ENZYMATIC UTILIZATION OF GLUCOSE BY A BASIDIOMYCETE

H. P. MELOCHE, JR.1Northern Regional Research Laboratory,2 Peoria, Illinois

Received for publication October 23, 1961

ABSTRACT

MIELOCHE, H. P., JR. (Northern RegionalResearch Laboratory, Peoria, Ill.). Enzymaticutilization of glucose by a basidiomycete. J.Bacteriol. 83:766-774. 1962.-Cell-free extractsof acetone-dried Lactarius torminosus NRRL 2900were prepared. These extracts contained hexo-kinase. They also contained triphosphopyridinenucleotide (TPN)-specific glucose-6-phosphatedehydrogenase and catalyzed the reduction ofTPN in the presence of D-fructose-6-phosphate,6-phospho-D-gluconic acid (6PG), and D-ribose-5-phosphate (RSP). Aged preparations oxidizedD-glucose-6-phosphate (G6P) to 6PG, whereasfresh preparations oxidized G6P to a pentose withthe uptake of 1 ,umole of 02 and the evolution of1 ,umole of CO2 per ,umole of G6P. Evidence forthe action of transketolase in the metabolism ofR5P by cell-free extracts was obtained.

Cell-free preparations lacked hexosediphos-phate enzymes. Triosephosphate isomerase andF6P kinase could not be demonstrated; however,aldolase activity was present.Evidence is presented for the conversion of

D-glyceraldehyde-3-phosphate to pyruvate. Inaddition, phosphohexoisomerase was demon-strated.

It appears that a hexosemonophosphate path-way operates in L. torminosus extracts and maybe the major mechanism of glucose dissimilationin this organism.

Little is known about the intermediary carbo-hydrate metabolism of the more complex fungi.In studying the biosynthesis of a polyacetylenicantibiotic by basidiomycete B-841, Bu'Lock andGregory (1959) presented data that suggested theoperation of the Embden-Meyerhof and pentose-

1 Present address: Department of Biochem-istry, Michigan State University, East Lansing.

2 This is a laboratory of the Northern Utiliza-tion Research and Development Division, Agri-cultural Research Service, U. S. Department ofAgriculture.

phosphate systenms. In addition, evidence wasobtained for the decarboxylation of glucuronicacid to xylose and for the oxidation of ethanol.McKinsey (1959) showed that a wild type of

Ustilago maydis, the causative agent of cornsmut, contained enzymnes of the glycolytic path-way. Tracer experiments indicated that thepentose-phosphate pathway was the majoravenue of glucose (lissimilation.Newburgh, Claridge, and Cheldelin (1955)

showed that Tilletia caries, the causative agentof wheat smut, possessed all the enzymes of thepentose-phosphate patlhNway. The operation of theglycolytic scheme was also suggested. Newburghand Cheldelin (1958) also found that spores of thisorganism oxidize glucose via the Entner-Dou-doroff pathway.

In conjunction with investigations at theNorthern Laboratory, knowledge was needed ofthe path(s) of glucose dissimilation in repre-sentatives of the A garicaceae, i.e.,mushrooms andrelated species. Lactarius torminosus, a mushroomwhich can be growin in the laboratory in theasexual, mycelial form, was chosen for the initialexperiments.

MATERIALS AND METHODS

L. torminosus NRRL 2900 was grown at 28 Cfor 30 to 36 hr on a reciprocal shaker in themedium shown in Table 1. The pH was adjustedto 5.5 with NaOH before sterilization, and themedium was dispensed either 300 ml in 2.8-literFernbach flasks or 30 ml in 300-ml Erlenmeyerflasks. Sterile glucose (40%) was added asepti-cally to a final concentration of 4%. Stock cul-tures were maintained in a similar solid mediumexcept that the pH was adjusted to 7.0, and 2%of glucose was included before autoclaving.The inoculum was prepared by transferring a

loopful of culture from the stock medium to anErlenmeyer flask containing liquid medium,followed by incubation for 24 hr. The entire con-tents of the small flask were transferred to aWaring blendor and honmogenized to reduce thesize of mycelial pellets. The contents of the

