (0-004M) doubled the rate of acidproduction in the presence of FDP, and there was a corre-sponding increase in FDP utilization. Similar experiments

Table 10. Dehydrogenase activity in Tetrahymena pyriformis (GL)

(Thunberg tubes contained homogenate or cell-free preparation and 0-04M-phosphate buffer (pH 7-3), with 0-134,umolemethylene blue and substrate as indicated added from hollow stopper. Tubes evacuated and washed with pure N2 fourtimes; temp. 370; total volume, 2-5 ml. Figures give time (min.) for 90% decolorization.)

Additions

PreparationHomogenate (2 mg. N)Homogenate (3-9 mg. N)Cell-free preparation (1-8 mg. N)Cell-free preparation (1-1 mg. N)

Lithiumlactate

None (0-03M)45 3333 2780 37

150 65

Sodiumsuccinate(0-03M)

118-52537

Glucose(0-03M)

3723

FDP(0-008M)

61

VoI. 52 489

J. F. RYLEY

showed that the endogenous fermentation of the prepara-tion was insensitive to iodoacetate, but that acid formationin the presence of FDP, with or without arsenate, wasreduced to the endogenous level by the addition of 0-001 M-iodoacetate.As already stated, during glycogen fermentation by intact

cells of T. pyriformia (GL), lactic and succinic acids accumu-late. It seems probable that the production of these acidsis a means of reoxidizing dihydrocozymase, which is formedfrom cozymase during the oxidation of phosphoglyceralde-hyde. Table 10 gives the results obtained from a number ofThunberg tube experiments, using both whole and centri-fuged homogenates of the ciliate. Succinate accelerated thereduction of methylene blue fourfold, while lactate, glucoseand fructose diphosphate were less effective. The centri-fuged preparation alone did not respire, but in the presenceof 0-03 M-lactate, there was a vigorous 02 uptake (qo2 = 125;Fig. 4). On the other hand, succinate, which was shown inThunberg tube experiments to accelerate methylene bluereduction, did not increase the 02 uptake of homogenateswhen tested manometricaily, either in the presence or in theabsence of added cytochrome c or methylene blue.

DISCUSSION

The ciliate T. pyriformi.8 (GL) is able to maintainactive motility and respiration in the absence ofextracellular substrates over a wide range oftemperature, in contrast to the trypanosomes,flagellates which have become adapted to life in aspecific and constant environment on which theyrely for their source of energy. T. pyriformi8 (GL)respires with a qo2 of 480 at 370, and 225 at 250. Thenature ofthe intracellular substrates responsible forthis endogenous respiration is an interesting matterfor speculation. The present study indicates thatrespiration is relatively independent of the intra-cellular glycogen reserve. A respiratory quotient of0-85 suggests that the ciliates may be oxidizing fator protein, rather than carbohydrate. Pace &Ireland (1945) demonstrated the presence of fatdroplets in the ciliate by staining techniques, whileWilber & Seaman (1948) showed that the organismscontain lipid, and a small amount of phospholipin.However, Pace & Lyman (1947) were unable bymicroscopical methods to observe any utilization offat during the metabolism of T. pyriformi8 (W); itwould be interesting to confirm this, carrying outchemical estimations of cell lipid before and after aperiod of active respiration.Although T. pyriformi8 (GL) respires well in the

absence of extracellular substrates, a number ofcompounds are able to increase the oxygen uptake.Chaix et al. (1947) found that the endogenous respira-tion of this strain was stimulated by short-chainfatty acids, but was little affected by citrate, succin-ate or malate, substances associated with the tri-carboxylic acid cycle. The present study confirmsthese observations, except that the respiratorystimulations produced by fatty acids were larger;

