enzyme patterns in human tissues. i. methods for the ... · on tissues of many species, but with...

Enzyme Patterns in Human Tissues. I. Methods for theDetermination of Glycolytic Enzymes*

CARL E. SHONK AND GEORGE E. BOXER(Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey)

SUMMARY

The prospective aim of these studies is the development of consonant procedures forthe determination of comparative enzyme patterns in human malignant and non-malignant tissues. Based on the well established principle of using nicotinamideadenine dinucleotides to follow reaction rates, improved methods and a unified program for the determination of all the glycolytic and some related enzymes hi crudetissue homogenates have been described. With rat tissues used as models, the precision of the measurements and the stability of the enzymes to storage in the intacttissue at â€”20Â°C.have been established.

The consonant methodology permits direct comparison of the activities of all theenzymes. Although such enzyme patterns cannot yield information on the actualrates of metabolic processes in vivo, they do give information on the potential rates ofmetabolite flow, the steps in an enzymic reaction chain that are likely to be rate-limiting, and the potential for alternate metabolic pathways.

In studies on the biochemistry of human malignanttissues, it would be desirable to obtain information ondirection and rates of over-all metabolic processes. Considerable information along these lines is available formalignant tissues from rodents, but the methods usedrequire the most exacting simultaneous controls in normaland tumor-bearing animals, measurements on samples atcarefully timed intervals, use of very hot isotopes, etc.It is doubtful that methodology of this type can berefined to a point where application to human malignanttissues could even be considered ; thus, it becomes essentialto search for other methods that might validate the extrapolation from observations on rodent tumors to humantumors.

Single enzyme reactions or groups of enzyme reactionscan, however, be measured in human tissues. Suchstudies cannot yield information on metabolic processesas they occur in vivo, but they can give information onthe potential rates of metabolite flow, the steps in anenzymic reaction chain that are likely to be rate-limiting,and the potential for alternate metabolic pathways hidifferent tissues be they normal or malignant.

The glycolytic pathway has been of enduring interestin studies of the metabolism of malignant tissues eversince the original observation of Warburg et al. (37) thatmost malignant tissuesâ€”rodent as well as humanâ€”showa high rate of aerobic glycolysis. Thus, a study of theactivities involved in this pathway was undertaken.

* This investigation was supported by the Cancer Chemotherapy National Service Center, National Cancer Institute,under the National Institutes of Health, Contract #SA-43-ph-1886.

Received for publication December 16, 1963.

If enzymes are studied individually they should bemeasured under optimal conditions of substrate concentration, temperature, pH, etc. The physiological meaningof the measurement of an individual enzyme will remainobscure, unless it can be related to other enzymes,especially in regard to pathways involving a long sequenceof enzyme reactions, for here the activities of the enzymesrelative to one another are likely to be of greater metabolicsignificance than the values for optimal absolute activities.For example, comparison of the optimal activity of oneenzyme determined at one pH with the optimal activityof another enzyme measured at another pH cannot readilyyield information on the metabolic potentials of theseenzymes in a given tissue in vivo.

The basic methodology for the determination of theglycolytic enzymes depends in essence on coupling thereactions to the oxidation or reduction of the nicotinamideadenine dinucleotides and has been extensively used inmany investigations. In applying this basic methodologyto a variety of tissues of rodents and man, a series ofimprovements and precautions hi technic proved to beessential in order to get data that were reproducible andpermitted direct comparisons of enzyme activities withone another. This paper describes the methodologydeveloped and the extent of its applicability as exemplifiedhi rodent tissues. Subsequent papers1 (32) describe the

application of the methodology to normal as well asmalignant human tissues. Glycolytic enzymes have beenmeasured in a variety of tissue both malignant (for

1C. E. Shonk, B. Koven, H. Majima, and G. E. Boxer. EnzymePatterns in Human Tissues. III. Glycolytic Enzyme Patternsin Malignant Human Tissues (in preparation for Cancer Res.).

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710 Cancer Research Vol. 24, May 1964

a review see [1]) and normal by numerous investigators.Unfortunately, most of these studies involve single enzymesor a few enzymes (and sometimes these are unrelated)measured under different conditions. Recently, however,there have appeared a limited number of studies involvingcongruent measurements of related enzymes in animaltissues. Among these have been the studies of DelbrÃ¼cket al. (8) on tissues of the rat and mouse; Pette and BÃ¼cher(20), Pette, Luh, and BÃ¼cher(23), and Vogell et al. (36)on tissues of many species, but with particular emphasison enzyme patterns of striated muscles; Fellenberg et al.(14) on tissues of the rat and mouse; Wu and Racker (39)on the Ehrlich ascites cell ; and Schmidt and Schmidt (29)on human tissues.

Much of the success obtained in this area can be attributed to the studies of Beisenherz (2) et al., who introducedethylenediamine tetraacetate (EDTA) in all solutionsinvolved in the preparation, isolation, and handling ofthese enzymes. The measurements in this metabolicarea depend on the systematic use of auxiliary enzymes asreagents (25) which are now commercially available incrystalline form of high purity, activity, and stability.

MATERIALS AND METHODSBuffer.â€”The buffer used in all systems was triethanol-

amine buffer, pH 7.6, 0.05 M, containing Na2H2EDTA,0.006 M.

Homogenization or dilution medium.â€”This was anaqueous solution containing KC1, 0.15 M; KHC03) 0.05 M;Na2H2EDTA, 0.006 M. This medium provided maximalextraction with minimal interferences for this series ofenzymes. In particular, a-glycerolphosphate dehydro-genase and phosphofructokinase required a slightlyalkaline medium for extraction from the tissue.

Reagents.â€”It is essential that all reagents be as free aspossible from related interfering substances. Thoselisted here have worked well, but variations in differentbatches occur and must be looked for by the internalchecks described below.

a. Substrates: FDP,2 F-6-P, 6-PGA, 3-PGA, and2-PGA were products of C. F. Boehringer & Soehne,distributed by Calbiochem. FDP was obtained as thetrisodium salt, and aqueous solutions of it were useddirectly. The F-6-P, 6-phosphogluconate, 3-PGA, and2-PGA were obtained as barium salts; barium was removedby the addition of stoichiometric amounts of K2S04 andcentrifugation. G-6-P was the product of Sigma ChemicalCorporation, and solutions of the potassium salt wereused directly. Oxalacetic acid, DHAP, PEP, and pyruvicacid were obtained from Calbiochem. Solutions ofoxalacetate are unstable, and a neutralized (KHCO3)0.2 M solution was prepared daily and diluted twentyfold

2The following abbreviations are used:Coenzymes.â€”ATP, adenosine triphosphate ; ADP, adenosine

diphosphate; NAD+, nicotinamide adenine dinucleotide ; NADH,reduced nicotinamide adenine dinucleotide; NADP+, nicotinamideadenine dinucleotide phosphate.