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TABLE 1. Medium used for growthof Lactarius torminosuts

Component Concn per liter

KH2PO4 200 mgMgSO4 700 mgNH4NO3 1,500 mg(NH4)2SO4 3,000 mgCaC12 250 mgNaCl 10 mgZnSO4 1 mgMnC12 1 mgH3BO3 1 IngFeC12 500 i.gCuS04 100 g

KI 100lugThiamine-HCl 50,g

blendor were then transferred to a Fernbachflask and again incubated as described. Cells were

harvested by centrifugation, washed twice withdistilled water, and dried by the addition of 10volumes of acetone at 0 C. The dried cells were

refrigerated with a dessicant until used. About4 to 6 g of acetone-dried cells were obtained per

liter of culture medium.Preparation of extracts. Initial attempts to

extract fresh cells by ultrasonic oscillation, bygrinding with alumina, and by use of a homog-enizer were unsuccessful because of the physicalcharacteristics of the mycelial pellets. However,acetone-dried cells could be extracted very rapidlyby sonic oscillation. Acetone-dried cells were

suspended (4 g per 50 ml of either water or

aqueous buffer) and treated for 3 min in a

Raytheon 10-ke magnetostrictive oscillator.Cell debris was removed by centrifugation at30,000 or 136,000 X g in an ultracentrifuge.No difference in activity of the two preparationswas observed. All operations were carried outat 2 C. The supernatant solution was dialyzedand stored at -20 C. The preparations con-

tained about 10 mg of protein per ml.Materials. Ba-6-phospho-D-gluconate was a

gift of R. D. DeMoss. All other substrates andcofactors were commercial products. Those ob-tained as barium salts were put into solution bybatchwise treatment with IR 120 (H+) until acidto congo red. The supernatant and washingswere neutralized with KOH and made to volume.

Glyceraldehyde-3-ph osph ate dehydrogenaseand lactic dehydrogenase were obtained as

crystalline preparations from the Sigma ChemicalCo., St. Louis, Mo.

The D-fructose-1, 6-diphosphate (FDP) usedin this study was found by paper chromatographyto possess only two organic phosphate spots; onecontiguous with D-fructose-6-phosphate (F6P)and the other presumably FDP. By enzymaticassay (phosphohexoisomerase and glucose-6-phosphate dehydrogenase) and organic phos-phate analysis, the preparation was found to beabout 15% F6P, and presumably 85% fructose-1,6-diphosphate.

Analytical methods. Substrates were assayed fororganic phosphate with the method of Fiske andSubbaRow (1925) after the digestion as describedby Cardini and Leloir (1957).

Pyridine-nucleotide oxidation or reductionwas followed at 340 m,u (OD340) with a Beckmanmodel DU spectrophotometer. Oxygen uptakeand CO2 evolution were measured manometri-cally by means of standard Warburg techniques.All enzyme experiments were carried out at30 C.Chromatography for the detection of phos-

phates was carried out by the descending tech-nique. Phosphates were spotted on HCl-ethylene-diaminetetraacetic acid-washed Whatman no. 1filter paper. The t-butanol-water-picric acidsolvent of Wilson (1954) was employed. Phos-phates were detected with the reagent of Ban-durski and Axelrod (1951).

Fructose was detected colorimetrically by thecysteine-carbazole test (Dische and Boren-freund, 1951). The oreinol method of Mejbaum(1939) was used for the qualitative identificationof pentose.

RESULTS

Manometric experiments. Oxygen uptake cat-alyzed by L. torminosus extracts occurred withD-glucose-6-phosphate (G6P) and triphospho-pyridine nucleotide (TPN) as substrates. Pyri-dine-nucleotide oxidase, as the particulate frac-tion of Azotobacter vinelandii (Cotta-Robles,Marr, and Nilson, 1958), was required to couplethe reaction to atmospheric oxygen. Figure 1shows that 107 ,liters of 02 were taken up in thepresence of 5 ,moles of G6P. The respiratoryquotient was 1.13. These data correspond to theuptake of 1 ,umole of 02 per ,umole of G6P, withthe evolution of 1 Mmole of CO2.At the cessation of oxygen uptake, the contents

of the Warburg vessel were deproteinized, and a

sample was reacted with orcinol. The resultingspectrum (read against similarly treated endog-

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-~75

o 50 oSubstrate - 5,umoles G6P

25

0

'0 15 30 45 60Time (min.)