this may well be due to the higher temperature usedin the present work. In contrast to these findings,Seaman (1949) reported that the endogenousrespiration of the S strain of T. pyriformis wasdoubled in the presence of 0-02M-succinate, oc-keto-glutarate, fumarate, malate or oxaloacetate, al-though citrate was without effect, and that theincrease in respiration produced by pyruvate wasreversed by malonate, this malonate inhibitionbeing antagonized by fumarate. On the basis ofthese observations, Seaman (1949) suggested thatrespiration in the ciliate involved the tricarboxylicacid cycle. With the GL strain of T. pyriformi8 usedin the present study it has not been possible to ob-tain such effects. Moreover, unlike Seaman (1950,1951), we could also find no evidence for the oxida-tion ofsuccinate by cell homogenates prepared fromthe ciliate. No oxidation of succinate was observedwith cell homogenates or particle-free preparations,either in the presence or in the absence of addedcytochrome c; such preparations, however, readilyoxidized lactate.According to Seaman (1950), T. pyriformi8 (S)

also attacks added acetate, partly by oxidizing it,and partly by converting it to intracellular lipid andcarbohydrate. However, since his carbohydrateanalyses were carried out on filtrates prepared fromcell suspensions deproteinized with barium hydr-oxide and zinc sulphate, it is difficult to assess therelation between these results and the total carbo-hydrate content of the cells. It would appear fromSeaman's data, that the initial 'carbohydrate'content ofthe cells was ofthe order of 0- 1 %, a valuewhich contrasts with a content of 20% glycogendemonstrated by us in the case of T. pyriformi8 (GL)(Ryley, 1951; Manners & Ryley, 1952).In the present study, it was found that the respir-

ation of T. pyriformis (GL) was not affected by thepresence of carbon monoxide (CO/O2= 19) in the gasphase, and that cyanide at a concentration of0-46 x 10-3M inhibited endogenous respiration byonly 18%. Lwoff (1934) found that respirationof the GL strain in a peptone solution was cyanide-sensitive, but that in Ringer--glucose solution,0-0022M-cyanide stimulated respiration by 36 %.Using the H strain of T. pyriformi8, Hall (1941)observed that respiration was inhibited 88% by0-OO1M-cyanide, or 35% by 0-OOO1M-cyanide.Baker & Baumnberger (1941), using the T strain ofT. pyrtformis, observed that respiration wasblocked, and oxidation of the cytochrome bandsprevented, in the presence of an unspecified con-centration of cyanide. They also found that respira-tion was sensitive to carbon monoxide, but thiseffect was only observed at very high partialpressures of carbon monoxide (CO/02 = 45). Itwould seem that on the whole, while cyanideprobably has some effect on the respiration of T.

490 1952

METABOLISM OF TETRAHYMENA PYRIFORMISpyriformi8, the cyanide-sensitivity of the process isfar below that of other cells with typical cytochromeoxidase activity. These facts, and the absence ofanyp-phenylenediamine oxidase activity in cell homo-genates, are in agreement with the spectroscopicobservations reported. We observed very littleabsorption at 605 m,u., the location ofcytochrome a,usually involved in cyanide-sensitive respiration,but in contrast to this there was intense absorptionin the green part of the spectrum. This was con-sidered to be due to a strong cytochrome e band,with a weaker b band, and a weak c band. Theseresults are in good agreement with the spectroscopicobservations of Baker & Baumberger (1941) on theT strain, but they attributed their band at 552 mlu.to cytochrome c, rather than to cytochrome e. Theyfound absorption in the region 587-592 m,u., and insome cases at 616-617 m,., but they were not ableto observe the oc-band of oxyhaemoglobin, whichappears at about 582 m . under conditions of goodaeration.