Substrates and reagents.â€”DHAP, dihydroxyacetone phosphate;FDP, fructose-1,0-diphosphate; F-6-P, fructose-6-phosphate;GAP, glyceraldehyde-3-phosphate ; G-6-P, glucose-6-phosphate;3-PGA, 3-phosphoglycerate; 2-PGA, 2-phosphoglycerate; PEP,phosphoenolpyruvate.

just prior to addition to the reaction mixture. Solutionsof pyruvic acid were neutralized by the addition of solidKHC03. DHAP was prepared from the dicyclohexyl-ammonium salt of the dimethylketal according to themanufacturer's instructions. Solutions of this compound

were adjusted to pH 6.0 by the cautious addition of solidKHC03. This partially neutralized solution can be keptin the frozen state for at least 2 weeks, but its concentration should be determined enzymically before eachuse. Aqueous solutions of the trisodium salt of PEPwere used directly.

GAP was obtained as a diethylacetal from SchwarzBioResearch. The GAP was released according to themanufacturer's instructions, and the resulting solution

was adjusted to pH 6.0 by the addition of solid KHCOs.Such solutions can be used for several days if stored frozenat â€”20Â°C. The actual concentration of this substance

should be determined enzymically each time it is used.b. Coenzymes: ATP, ADP, NAD+, NADP+, and

enzymically reduced NADH were obtained from PabstLaboratories. Solutions of ATP, ADP, NAD+, andNADP+ were adjusted to pH 6.5 by the cautious additionof solid KHC03. These solutions were sufficiently stablefor 1 day's work if kept in an ice bath. Freshly prepared

solutions of NADH were used directly.c. Enzymes: All crystalline enzymes were products of

C. F. Boehringer & Soehne, distributed by Calbiochem.The following crystalline enzymes were required: glucose-6-phosphate dehydrogenase ; aldolase; a-glycerolphosphatedehydrogenase ; a-glycerolphosphate dehydrogenase-triose-phosphate isomerase mixture; glyceraldehyde phosphatedehydrogenase; phosphoglycerate kinase; phosphoglyc-erate mutase; enolase; pyruvate kinase; and lactatedehydrogenase. The ammonium sulfate suspensions ofthe crystalline enzymes were diluted twentyfold in thehomogenization medium. The dilute solutions werereasonably stable and could be stored at â€”20Â°C.for 3 or

4 days.Animals.â€”Holtzman rats of both sexes, weighing

between 150 and 250 gm., were killed by decapitationand exsanguination. All data reported here were obtainedon animals fed ad libitum up to the time of killing.

Tissue storage and homogenization.â€”Tissue piecesweighing between 100 mg. and 1 gm. were stored frozenat â€”20Â°C.until the enzymic assays could be performed.

After small slices were removed from the frozen tissue forhistological controls, the tissue was weighed rapidly, andhomogenization was begun on the still frozen tissue, sothat thawing occurred during tissue disintegration. Inall cases, a chilled homogenization medium was used anda 5 or 10 per cent homogenate was prepared.

In most cases the tissues were homogenized for 2 minutesat top speed in the Virtis 45 homogenizer; however, sometimes a Potter-Elvehj em-type homogenizer with a Teflonpestle was used. In either case the enzyme activities wereidentical. The main advantage of homogenization withthe Virtis homogenizer is that it provides a smoothermore uniform homogenate even with fibrous humantissues, thus permitting more accurate determinations ofthe chemical constituents. Enzyme determinations weremade on appropriate dilutions of this homogenate, the



SHONKANDBOXERâ€”EnzymePatterns in Human Tissues. I 711

diluent being the homogenization medium. All operations were carried out in vessels immersed in an ice bath.

Since the process of measuring fifteen to twenty enzymescannot be done instantaneously and the stabilities of theindividual enzymes vary considerably in homogenates, itis necessary to set up an enzyme assay program in whichthe most labile enzymes are measured first. The orderin which these enzymes were measured is indicated by theorder in which the enzymes are listed in the assay procedures. Except for the first four enzymes this programneed not be rigidly adhered to, but it is essential thatglyceraldehyde phosphate dehydrogenase be measured assoon as possible following homogenization. The otherthree enzymes, although considerably more stable inhomogenates than glyceraldehyde phosphate dehydrogenase, should be measured shortly thereafter. Eventhough some of the enzymes at the end of the compilationare stable in stored frozen homogenates, the completesequence of enzymes was measured immediately followinghomogenization because on prolonged standing or storagemany homogenates (and also supernatant fractions) losetheir homogeneity owing to aggregation, clumping, orgelling, a fact which makes accurate pipetting difficult.

All enzyme determinations were carried out in 0.05 Mtriethanolamine buffer, pH 7.6, at room temperature,24 +1Â°C., and all enzymic activities are expressed in

terms of the unit recommended by the Commission onEnzymes of the International Union of Biochemistry(26). One unit (U) of any enzyme is defined as "that

amount of enzyme which will catalyze the transformationof 1 Â¿uÃ±Ã³leof substrate per minute." This permits direct

comparison of the activities of all the enzymes.

GENERALENZYMEASSAYPROCEDURE

Since all the enzymic assays were coupled to systemsinvolving the oxidation or reduction of one of the nico-tinamide adenine dinucleotides, the enzymic measurements were made by recording rates of change in absorb-ance at 340 m/i on the Gary spectrophotometer. Thekinetics were zero order in all instances, and the concentration of substrate, auxiliary enzymes, coenzymes, andions were chosen to give maximal rates. (The oneexception was triosephosphate isomerase, where a sub-optimal, though reproducible, substrate concentration waschosen for practical reasons given below.)

In general, 3 ml. of triethanolamine buffer containingEDTA were placed in each cuvette. After balancing ofthe instrument, the other components in as small a volumeas practical were individually added to the sample cuvette.Although the final volume of the reaction mixture wasconstant for each particular assay system, the volume forthe different assay systems varied between 3.3 and 3.6 ml.Mixing was not done in the cuvettes but by pouring froma cuvette into a small test tube which already containedthe next additive and repouring back ulto the cuvette.This insured complete, rapid mixing and avoided difficulties due to schlieren effects which frequently appearedupon the addition of a small amount of a concentratedreagent to a dilute solution in the cuvette. Combinationof reagents into single solutions for addition was generallynot done for two reasons: (a) to avoid interactions leading

to instability of the reagents and (o) to observe the effectsof the addition of each individual component as describedbelow. By keeping the recording chart running whileinterrupting the light measurement, a record of the timeinvolved in the addition of reagents was obtained. Insome assay systems the number of components was solarge that all the desired information could not be obtainedin a single run. In these cases it was necessary to rerunthe system with the components added in a differentorder. The essential advantage of the stepwise monitoringof the assay system was that interferences were immediately observed and that any necessary adjustments,corrections, or rejections could be made. In additionslight modifications of the assay systems permitted thedetermination of concentrations of substrate solutions andthe activity of ancillary enzymes. The disadvantageswere that an operator was kept in constant attendance,and only single determinations could be made at a time. '