FIG. 1. Oxygen uptake and CO2 evolution catalyzed by fresh cell-free extract in the presence of 5,umoles of G6P. Components of the system were 0.6 ml of cell-free extract (in 0.2 M phosphate buffer atpH 7.0, centrifuged at 30,000 X g), 0.2 ml of pyridine-nucleotide oxidase, 100 ,umoles phosphatebuffer at pH 7.0, 1 j,mole TPN, and 5 ,umoles G6P. Total volume: 3.2 ml. Center well contained 0.2 ml20% KOH in 02-uptake experiment only. Endogenous (without G6P) uptak-e subtracted.

enous flask contents) was that of a pentose.Thus, our results suggest the G6P is oxidized topentose-phosphate and CO2 by the enzymes,glucose-6-phosphate dehydrogenase and 6-phos-phogluconic dehydrogenase. Preliminary experi-ments showed that oxygen uptake would notoccur with glucose as substrate in either thepresence or absence of pyridine nucleotide andpyridine-nucleotide oxidase. These results indi-cate that neither typical glucose oxidase nortypical glucose dehydrogenase plays a significantrole in the utilization of glucose by L. torminosusextracts. Consequently, one would anticipatethat the first step in glucose metabolism wouldbe a phosphorylation to G6P catalyzed by hexo-kinase. That hexokinase is indeed present will beshown in a later section of this paper.

In subsequent experiments, it was observedthat the degree of G6P oxidation was less whenold cell-free preparations were used (Fig. 2).In the presence of 5 Amoles of G6P, about 50,uliters of 02 were consumed. This consumptioncorresponds to 0.5 ,umole of 02 per ,umole of sub-strate. Oxidation was not reinitiated by addi-

tional aged extract. HowA-ever, upon the introduc-tion of fresh enzyme, the uptake of another 50,uliters of 02 occurred. These results suggest thatL. torminosus preparations contain a labile6-phosphogluconic dehydrogenase. Thus, it canbe hypothesized that aged cell extract oxidizesG6P to 6-phospho-D-gluconic acid (6PG), whichaccumulates. The accumulated ester is rapidlyoxidized (presumably to pentose-phosphate andC02) when fresh cell extract is added.That 6PG indeed does accumulate when aged

cell-free extract is used was shown in an experi-ment in which a total of 60 ,umoles of G6P wereoxidized in a series of Warburg flasks. Whenoxidation ceased, the contents of the flasks werepooled and deproteinized; then barium salts wereformed and isolated. The barium salts were con-verted to the potassium form and analyzed. Thepotassium salt reduced TPN in the presence offresh, but not of aged, cell extract. The potassiumsalt also reduced TPN at the same rate as anequivalent amount of 6PG in the presence ofknown 6-phosphogluconic dehydrogenase. (Thisenzyme was a gift from W. A. Wood.) The salt

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124

0. 6= U6O "Aged"C4 ~ enzyme

oAdd fresh enzyme

30

Substrate - 5j,moles G6P

0 / l l0 10 20 30 40 50 60Time (min.)

FIG. 2. Oxygen uptake catalyzed by aged cell-free extract in the presence of 5 ,umoles of G6P and theeffect of the addition of fresh cell-free extract upon the degree of oxidation. Components of the sys-tem: 0.5 ml aged cell-free extract (in 0.2 M phosphate buffer at pH 7.0, centrifuged at 30,000 X g), 0.2ml pyridine-nucleotide oxidase, 100 ,umoles phosphate buffer at pH 7.0, 1 ,umole TPN, and 5,umoles G6P. Total volume: 3.2 ml. Add 0.5 ml fresh cell-free extract (in 0.2 M phosphate buffer atpH 7.0, centrifuged at 30,000 X g) where indicated. Center well contained 0.2 ml 20% KOH in oxygen-uptake experiment only. Endogenous (without G6P) uptake subtracted.

contained 1 ,mole of organic phosphate per,umole of gluconate; gluconate was determined bythe formation of reduced triphosphopyridinenucleotide (TPNH). Lastly, paper chromatog-raphy disclosed a phosphate spot contiguous withthat of authentic 6PG. No other organic phos-phate spot was observed.