It has been noted (Experimental section) thatglucose stimulated respiration to a small extent, andthat under aerobic conditions appreciable amountsof sugar were removed from the medium. Kidder &Dewey (1945) noted that most strains of T. pyri-formi8 examined were able to ferment glucose,fructose, mannose, maltose, starch, dextrin andglycogen, but no other carbohydrates tested. Nielet al. (1942) examined the glucose metabolism oftheT strain under anaerobic conditions, and showedqualitatively the production of succinic, lactic andacetic acids. They were able, by the use of radio-active carbon, to demonstrate the assimilation ofcarbon dioxide, and its incorporation in thecarboxyl groups of succinic acid. The high intra-cellular carbohydrate content of Tetrahymenaappears to have escaped the notice of all previousinvestigators, nor have they realized the significanceof the intracellular carbohydrate reserve for theanaerobic metabolism of the ciliate. The presentstudy shows that under anaerobic conditions, theciliate T. pyriformis (GL) maintains a normal degreeof motility at the expense of its vast glycogen store.At room temperature, under strictly anaerobic con-ditions, the ciliate maintained normal motility inthe absence of any extracellular nutrient for at least72 hr. Although it seems doubtful that the ciliatewill reproduce under anaerobic conditions, yet,contrary to the findings of Pace & Ireland (1945)with the W strain, the organism will survive. It isinteresting to compare these ciliates with the haemo-flagellate Trypanosoma lewi8i (Ryley, 1951); thelatter organism is able to survive in the presence ofextracellular substrate under anaerobic conditions,but motility is much less than that under aerobicconditions. With Tetrahymena pyriformis (GL),motility under anaerobic conditions is just as

vigorous as under aerobic conditions. There is astriking similarity between the anaerobic metabolicprocesses ofthe two organisms; in the trypanosome,extracellular glucose is fermented to a mixture ofsuccinic, lactic and acetic acids, with the simul-taneous assimilation of carbon dioxide; with theciliate, intracellular glycogen is fermented with theformation of exactly the same products.

In cell-free preparations of the ciliate, the pre-sence of phosphorylase, phosphoglucomutase andoxoisomerase has been demonstrated. The phos-phorylase of the ciliate acts upon starch as well asglycogen. Hydrolytic enzymes capable ofdegradingthese polysaccharides have also been demonstrated.Cell-free preparations of T. pyriformi8 (GL) readilyconvert fructose diphosphate to triosephosphate,by an enzyme system which does not seem to besensitive towards 2:2'-dipyridyl. In this respect, thealdolase of the ciliate resembles the animal, ratherthan the yeast type ofenzyme (Warburg& Christian,1943). The occurrence of phosphorylative mechan-isms in carbohydrate breakdown by T. pyriformis(GL) is also supported by the observations relatingto the formation of lactic acid from fructose di-phosphate by cell-free enzyme preparations, thestimulation of this process by arsenate, and itsinhibition by iodoacetate.

SUMMARY1. The free-living ciliate Tetrahymena pyriformiw

(Glaucoma piriformi8) maintains metabolism andmotility in the absence of extracellular nutrients forconsiderable periods oftime at room temperature orat 370.

2. Under aerobic conditions at 370, the ciliaterespires with a q°a of 480 and an R.Q. of 0 85. Thisendogenous respiration is practically independent ofthe intracellular glycogen, is only slightly stimu-lated by added glucose and a number ofdicarboxylicacids, but is almost doubled by 0 01M-acetate, pro.pionate or butyrate.

3. High concentrations of azide or 2:4-dinitro-phenol inhibit endogenous respiration, while lowconcentrations stimulate it; in all cases, motility inthe presence of these substances is reduced.

4. The cytochrome system of the ciliate is com-posed of cytochromes b, c and e, but very little a;homogenates display no cytochrome oxidaseactivity towards p-phenylenediamine. Respirationof the ciliate is comparatively resistant to cyanideand carbon monoxide.

5. T. pyriformis contains a heat-labile peroxidase(purpurogallin number, 0-033); in the presence ofhydrogen peroxide this peroxidase is able to oxidizea number of phenolic compounds, and reducedcytochrome c.

6. Anaerobic metabolism and motility depend onthe fermentation of the intracellular glycogen; in

VoI. 52 491

492 J. F. RYLEY 1952the absence of extraneous carbohydrate, the ciliateproduces large amounts of succinic acid, withsmaller quantities of lactic and acetic acid, andassimilates some carbon dioxide.

7. Cell-free, centrifuged homogenates containenzymes capable of hydrolysing starch, glycogenand maltose to glucose; they are also able to phos-phorylate glycogen and starch to form glucose-l-phosphate and contain phosphoglucomutase andoxoisomerase.

8. Cell-free preparations convert fructose-1:6-diphosphate to triosephosphate, which is fermented

to lactic acid. This process is stimulated by arsenate,and inhibited by iodoacetate.