In most tissues the rate of endogenous oxidation ofNADH was insignificant unless the enzyme to be measuredwas present in such low levels that relatively large quantities of tissue homogenate had to be employed. In allcases the rate of endogenous oxidation of NADH wasmonitored, and when it became significant it was subtracted from the final enzyme measurement. In homogenates from a few freshly excised rodent tissues, particularly heart and kidney and to a lesser extent brain, thelevel of this "NADH oxidase" was too high to permit

accurate measurements of many enzymes. This particulardifficulty could be avoided by storing these tissues frozenat â€”20Â°C.for 7-10 days, during which time the "NADHoxidase" activity disappeared, whereas the activities of the

glycolytic enzymes remained unchanged under the sameconditions. The "NADH oxidase" activity of the brain

was the most labile, disappearing from a homogenate offresh brain in about 1 hour, but in heart and especiallykidney this activity was much more tenacious and requiredthe indicated storage conditions. Preincubation of theintact tissues at room temperature for several hours didnot appear to hasten this process. Surprisingly, this typeof interference has never been observed with humantissues.

Similarly, an appreciable endogenous "NADP+ reduc-tase" was sometimes observed in rodent tissue homog

enates when relatively large amounts of tissue samplewere required. In general this rate was small comparedwith the rate due to the enzyme being assayed, andappropriate corrections could be made.

After all the effects of the endogenous materials in thetissue extracts had been recorded, the enzymic reactionwas initiated by the addition of one of the substrates.In general, the rate of the enzymic reaction was recordedfor 1-3 minutes starting about 10 seconds after theinitiation of the reaction. Since the rates were zero orderlonger periods could be recorded, but the use of shortreaction times minimized interference of competing,subsequent, or simultaneous reactions. The followingdescriptions indicate the concentration of each componentin the final reaction mixture, and each reagent is listed inthe order in which it is added. In some cases it may bedesirable to repeat a particular run in a different sequence




in order to obtain some additional information. Thequantity of tissue employed is expressed as mg. wet weightand gives reaction rates that can be measured withprecision with most tissues. In some tissue samplesthe rates may be too slow or too fast, and the weight oftissue was then adjusted so that rates were recorded inthe range of 10-160 mamÃ³les of nicotinamide adeninedinucleotides oxidized or reduced per minute.

SPECIFICENZYMEDETERMINATIONSGlyceraldehyde phosphate dehydrogenase (GAPDH).â€”

This is the enzyme which must be measured as soon aspossible after homogenization. The activity has beenmeasured in both directions.

Assay system using reduction of NAD+ (6) : Triethanol-amine, 50 ITIM;EDTA, 5 HIM;arsenate, 12 HIM;NAD+,0.4 IBM;tissue, 1 mg; GAP, 0.6 HIM.

After the tissue sample was added, the absorbance at340 mp was recorded briefly, and the enzymic reactionwas initiated by the addition of the substrate, GAP.The recording chart remained activated during thisaddition so that the time measurement was not interrupted.The time required for the addition was then indicated onthe chart by the length of the line devoid of electronicnoise which was present during light measurements.This is essential, since the rate of the reaction in thisdirection decreases rapidly so that the enzyme activitieswere calculated from rates extrapolated back to zero time.

Assay system with oxidation of NADH (2, 9, 39):Triethanolamine, 50 HIM;EDTA, 5 niM; MgCl2, 10 HIM;NADH, 0.3 HIM;phosphoglycerate kinase, 7.1 U; sample,1 mg.; 3-PGA, 2.9 mM; ATP, 2.9 mm.

The rate of the reaction when run under these conditionswas equal to the rate observed when run in the oppositedirection. The concentration of Mg++ was critical, sinceit was required for activation of PGK but inhibitedGAPDH if added in excess. The crystallized enzymewas much more sensitive in this respect than the enzymein crude tissue homogenates.

The possibility was considered that a-glycerolphosphatedehydrogenase, present at relatively high levels in sometissue homogenates, might interfere with the measurementby oxidizing some of the NADH formed; but no interference was observed, because during the short time thatthe measurements were carried out insufficient triÃ³sephosphates were formed to produce measurable rates witha-glycerolphosphate dehydrogenase. Even the additionof a large excess of crystallized a-glycerolphosphatedehydrogenase to the assay system did not influence theinitial rate.

Glyceraldehyde phosphate dehydrogenase in freshlyprepared homogenates did not require activation bycysteine. Upon standing at 4Â°C.the enzyme activity in

homogenates decreased in about 1 hour. In the earlystages of inactivation, the activity could be restored bythe addition of cysteine, but it failed in the later stages.The inactivation of glyceraldehyde phosphate dehydrogenase occurred much more rapidly in dilute solutions ofthe crystallized enzyme than in crude tissue homogenates.Although full enzymic activity could be observed uponimmediate dilution of the crystalline enzyme in the

absence of cysteine, it was safer to add cysteine to systemscontaining the crystallized enzyme in dilute solution.

Pyruvate kinase (2, 39).â€”Triethanolamine, 50 niM;EDTA, 5 mat; MgCl2) 10 nut; LDH, 9.1 U; NADH; 0.3mM; PEP, 2.9 HIM;tissue, l mg.; ADP, 2.9 niM.

This reaction was initiated by the addition of ADPrather than PEP. This order of addition was necessary,because the substrate PEP invariably contained somepyruvate which had to be reduced to lactate beforeproceeding with the assay for pyruvate kinase. Theamount of contaminating pyruvate could be calculatedfrom the total change in absorbance at 340 imt followingthe addition of the PEP.

Phosphofructokinase (25, 39).â€”Triethanolamine, 50nui; EDTA, 5 nut, MgCl2, 10 mM; a-glycerolphosphatedehydrogenase-triosephosphate isomerase mixture containing 7.1 and 18.3 U, respectively; aldolase, 1.0 U;NADH, 0.3 mat; tissue, 1-10 mg.; ATP, 2.8 mat; F-6-P,2.8 mM.

Since this enzyme was present at low levels in mosttissues, 5-10 mg. of tissue were generally required. Onlyin samples of skeletal muscle could this enzyme be assayedwith 1 mg. of tissue.

It was important to add the ATP to the mixtureimmediately following the addition of the tissue sample,since ATP stabilized the enzyme and delay at this pointresulted in lower levels of activity. For this reason theenzymic reaction was initiated by the addition of F-6-Prather than ATP. This presented an additional difficulty,because samples of F-6-P contained some FDP, theconcentration of which had to be determined in a separatemeasurement employing the above assay system withoutATP and the tissue sample. Usually the amount ofFDP was so small that its effect in the phosphofructo-kinase assay system was noticeable only during the first30 seconds of the reaction and the reaction rate thereaftercorresponded to the phosphofructokinase activity. Theaddition of adenosine-3',5'-monophosphate (19) did not

enhance the reaction rates with the amounts of tissuerequired. Since 2 moles of NADH are oxidized per moleof substrate utilized, the observed rate has to be dividedby two in order to express the measurement hi terms ofthe international unit.