Evidence for the oxidation in L. torminosusextracts of G6P to pentose-phosphate and CO2via 6PG suggests that enzymes of the hexose-monophosphate pathway might be present.Consequently, further work was done to sup-port this hypothesis.Enzymes of a hexosemonophosphate pathway.

Cell-free extracts catalyze the oxidation ofG6P (Table 2), and this oxidation is specific forTPN. These results confirm those noted previ-ously that L. torminosus extracts contain glucose-

6-phosphate dehydrogenase. Table 2 also showsthat TPN is not reduced by glucose alone, butwhen adenosine triphosphate (ATP) is addedwith glucose, TPN is reduced. This reductionindicates that hexokinase is present as we previ-ously postulated. It can also be seen that TPNis reduced in the presence of 6PG, D-ribose-5-phosphate (R5P), and F6P.The oxidation of 6PG confirms the presence of

6-phosphogluconic dehydrogenase, a well-knownenzyme which oxidizes 6PG to pentose-phosphate(D-ribulose-5-phosphate) and CO2. This enzymeis associated with the hexosemonophosphatepathway.The formation of TPNH in the presence of

R5P (Table 2) indicates that R5P is cycled toG6P via hexosemonophosphate enzymes, namely,phosphoribose isomerase, D-ribulose-5-phos-

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TABLE 2. Initial rates of pyridine-nucleotidereduction in the presence of various

substrates*

Substrate Pyridine AOD,/5 min

G6P DPN 0G6P TPN 0.690Glucose TPN 0Glucose + ATP TPN 0.0186PG TPN 0.120R5P TPN 0.018F6P TPN 0.510

* Components of the system were 0.1 ml cell-free extract (in water, centrifuged at 136,000 X g),100 uAmoles tris (hydroxymethyl)aminomethane(tris) or phosphate buffer at pH 7.0, 1 ,umole phos-phate ester, 1 ,Amole glucose, 2,umole ATP, and1 ,umole pyridine nucleotide. Total volume: 3.0 ml.Endogenous (without phosphate ester) reductionsubtracted.

0.4 *...Product from

03- -

0.4

_̂°02\Product fron

CL~~~~

phate-3-epimerase, transketolase, and transaldo-lase. The cycle would be completed by phospho-hexoisomerase, which coilverts F6P to G6P. Thepresence of transketolase activity was furthersupported by an experimient in which the rate ofTPNH formation in the presence of R5P wasstimulated 1.7-fold upon the addition of diphos-phothiamine (DPT).The coaddition of Mg++ stimulated the reduc-

tion rate 3.1-fold. Phosphohexoisomerase, whichwould complete the cycle, was indicated byTPNH formation in the presence of F6P; i.e.,F6P was converted to GOP, which reduced TPN.Phosphohexoisomerase activity was confirmed byan experiment in which the OD560 (cysteine-carbazole test) was followed with G6P or F6Pas substrate (Fig. 3). With G6P as substrate, theOD560 increased rapidly with time; however, withF6P, the OD560 rapidly decreased. Since absorp-tion at OD560 in this test is specific for fructose,

0 5 10 15 20Time (min.)

FIG. 3. Formation of fructose from G6P and disappearance of F6P when G6P and F6P, respec-tively, are incubated with cell-free extract. Components of the system: 0.1 ml cell-free extract (inwater, centrifuged at 136,000 X g), 200 jmoles tris buffer at pH 7.0, 2 ,umoles G6P, and 2 ,imoles F6P.Total volume: 9.0 ml. Sampled 0.6 ml for cysteine-carbazole test. Read against appropriate blank after24 hr of reaction time.