9. Homogenates of the ciliate display bothsuccinic and lactic dehydrogenase activity; in thepresence of atmospheric oxygen, however, theyoxidize lactic, but not succinic, acid.

I should like to thank Prof. D. Keilin, F.R.S., Dr T. Mann,F.R.S., Dr P. Tate and Mr C. H. Chin for much helpfuladvice and discussion during the course ofthis work, and theMedical Research Council for the award of a ResearchStudentship.

REFERENCES

Baker, E. G. S. & Baumberger, J. P. (1941). J. cell. comp.Physiol. 17, 285.

Barker, S. B. & Summerson, W. H. (1941). J. biol. Chem.138, 535.

Buchanan, J. M., Sakami, W., Gurin, S. & Wilson, D. W.(1947). J. biol. Chem. 169, 403.

Chaix, P., Chauvet, J. & Fromageot, C. (1947). Antonie v.Leeuwenhoek, 12, 145.

Corliss, J. 0. (1952). Personal communication.Fiske, C. H. & Subbarow, Y. (1925). J. biol. Chem. 66, 375.Friedemann, T. E. & Haugen, G. E. (1943). J. biol. Chem.

147, 415.Good, C. A., Kramer, H. & Somogyi, M. (1933). J. biol.

Chem. 100, 485.Hall, R. H. (1941). Physiol. Zool. 14, 193.Keilin, D. & Ryley, J. F. (1951). Unpublished experiments.Kidder, G. W. & Dewey, V. C. (1945). Phy8iol. Zool. 18, 136.Lawrie, N. R. (1935). Biochem. J. 29, 2297.Lwoff, A. (1932). Recherches biochimiques sur la nutrition des

Protozoaires. Paris: Masson.Lwoff, M. (1934). C.R. Soc. Biol., Paris, 115, 237.Mann, T. (1946). Biochem. J. 40, 481.

Manners, D. J. & Ryley, J. F. (1952). Biochem. J. 52, 480.Nicloux, M., le Breton, E. & Doutcheff, A. (1934). Bull. Soc.

Chim. biol., Paris, 16, 1314.Niel, C. B. van, Thomas, J. O., Ruben, S. & Kamen, M. D.

(1942). Proc. nat. Acad. Sci., Wash., 28, 157.Pace, D. M. & Ireland, R. L. (1945). J. gen. Physiol. 28, 547.Pace, D. M. & Lyman, E. D. (1947). Biol. Bull. Woods Hole,

92, 210.Robbie, W. A. (1946). J. cell. comp. Physiol. 27, 181.Roe, J. H. (1934). J. biol. Chem. 107, 15.Ryley, J. F. (1951). Biochem. J. 49, 577.Seaman, G. R. (1949). Biol. Bull. Woods Hole, 96, 257.Seaman, G. R' (1950). J. biol. Chem. 186, 97.Seaman, G. R. (1951). J. gen. Physiol. 34, 775.Somogyi, M. (1945). J. biol. Chem. 160, 61.Speck, J. F. & Evans, E. A. jun. (1945). J. biol. Chem. 159,

71.Sumner, J. B. & Gjessing, E. C. (1943). Arch. Biochem. 2,

291.Warburg, 0. & Christian, W. (1943). Biochem. Z. 314, 149.Wilber, C. G. & Seaman, G. R. (1948). Biol. Bull. Woods

Hole, 94, 29.

Glyoxalase: the Role of the Components

BY E. M. CROOK AND K. LAWDepartment of Biochemi8try, University College, London

(Received 5 March 1952)

Since the discovery by Lohmann (1932) thatreduced glutathione (GSH) is the coenzyme ofglyoxalase, several workers have reported thatmethylglyoxal and GSH form a compound and havesuggested that this might be the true substrate forthe enzyme or an intermediate in the reaction. ThusJowett & Quastel (1932) observed that SH groupstitratable by iodine decreased when methylglyoxal

was added to GSH solution and suggested a reactionsequence:

CH3 CH3 CH3II

CO + (GSH) CO + H20 -+ CHOR + (GSH)II

CHO HC-OH COGH

(GS)