Enolase.â€”Triethanolamine, 50 mat; EDTA, 5 mai;MgCl2, 10 niM; lactate dehydrogenase, 9.1 U; pyruvatekinase, 16.0 U; NADH, 0.3 niM; tissue sample, 2 mg.;ADP, 2.9 mai; 2-PGA, 2.8 mM.

Phosphoglycerate kinase (2, 39).â€”Triethanolamine, 50mM; EDTA, 5 mM; MgCl2, 10 mM; NADH, 0.3 nut;glyceraldehyde phosphate dehydrogenase, 4.3 U; cysteine,2.8 nut; tissue, 1 mg.; 3-PGA, 2.8 mM; ATP, 2.8 mM.

The cysteine was essential to insure full activity of theancillary enzyme, glyceraldehyde phosphate dehydrogenase. This system was not as sensitive to Mg++concentration as the system for the determination ofglyceraldehyde phosphate dehydrogenase, since glyceraldehyde phosphate dehydrogenase was present inlarge excess and was not rate-limiting.

Glucokinase (25).â€”Triethanolamine, 50 mM; EDTA, 5mM; MgCl2, 10 nut; NADP+, 0.35 nut; glucose-6-phos-



SHONKAND BOXERâ€”EnzymePatterns in Human Tissues. I 713

phate dehydrogenase, 4.3 U; tissue, 5-10 mg.; glucose,2.8 mat; ATP, 2.8 mai.

It was essential to employ glucose-6-phosphate dehydrogenase that was free of glucokinase and 6-phospho-gluconate dehydrogenase activities in the glucokinaseassay system.

Glucokinase could also be measured by coupling to asystem involving pyruvate kinase and lactate dehydrogenase. In this case the ADP that was formed duringthe phosphorylation of glucose was used to drive thereaction catalyzed by pyruvate kinase which, in turn,regenerated ATP. Unfortunately, this system workedwell only in those few tissues in which ATPase activitywas very low.

With the levels of tissue homogenate required for theassay, some rodent tissues showed considerable rates ofendogenous reduction of NADP+ ("NADP+ reducÃase")

without the addition of glucose or ATP. Similarly, theeffect of each of these substances had to be tested separately by revereing the order of addition. Because6-phosphogluconate dehydrogenase is present in mosttissues, it has been generally assumed (10, 17) that thisenzyme participated in the assay system so that 2 molesof NADP+ were reduced per mole of glucose phosphory-lated. This has not been observed with the assay systemand conditions employed here. Studies with partiallypurified 6-phosphogluconate dehydrogenase from ratliver have shown that this enzyme required a rather highconcentration of 6-phosphogluconate for activity andduring the short interval (1-3 minutes), and at the levelsof GK activity measured the concentration of 6-phosphogluconate was not sufficient to activate measurably the6-phosphogluconate dehydrogenase. 6-Phosphogluconatedehydrogenase was present in tissues at a relatively lowlevel, but even if purified enzyme was added to the GKassay system, the rate remained unchanged owing toinsufficient substrate concentration. Only if the systemwas run over an extended period of time did the 6-phosphogluconate dehydrogenase activity become noticeable,and in experiments in which the measurements werecarried out for 20 minutes it was found that up to 1.5moles of NADP+ were reduced per mole of glucose phos-phorylated.

Recent reports by DiPietro et al. (10, 11) have shownthat rat liver glucokinase behaves anomalously in that itrequires an unusually high concentration of glucose formaximal activity. Studies by these investigators and bythe authors of this paper indicate that it is unlikely thatglucokinase in tissues other than rat liver requires suchhigh levels of glucose, but obviously this will have to bestudied further, particularly with respect to human liver.More recently Vinuela, Salas, and Sols (35) have separatedtwo glucokinases from rat liver, the one resembling theglucokinase of other tissues in respect to affinity to glucoseand the other requiring the unusually high levels of glucosereported by DiPietro et al. (10, 11).

With respect to the assay system employed in thecurrent paper, the glucose concentration should besufficiently high to measure both glucokinases in rat liverand if they exist in other tissues.

Glucose-6-phosphate dehydrogenase (9, 17).â€”Triethanol-

amine, 50 mat; EDTA 5 mai; MgCl2, 10 mat; NADP+,0.36 niM; tissue, 5-10 mg.; G-6-P, 3 mat.

In the short interval in which this assay was run, tissue6-phosphogluconate dehydrogenase did not add to thereduction of NADP+ (see glucokinase).

6-Phosphogluconate dehydrogenase.â€”-Ibid,for glucose-6-phosphate dehydrogenase, except that 6-PGA was substituted for G-6-P.

Phosphoglucomutaseâ€”Triethanolamine, 50 niM; EDTA,5 mai; MgCl2, 14 mat; NADP+, 0.36 mat; glucose-6-phosphate dehydrogenase, 4.3 U; tissue sample, 1 mg.;G-l-P, 3 mai.

In tissues such as liver, skeletal and heart muscle, theactivity of this enzyme was high enough to require suchsmall amounts of tissue sample that the levels of endogenous reduction of NADP+ were not significant, but inmany other tissues, particularly malignant tissues,considerably more tissue sample was required so that thelevel of endogenous reduction of NADP+ became appreciable. Consequently, it was necessary to record theabsorbance for about 3 minutes following the addition ofthe tissue sample and prior to the addition of the substrate.

This short preincubation also permitted activation ofthe enzyme by magnesium ions (27), even though theinterval was shorter than that suggested by Robinson andNajjar (28) who indicated that imidazole, histidine, orother metal-binding agents (18) were also required. Noadditional enhancement of activity even after prolongedpreincubation was observed upon the addition of imidazoleinto the reaction mixture without the substrate. Thislack of an effect was probably due to the fact that EDTApresent in both the homogenization medium and the assaybuffer fully activated the enzyme (4). Adequate catalyticamounts of glucose-l,6-diphosphate were apparentlypresent in the reaction mixture.

Lactate dehydrogenase (2, 3).â€”Triethanolamine, 50 mai;EDTA, 5 mat; NADH, 0.3 mat; tissue sample, 1 mg.;pyruvate, 6.2 mat.

In general, the absorbance at 340 m/i was recorded for ashort interval following the addition of the tissue sampleto ascertain the level of endogenous NADH-oxidizingsubstances which might be present hi the tissue sample.Most frequently there was little or no endogenous oxidation of NADH observed with the quantities of tissuerequired to measure lactate dehydrogenase activity, exceptions being rodent heart and kidney homogenates prepared from freshly excised tissues.