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Add more DPN (1 Aumole)

x -- F6P + ATPg 0.5- ---0 FDP

0.0 x.xM x.x__.x

''--Add cell-free enzyme

-10 0 15 30 45 60Time (min.)

FIG. 4. Assay for FDP aldolase, F6P kinase, and triosephosphate isomerase. Components of thesystem are 0.1 ml of cell-free extract (in water, centrifuged at 136,000 X g), 50 ,ug G3P de-hydrogenase, 33.3 j.moles cysteine, 75 ,umoles glycylglycine buffer at pH 7.5, 40 iAmoles Na2HAsO4, 10,umoles MgC12, 1 ,umole DPN, 0.725 ,umole FDP, 2 ,Amoles ATP, and 0.324 ,Amole F6P. Total volume: 3.0ml. An additional 1 ,imole DPN was added where indicated. Endogenous (without FDP or F6P)reduction subtracted.

these data suggest that G6P was converted toF6P and that the reaction was reversible. Further,the spectrum of the material formed from G6Pwas compared to known F6P, and the two spectrawere identical.The data to this point suggest that L. tor-

minosus extracts catalyze the TPN-specificoxidation of G6P to pentose-phosphate and CO2via 6PG. The pentose-phosphate is convertedto F6P by enzymes of a hexosemonophosphatepathway, and the F6P is cycled to G6P by phos-phohexoisomerase. Further work was done toestablish whether enzymes of the hexosediphos-phate (Embden-Meyerhof) pathway were pres-ent in L. torminosus extracts.Enzymes of the hexosediphosphate pathway.

Current concepts of enzyme systems dictate thatthe unique enzymes of the hexosediphosphatepathway are fructose-6-phosphate kinase, aldo-

lase, and triosephosphate isomerase (Gunsalus,Horecker, and Wood, 1955). The assays for theseenzymes are shown in Fig. 4. The reactions werefollowed by measuring diphosphopyridine nucleo-tide (DPN) reduction catalyzed by added glyc-eraldehyde-3-phosphate dehydrogenase in sys-tems containing cell-free extract and substrates.No DPN reduction occurred in the system con-taining F6P and ATP, indicating the absenceof fructose-6-phosphatekinase; however, DPNwas reduced in the system containing FDP,indicating the presence of aldolase in L. tormino-sus extracts. Surprisingly, the molar ratio ofDPN reduced to FDP added was 1.01. In thepresence of triosephosphate isomerase, the ratiowould be 2.0. It should be noted that the purityof the FDP was established (see Materials andMethods).The absence of fructose-6-phosphate kinase

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Time (min.)FIG. 5. Reduction of pyridine nucleotide by cell-free extract in the presence of D-G3P. Components

of the system: 0.1 ml cell-free extract (in 0.2 M tris buffer at pH 8.8, centrifuged at 30,000 X g), 100jumoles pyrophosphate buffer at pH 8.8, 1 .umole glutathione, and 0.5 ,umole DL-G3P. Total volume:3.0 ml. Endogenous (without GSP) reduction subtracted.

was substantiated in an experiment in which5 ,moles of F6P and 15 ,moles of ATP wereincubated with 0.1 ml of cell-free extract. After2 hr, no decrease in 7-min acid-hydrolyzablephosphate was observed, indicating no ATPutilization. Further, incubating 0.1 ml of cell-freeextract with 5 ,umoles of FDP for 1 hr resulted inno liberation of free orthophosphate, indicatingthe absence of phosphatase activity. Conse-quently, the negative kinase assay is not an arti-fact resulting from competition between aldolaseand fructose-1 , 6-diphosphate phosphatase for theFDP formed.The absence of triosephosphate isomerase was

substantiated by an experiment in which 0.1 mlof cell-free extract was incubated with 10 ,umolesof DL-glyceraldehyde-3-phosphate (G3P). Withtime, no increase in OD660 was observed in thecysteine-carbazole test, showing that fructose

was not formed. Equilibrium would favor FDPformation. These facts indicate that eitheraldolase or triosephosphate isomerase was absent.The presence of aldolase had already been demon-strated (Fig. 4); therefore, the enzyme prepara-tion must lack triosephosphate isomerase.Enzymes utilizing glyceraldehyde-3-phosphate.