The enzymic reaction was initiated by the addition ofthe substrate, pyruvate, and the absorbance was recordedfor 1-3 minutes. This assay system was designed to givemaximal rates with the skeletal muscle type lactatedehydrogenase and therefore may not give the maximallactate dehydrogenase activity in all tissues. Our studiesindicated that most tissues, heart muscle being the outstanding exception, contained a large enough proportionof the muscle type LDH so that the activity measured inthis system represented the characteristic LDH activityof the tissue.

Glycerolphosphate dehydrogenase (2, 3).â€”Triethanol-




amine, 50 ITIM;EDTA 5 mw; NADH, 0.3 HIM; tissuesample, 1-10 mg.; DHAP, 1.2 niM.

The reaction was initiated by the addition of the substrate after first recording the level of endogenous oxidation of NADH. Tissues in which the enzyme activitywas high so that only small amounts were requiredgenerally showed no appreciable rates, with the exceptionof rodent heart and kidney. In the cases where glycerol-phosphate dehydrogenase activity was low and largeramounts of tissue were employed, endogenous oxidation ofNADH was sometimes appreciable.

Since the substrate was labile and also was not directlyweighed but had to be converted from a derivative, itsconcentration had to be measured before it was used in theenzyme assay system. This was rapidly accomplished bythe addition of small increments of the diluted substrateto an assay system consisting of triethanolamine, 50 mM;EDTA, 5 mM; a-glycerolphosphate dehydrogenase, 7.1 U;and NADH, 0.3 mM.

Emmelot and Bos (12) reported that potassium cyanideat a concentration of 5 X 10~4 M resulted in a two- to

fivefold increase in the a-glycerolphosphate dehydrogenaseactivity of rat liver and also resulted in the appearance ofsubstantial a-glycerolphosphate dehydrogenase activityin rat hepatomas in which this enzyme was not detectablein the absence of this compound. This effect was neverobserved with DHAP as the substrate. A recent observation of Pette and Ruge (24) demonstrated that thisapparent stimulation was due to an experimental artifactwhen mixed triÃ³se phosphate esters were used as thesubstrate.

Aldolase (2, 9, 25).â€”Triethanolamine, 50 mM; EDTA,5 mM; NADH, 0.3 mM; a-glycerolphosphate dehydro-genase-triosephosphate isomerase 7.1 and 18.3 U, respectively; tissue sample, 1-10 mg.; FDP, 3 mM.

Because of the relatively low activity of this enzyme,5-10 mg. of tissue were generally required (only 1 mg. isrequired for skeletal muscle). With these amounts oftissue the endogenous rate of oxidation of NADH wasappreciable and had to be measured prior to the additionof FDP in order to obtain the appropriate corrections.In addition, the FDP solution had to be checked for thepresence of triÃ³se phosphates. This was easily accomplished by employing the above assay system without thetissue sample. In order to get valid aldolase measurements, it was essential to use batches of FDP that werelow in triÃ³se phosphate. Since 2 moles of NADH wereoxidized per mole of substrate utilized, the observed ratehad to be divided by two in order to express the measurement in terms of the international unit (see also phos-phof ructokinase).

Triosephosphale isomerase (9).â€”Triethanolamine, 50HIM;EDTA, 5 mM; NADH, 0.3 mM; a-glycerolphosphatedehydrogenase, 7.1 U; tissue, 0.1 mg.; GAP, 0.62 mM.

This enzyme was so active in all tissues and the quantityof tissue employed was so small that endogenous oxidationof NADH was never a problem. It was, however, important that the a-glycerolphosphate dehydrogenase used asan ancillary enzyme was free of triosephosphate isomeraseactivity. This could be readily ascertained by recording

the rate of change of absorbance at 340 m/i in the assaysystem in the absence of the tissue sample.

Triosephosphate isomerase activity always had to bemeasured at the same substrate concentration, becausethis was the only enzyme in this series of enzymes whichwas not measured at saturating substrate concentration,since it was difficult to prepare sufficiently concentratedGAP solution to saturate the enzyme. The substrateconcentration could be determined in this system bymeasuring the total change in absorbance on the additionof small increments of the diluted substrate solution to anassay system similar to the above but supplemented with18 U of triosephosphate isomerase. The rate observedwith the indicated concentration of GAP was reportedthroughout the tables and was about 65 per cent of themaximal rate.

Phosphoglycerate mutase.â€”This enzyme was measuredin both directions.

Assay system for forward direction: Triethanolamine,50 niM; EDTA, 5 mM; MgCl2, 10 mM; NADH, 0.3 mM;lactate dehydrogenase, 9.1 U; pyruvate kinase, 16 U;enolase, 2.6 U; sample, 2 mg.; ADP, 2.7 HIM; 3-PGA,2.7 niM.

Assay system in reverse direction: Triethanolamine, 50niM; EDTA, 5 mM; MgCl2, 10 mow; NADH, 0.3 HIM;glyceraldehyde phosphate dehydrogenase, 4.3 U; phos-phoglycerate kinase, 7.1 U; cysteine, 2.7 mM; tissue, 2mg.; 2-PGA, 2.7 mM; ATP, 2.7 mM.

In both cases it was essential that the substrates andenzymes were relatively free of related substances whichinterfered in the enzymic assay. This could be checkedquickly by measurements without the tissue samples.The reaction in the reverse direction was sensitive toMg++ concentration. Addition of 2,3-diphosphoglyceratedid not change the rate of the reaction, catalytic quantitiesbeing apparently present in the ancillary enzymes.

Phosphoglucoisomerase (9).â€”Triethanolamine, 00 m.\i;EDTA, 5 IHM;MgCl2, 10 mM; NADP+, 0.36 HIM;glucose-6-phosphate dehydrogenase, 4.3 U; tissue, 0.1 mg.;F-6-P, 3 HIM.

Since all samples of F-6-P contained some G-6-P, it wasessential to measure the latter by employing the aboveassay system in the absence of tissue. In general, withthe assay system indicated all G-6-P in F-6-P was consumed in the first 20-30 seconds of the reaction, and therate then became linear and representative of the phos-phoglucoisomerase activity.

Malate dehydrogenase.â€”Christie and Judah (5) andDelbruck et al. (8) showed that this enzyme existed inboth the mitochondrial and extra-mitochondrial compartments of the cell and that these are two distinct enzymes(7, 13, 33, 34, 38). It has now been observed that onlypart of the malate dehydrogenase activity is inactivatedin 50 per cent ethanol at room temperature. Studies withcentrifugally separated fractions of rat liver indicated thatthe malate dehydrogenase activity which is inactivatedby ethanol is primarily extra-mitochondrial and that theenzyme derived from the mitochondrial fraction is resistantto ethanol. A small part of the malate dehydrogenaseactivity of isolated mitochondria proved, however, to beunstable in ethanol. At this time it is not certain whether




this represents contamination of the mitochondria withcytoplasmic malate dehydrogenase or whether a portionof the mitochondrial MDH is intrinsically unstable inethanol. In the tables the ethanol-unstable malatedehydrogenase is recorded as cytoplasmic malate dehydrogenase (extra-mitochondrial malate dehydrogenase) andthe ethanol-stable one as mitochondrial malate dehydrogenase (mitochondrial malate dehydrogenase). Detailson the differential stability of the two MDH's in mixed

solvents will be published separately.Assay system for total malate dehydrogenase activity:

Triethanolamine, 50 HIM;EDTA, 5 mm; NADH, 0.3 nut;tissue, 1 mg.; oxalacetate, 0.31 mm.