The oxidation of D-G3P catalyzed by L. tormino-sus extracts was specific for DPN and proceededat a slow rate (Fig. 5). Preliminary experimentsshowed that this reaction had maximal activityat pH 8.8. The results indicate the presence of aDPN-specific glyceraldehyde-3-phosphate de-hydrogenase in the crude enzyme preparations.The conversion of 3-phospho-D-glyceric acid

(3PGA) to pyruvate (Fig. 6) was followed bymeasuring reduced diphosphopyridine nucleotide(DPNH) oxidation catalyzed by lactic dehydro-genase added to a system containing cell-free

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1.0

0.9k_

.81--

0. E- cO*_ C"I 0CL .7

0.6 kSubstrate

,4-Add 0.5,umole ADP

A.,mole DPNH oxidized= 0 92

,umole 3PGA added

0.242,umole 3PGA -Cuw qI I I

-20 -10 0 10 20 30 40 50Time (min.)

FIG. 6. Assay for the conversion of SPGA to pyruvate. Components of the system: 0.1 ml cell-free extract (in water, centrifuged at 30,000 X g), 704 units of crystalline lactic dehydrogenase dis-solved in 0.01 M NaCI, 50,imoles tris buffer at pH 7.5, 10 uimoles MgC12, 0.242 Amoles SPGA, and0.75 ,umoles DPNH. Total volume: 3.0 ml. Endoqenous (without SPGA) reduction subtracted. Add0.5,mole ADP where indicated.

extract and 3PGA. If 2, 3-diphosphoglycericmutase, enolase, and pyruvic kinase are present,3PGA would be converted to pyruvate upon theaddition of adenosine diphosphate (ADP). Thelactic dehydrogenase present would catalyze thereduction of pyruvate to lactate with the con-comitant oxidation of DPNH. Since 1 ,umole ofpyruvate should arise from 1 ,umole of 3PGA,the ratio of oxidized DPNH to 3PGA would be1/1. Upon the addition of ADP, DPNH oxidationdid occur. The molar ratio of DPNH oxidized to3 PGA added was 0.92. These results suggest theformation of pyruvate from 3 PGA. Figures 5and 6 indicate that D-G3P was converted topyruvate by extracts of L. torminosus.

DISCUSSION

The occurrence of glucose-6-phosphate de-hydrogenase and 6-phosphogluconic dehydro-genase in extracts of L. torminosus suggests thatenzymes of a hexosemonophosphate pathwayalso may be present. Such enzymes are phos-phoribose isomerase, D-ribulose-5-pihosphate-3-

epimerase, transketolase, and transaldolase. Thathexosemonophosphate enzymes indeed do occurin L. torminosus extracts is supported by TPNreduction in the presence of R5P. This reductionwould occur if R5P were cycled to G6P by hexose-monophosphate enzymes and phosphohexo-isomerase. Phosphohexoisomerase has beendemonstrated in our extracts. Transketolase,one of the hexosemonophosphate enzymes, isknown to be stimulated by DPT and Mg-.The rate of TPNH formation in the presence ofR5P catalyzed by cell-free extract is increased bythe addition of DPT. The reduction rate isfurther increased by the coaddition of Mg++.Further evidence for hexosemonophosphateenzymes is presented in another paper (Meloche,1961).The lack of active fructose-6-phosphate

kinase and triosephosphate isomerase in theseextracts suggests that the hexosediphosphatesystem does not operate. Unfortunately, acetonedrying of the cells may have damaged either orboth of these enzymes. The cells were so treated