Although only freshly prepared oxalacetate solutionswere used, it was necessary to measure the pyruvate content of this substrate by adding it to a reaction mixtureconsisting of buffer, NADH and lactate dehydrogenase.

Ethanol-resistant malate dehydrogenase.â€”The assaysystem was the same, except that the reaction mixture was2.7 M in ethanol. This assay was run in the followingmanner: The tissue sample was added to a solutionconsisting of 0.5 ml. ethanol and 0.5 ml. 0.05 Mtriethanol-amine buffer, pH 7.6, and permitted to stand at roomtemperature (24Â°C.)for 3 minutes, after which 2 ml. of

triethanolamine buffer was added. The assay was thencarried out by the addition of NADH followed by oxalacetate. The time required for inactivation was notcritical; identical results were obtained when the time wasextended to 15 minutes.

Fructosediphosphatase.â€”Triethanolamine, 50niM;EDTA,5 niM; MgCl2, 10 nui; NADP+, 0.36 nui; glucose-6-phosphate dehydrogenase, 4.3 U; tissue sample, 5-10 mg. ;FDP, 0.3 nui.

Since nearly all samples of FDP contain some F-6-P,this had to be measured, and only the FDP samples lowin F-6-P content were used in the assay.

Adenosine triphosphatase.â€”Triethanolamine, 50 HIM;EDTA, 5 nui; MgCl2, 10 nui; NADH, 0.3 nui; pyruvatekinase, 16 U; lactate dehydrogenase, 9.1 U; PEP, 2.8 U,tissue, 5 mg. ; ATP, 2.8 niM.

It was essential to consume all pyruvate present in thePEP before proceeding further with the assay (see assayfor pyruvate kinase). This measurement was employedto determine the feasibility of determining glucokinase bycoupling to pyruvate kinase (see assay for glucokinase),but was not intended to be a measurement of either thetotal ATPase or any individual ATPase in the tissue.

APPLICATIONOF ASSAYPROCEDURESTO TISSUES

From a practical point of view determinations ofglycolytic enzymes in human tissues will depend to a greatextent on their stability at room temperature for a shorttime following surgical removal and to storage in thedeepfreeze for several days. The conditions illustrated inTables 1-3 were designed to establish the permissiblelimits of collection and storage on the model system of rattissues. Table 1 presents data showing the stability ofselected enzymes in rat liver kept at room temperature(24Â°C.). Included in this selection are some of the more

labile enzymes.Surprisingly, none of these enzyme activities decrease

TABLE 1EFFECT OF STORAGEOF RAT LIVER AT 24Â°C.

ExZYUE

ENZYMEACTIVITYop RATLIVEÂ»STOKEDINTACT AT 24Â°C. FOR:

a-Glycerolphosphate dehydrogenaseGlyceraldehyde

phosphate dehydrogenaseLactatedehydrogenasePhosphoglyceratekinasePyruvate

kinase0.5

hrs.827017894231.5hrs.775716679262.5hrs.836117691254.5hrs.835818591256.5hrs.796916723

Enzyme activity is expressed as U/gm wet weight of tissue.Two rat livers were cut up in small pieces and pooled. An attempt was made to select portions for study randomly.

TABLE 2ENZYMEACTIVITIESOF PORTIONSOF SOMERAT LIVER STORED

FROZENAT -20Â°C.

ENZYME

Glyceraldehyde phosphate dehydrogenaseLactatedehydrogenasea-GlycerolphosphatedehydrogenaseAldolasePhosphoglucoisomerasePhosphoglycerate

kinasePyruvatekinase18

hrs.62118426.5528919186hrs.65106595.5587915

ACTIVITYOF RATLIVERSTOREDFROZENAT â€”20Â°C.FOR:

Activities are expressed as U/gm wet weight of tissue.

TABLE 3ENZYMEACTIVITIESAFTERSTORAGEOF 30,000 X g RAT LIVER

SUPERNATANTFRACTIONS

Lactatedehydrogenasea-Glycerolphosphatedehydro

genasePyruvatekinaseGlyceraldehyde

phosphate dehydrogenaseINITIALACTIVITY136551959ACTIVITY

AFTER18HRS.At

4"C.12944417At-20Â°C.131451227

Enzyme activities are expressed as U/gm wet weight of tissue.

when relatively large pieces of rat liver were kept at roomtemperature for the indicated times. Table 2 indicatesthe stability of some of the more labile glycolytic enzymesin 1-gm. pieces of rat liver stored at â€”20Â°C.for more than

a week.Homogenates of tissues or their supernatant fluids

cannot be stored for any appreciable length of time eitherat ice-bath temperature or â€”20Â°C. This is illustrated in

Table 3 by the substantial loss of glyceraldehyde phosphate dehydrogenase and pyruvate kinase activity on




TABLE 4

COMPARISONOPSOMEENZYMEACTIVITIESIN RATLIVERSOFANIMALSOFA SINGLEGROUPWITHTHOSEOF A CUMULATIVEGROUP

ENZYMEAldolaseGlyceraldehyde

phosphatedehydrogenaseTotal

malatedehydrogenaseMitochondrialmalatedehydrogenaseExtra-mitochondrial

malateSINGLE

GROUT(Â»*â€¢â€¢5)Activity(U/gm)4.954309114195StandarddeviationfÂ±0.6Â±6.8Â±21Â±10Â±20Coefficientof

variation^6.112.66.88.829CUMULATIVE

GSODP (Â«â€”12)Activity(U/gm)5.863388129258StandarddeviationÂ±2.9Â±8.2Â±119Â±25.6Â±105Coefficientof

variation50.51330.719.840.7

* TI= the number of determinations.x = the mean or average.x = the individual determinations.

t The standard deviation, a, = - *>'

_

ÃŽCoefficient of variation = - X 100.x

storage for 18 hours. The order of enzyme assays suggested were based on these and similar data. The instability of a number of glycolytic enzymes in homogenatesbut not whole pieces of tissue raised the question as to thesmallest blocks of tissue that could be stored safely atâ€”20Â°C.Investigations on blocks of tissue of varying

sizes indicated that pieces of the order of 100 mg. can besafely stored for long periods, but smaller pieces, 40-100mg., must be worked up without prolonged storage.Measurements were also made on very small pieces oftissue (5-20 mg.), frozen at a controlled rate in media usedfor the preservation of tissues for cell culture. Invariably,extensive losses of glycolytic enzyme activities occurredunder these conditions.