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to facilitate preparation of extracts (see Materialsand Methods). The importance of this pathwayin vitro would have to be determined using wholecells and labeled substrates. Our experimentswith whole cells have indicated that such studieswould be complicated by very high endogenousactivity. Since the rate of glucose-6-phosphatedehydrogenase is so much greater than that ofglyceraldehyde-3-phosphate dehydrogenase inthese extracts, a hexosemonophosphate systemmay be the major avenue of glucose dissimilation.Acetone-dried L. torminosus cells provide a

source of triosephosphate isomerase-free aldolase.The cycling of R5P to G6P, demonstrated in

these extracts, is worth consideration. To our

knowledge, the operation of either a pentose-phosphate or a hexosemonophosphate "cycle"in vivo is indeed a rare phenomenon, if it occurs

at all. The postulation of a cyclic mechanism is alogical outgrowth of hexosemonophosphateenzymes and phosphohexose isomerase. In thecell, however, there exists competition betweenthe cycle and other enzyme systems for theproducts formed: as examples, competition of6-phosphogluconic dehydrase for 6PG, phospho-ketolase for D-xylulose-5-phosphate, the systemconverting D-G3P to pyruvate and also the tri-carboxylic-acid cycle for D-G3P, and fructose-6-phosphate kinase and the hexosediphosphatesystem for F6P. Our data suggest that some ofthese competitive systems either are of a lowactivity or are absent in cell-free extracts. Con-sequently, L. torminosus whole cells may presentone of those rare instances where the operationof a cycle might be demonstrated. In early ex-

periments, assays for 6-phosphogluconic de-hydrase and 2-keto-3-deoxy-6-phosphogluconicaldolase were negative.

It should be emphasized that the cells used inthis study are not necessarily comparable tomushrooms found in nature. These cells are

asexual and mycelial; the native mushroom is a

sexual form containing organized tissue systems.

LITERATURE CITED

BANDURSKI, R. S., AND B. AXELROD. 1951. Chro-matographic identification of some biologi-

cally important esters. J. Biol. Chem. 193:405-410.

BU'LOCK, J. O., AND H. GREGORY. 1959. Path-ways of sugar metabolism in relation to thebiosynthesis of polyacetylenic antibiotics.Experientia 25:420-421.

CARDINI, C. E., AND L. F. LELOIR. 1957. Generalprocedure for isolating and analyzing tissueorganic phosphates, p. 844. In S. P. Colowickand N. 0. Kaplan [ed.], Methods in enzy-mology, vol. 3. Academic Press, Inc., NewYork.

COTTA-ROBLES, E. H., A. G. MARR, AND E. H.NILSON. 1958. Submicroscopic particles inextracts of Azotobacter agilis. J. Bacteriol.75:243-252.

DISCHE, Z., AND E. BORENFREUND. 1951. A newspectrophotometric method for the detectionand determination of keto sugars and trioses.J. Biol. Chem. 192:583-587.

FISKE, C. H., AND Y. SUBBAROW. 1925. Thecolorimetric determination of phosphorous.J. Biol. Chem. 66:375-400.

GUNSALUS, I. C., B. L. HORECKER, AND W. A.WOOD. 1955. Pathways of carbohydratemetabolism in microorganisms. Bacteriol.Rev. 19:79-128.

McKINsEY, R. D. 1959. Glucose dissimilation inUstilago maydis. Am. J. Botany 46:566-571.

MEJBAUM, W. Z. 1939. tber die Bestimmungkleiner Pentasemengen, insbesondere inDerivaten der Adenysaure. Z. physiol. Chem.,Hoppe-Seyler's 258:117-120.

MELOCHE, H. P. 1961. The metabolism of ribose-5-phosphate by cell-free extracts of Lactariustormitnosus. Biochimr. et Biophys. Acta 51:586-588.

NEWBURGH, R. W., C. A. CLARIDGE, AND V. H.CHELDELIN. 1955. Carbohydrate oxidationby the wheat smut fungus, Tilletia caries.J. Biol. Chem. 214:27-35.

NEWBURGH, R. W., AND V. H. CHELDELIN. 1958.Glucose oxidation in mycelia and spores ofthe wheat smut fungus Tilletia caries. J.Bacteriol. 76:308-311.

WILSON, A. T. 1954. Ph.D. Thesis, Universityof California. Quoted in: Benson, A. A. 1957.Sugar phosphates, paper and column chro-matography, p. 118. In S. P. Colowick andN. 0. Kaplan [ed.], Methods in enzymology,vol. 3. Academic Press, Inc., New York.

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