The evaluation of any data on enzyme activities bringsup the question as to the precision of the measurements.The answer is complex because of the large number andkinds of enzymes and tissues involved. In a comparisonof duplicate determinations of the activities of eighteendifferent crystallized enzymes the coefficients of variationaveraged 1.6 per cent, with a standard error of the meanof the coefficients of variation equal to 0.16 per cent.In a similar series of duplicate measurements on tendifferent enzymes involving four human and two rattissues, the coefficients of variation of all the measurements averaged 1.2 per cent, with a standard error of themean equal to 0.13 per cent of the coefficients of variation.In a series of 84 duplicate determinations of aldolase in avariety of human tissues of both malignant and non-malignant origin in which the aldolase activities variedbetween 1 and 80 U/gm of tissue, the coefficient of variation was 1.3 per cent throughout, with a standard error ofthe mean of the coefficient of variation equal to 0.16 percent. These results indicate that the precision of theenzyme activity measurements were similar whetherapplied to highly purified enzymes or to crude homogenates. They also indicate that the same precision wasobserved over a wide range of absolute enzyme activities.

TABLE 5AVERAGEPATTERNIN NORMALRAT LIVERS

EnzymeGlucokinasePhosphofructokinaseAldolaseGlyceraldehyde

phosphatedehydrogenasePhosphoglycerate

kinaseEnolasePyruvate

kinaseLactatedehydrogenasePhosphoglucomutaseGlucose-6-phosphate

dehydrogenasea-Glycerolphosphate

dehydrogenaseTriosephosphateisomeraseFructose-1,6-diphosphataseTotal

malate dehydrogenaseMitochondrial

malate dehydrogenaseExtra-mitochondrial

malatedehydrogenaseH12121212121212126121277121212Activity

(U/gm)1.22.05.863759.623.6162192.565.14763.6388129258StandarddeviationÂ±0.75Â±0.95Â±2.9Â±8.2Â±14Â±3.5Â±5.7Â±74Â±1.1Â±1.6Â±13.5Â±193Â±1.8Â±119Â±25.6Â±105Coefficientvariation62.547.350.51318.636.524.245.65.96420.740.550.530.819.840.6Standard

error ofmean*Â±0.22Â±0.28Â±0.84Â±2.36Â±0.40Â±1.0Â±1.65Â±21.30.450.46Â±3.9Â±73Â±0.69Â±34.4Â±7.4Â±30.2

* The standard error of the mean VÂ«'

These data are, however, concerned only with the precision of the enzymic assay and do not have a directbearing on the variations involved in the selection of thetissue sample.

Tables 4 and 5 are concerned with the variations to beexpected from the selection of the tissue sample. Table4 compares the values of four selected enzymes measured




in the livers of five rats killed at the same time (singlegroup) with values for the enzymes measured on liversof rats killed at different times over the period of about 1year (cumulative group). The coefficient of variation foraldolase in the single group was considerably less than inthe cumulative group. A similar though less pronounceddifference was observed for malate dehydrogenase. Incontrast, the coefficient of variation for glyceraldehydephosphate dehydrogenase was the same for both groups.

The small variation of glyceraldehyde phosphatedehydrogenase is even more apparent if the larger selectionof glycolytic enzymes in Table 5 is considered, whichagain were obtained on livers of animals that were killedat various times over a period of about 1 year. In comparing enzyme activities of the glycolytic chain it is usefulto select one enzyme as the reference standard (32).Glyceraldehyde phosphate dehydrogenase serves thispurpose best from an analytical point of view and alsobecause it is ubiquitous in distribution. Phospho-glucomutase could not be used, since it is essentiallyrestricted to a few tissues such as liver, skeletal muscle,and heart.

The enzyme pattern for the whole series of enzymesdiscussed is given for seven rat tissues in Table 6. Gluco-kinase in all these tissues shows the lowest activity of thestraight glycolytic pathway and, therefore, is not only thefirst but also potentially the rate-limiting step. Brainhas the highest glucokinase activity, which is in accordwith glucose as primary energy source for this tissue. Insurprising contrast is the low glucokinase activity ofskeletal muscle, which is even more pronounced if thevery high activities of the other glycolytic enzymes and,in particular, phosphofructokinase are considered. Studyof this limiting glucokinase potential in the organ thatreputedly derives such large parts of its energy fromcarbohydrate metabolism deserves further metabolicinvestigations.

Although data on enzyme activities cannot by themselves be used as a measure of metabolite flow throughalternate pathways, they do provide information of thelimiting potential of competing pathways. For example,this applies as far as the data in Table 6 are concerned inconsideration of the metabolite flow through the Embden-Meyerhof path versus the oxidative shunt. In thisrespect the ratio of phosphofructokinase to glucose-6-phosphate dehydrogenase will be of major significance,since it is unlikely that phosphoglucoisomerase, which ispresent in activities at least one order of magnitude higherthan phosphofructokinase or glucose-6-phosphate dehydrogenase, can influence the direction of metabolite flow. Inboth heart and skeletal muscle, and probably also inbrain, the activity of glucose-6-phosphate dehydrogenaseseems too low compared with that of phosphofructokinaseto permit a major part of the carbohydrate metabolism toflow through the shunt. On the other hand, in kidneyand adipose tissue the two activities are of the sameorder of magnitude, so that other factors such as theavailability of ATP and the rate of reoxidation of NADPHcould have a predominant influence on the direction ofmetabolite flow. The activities measured in spleen aremore difficult to interpret, since the high blood content of

this organ tends to distort the enzyme pattern, as will bediscussed in more detail in a subsequent paper (32).

An alternate pathway for G-6-P is also provided byphosphoglucomutase. In all the tissues listed in Table 6this enzyme is, at least potentially, active enough tocompete for the G-6-P. As far as gluconeogenesis andglycogenesis is concerned, not only phosphoglucomutase isof importance but also the relative activities of phosphofructokinase and fructose diphosphatase. In liver andkidney the activity of fructose diphosphatase is sufficientto permit reversal of this step, whereas in heart and skeletalmuscle and in brain fructose diphosphatase activity issubstantially lower than the phosphofructokinase activity.

DEPENDENCEOF ENZYMEACTIVITIESON pHAll enzyme activities in this and subsequent papers1

(32) were measured at pH 7.6 and are consequentlydirectly comparable to one another. Since many valuesin the literature were obtained at other pH's it seemed

desirable to consider the effects of pH on the activities ofthese enzymes, keeping other conditions constant. InTable 7 the activities of the commercially availablecrystallized enzymes were measured at 24Â°C.in 0.05 M

triethanolamine buffer containing 0.006 M EDTA andsufficient HC1 to provide the desired pH value. Theactivities are expressed as per cent of the activity at pH7.6, which is given in units (U) per mg. of protein. Theoptimal substrate concentration at pH 7.6 was employedover the pH range studied. No attempt was made atthis time to determine whether changes in pH resulted inalterations in the optimal substrate, coenzyme, or cofactorrequirements.

Since phosphofructokinase is the only glycolytic enzyme not available in crystalline form, it is missing fromthe compilation; however, the activities of this enzymein a crude homogenate of human skeletal muscle wasstudied at several hydrogen ion concentrations, and theseresults are included as a footnote to Table 7.

The data in Table 7 indicate that, except for the heartmuscle-type lactate dehydrogenase, the enzyme activitiesmeasured at pH 7.6 were not far removed from the maximally observed activities, usually within 20 per cent. Inaddition to the heart-type lactate dehydrogenase, phosphoglucomutase and phosphoglucoisomerase were outside thislimit, the maximal activity being 40 and 30 per cent, respectively, higher than at pH 7.6. Studies with crude homog-enates of human tissues have shown that the responses ofhuman liver and skeletal muscle lactate dehydrogenase tochanges in pH were similar to that observed with rabbitskeletal muscle lactate dehydrogenase, whereas theresponse of human heart muscle lactate dehydrogenaseresembled the pattern recorded for bovine heart musclelactate dehydrogenase.

The data as recorded in Table 7 should permit theiruse as factors for the translation of enzyme activitiesmeasured at pH 7.6 to other pH values. This depends onthe assumption that enzymes in tissue homogenatesexhibit the same response to changes in pH as the crystallized enzymes included in Table 7. Additional studiesemploying crude human and rat tissue homogenates asmodel systems indicate that this is a reasonable assump-



718 CÃ¡ncerResearch Vol. 24, May 1964

a

Â£B1O.Lfl"ua4)"31v*Ã¼9ae

sK'Spa|2SS!sICJ-HCN1-H0co.CO

rfO-H1-HcoCDCNd-tÃ¬OS0S1IOrjiO-H>o"*>o1-HCN-tÃ¬1-Hif5Ã•o-HÅ“coc71-HSd-tÃ¬CN1-HGlucokinaseCO,co

coo-HCNCO1-Hg1-H^S1-HIO

91-Hoi-Ho!SCNi-HPhosphoglucoisomeraseSoo-H_

,.c0sS-0o-Ho>

ci^~.,_*if^yoco^JÂ»IOdÃ²O-tÃ¬"C**IOoÂ»1-Hâ€¢HIO^HTNi-H-HO)cic7Â»â€¢Hgo-tÃoci9sCC

â€¢frj*cfI0oâ€¢Hâ€”ot^C-Io-tÃ¬IMcipi'o>U9-H1-HU3o<Ni-H-tÃ¬o1-HTCO0-H>o00g.1â€”1Â«5ci-HocoCisoâ€¢tÃ00â€¢0AldolaseCiSâ€¢tÃ1CÃ¯oof-H-tÃ¬gCNCNC-HC0,-HSâ€¢*coC4CO-H00IO^Â£>â€¢coâ€¢tÃcoâ€¢*sTriosephosphate

isomeraigceo-Hepoet.ÎO-tÃ¬ci^T-HCOCO-H3>oco^-Hg5â€¢o0â€¢tÃ00o1â€”1IOo-tÃ¬scoÂ«ciâ€¢tÃÂ§X"3"CDGlyceraldehydephosphat

drogenaseco.e.o-HIO1â€”1cooco-tÃ¬oIOoco>-HÂ§1-H^^*uj-HgcoIOCO-Hco

Â»^î*CNcoâ€¢HcSs*Hooâ€¢tÃ¬IO(SBPhosphoglycerate

kinaseco.(o-tÃcoci1â€”1"Â»HS1-HSoC-Ioco-HSo1-HscooCN-HcoPhosphoglyceratemutaseQ-1-HOâ€¢HP^coe;CNCNci-tÃ1-H

|>.lÂ£ooi-H-HSC?SSci-Hocort-H"2,cocoâ€¢tÃ1

-c71-^tâ€”t-HCOCÂ»Enolase52-SS0-H^cos-HS-rgâ€¢tÃÂ£corHCO-Hgo1-HCOCOo-tÃ¬oo1-Hco"1-Hco-Hco-Ã‘"â€”

Hco1-Ã•-tÃc^Pyruvate

kinase00-HSoo-tÃCNCOcosâ€¢tÃlooC<1"T'-tÃ¬COcoco-tÃ¬sCNcoIOIO-tÃ¬sco,_,co-H09

00IM1-Hcoe*â€¢HCi^^Lactate

dehydrogenaseGco0â€¢tÃ!_,coC-I".0Â¿-HIO^-1â€”

trÂ»uÃ â€¢HoÂ»Ã•Ã•?t*.rJ-tÃ¬i-HÂ»M3oâ€¢Â«o*â€¢"^Ã•,ci-HÂ«S

CNc?^HOÂ»co-tÃ¬Â§o"3^r*;a-Glycerolphosphate

de

genÃ¤se52-8C-H(Mr-HOCN^HO-tÃo1-HJjjCOcoâ€¢tÃScoIOo-tÃ¬^**CNeoâ€¢tÃâ„¢coso-H1â€¢*toU5e-HO>1-HPhosphoglucomutasecoet0-tÃ¬00oM***0Vôoâ€¢tÃtÂ»QCN3o-HÂ°Ã¬1-H-^80-Hâ€¢*co_t*Â»o

co0-HCOco92Od1

p.1

o,â€¢3ciâ€¢Â«a

Osklr-<1-HCOo-Hâ€¢9o0_<co

co0-i0VcotÂ»o-Ht-:ocoS3o-HCO"Â¡2coo-tÃ¬frw*HCÃŽ1-H<Oi-Hâ€¢tÃ¬â€¢oci2â€¢e>,-CGlueose-6-phosphate

de

genÃ¤sec7^H8oâ€¢Hi-Â¡^ôrÂ¿~^rt6-phosphogluconate

del

genÃ¤se{O,00o-H^^S00ciâ€¢tÃ-fTflâ€¢o^-Hs00SJ-HS?0

1-HSg-tÃ¬scooo.o-H0C<lco-H08coCDESTotal

malatedehydrogemJO,So-H1-Hciso1-Hâ€¢tÃ3wÂ£^^1HCOâ€¢HÂ§i-HSi-H-H0seÂ»â€¢*-H1-HcoSÂ»oui-HSeSi-H*^-HSÂ»â€”

*Ã³jTjS_tÃMitochondrial

malatedel

genÃ¤seSOÃo-H1-HoÃ¢sOS1â€”

t-HÂ«T}4Ci'(Pâ€¢tÃSooc^-H000c?s-HÃœs1-1â€¢tÃ009CNs-H00Â¿T3Jâ€¢sExtra-mitochondrial

mali

hydrogenase




TABLE 7RELATIVEACTIVITYOF CRYSTALLIZEDGLYCOLYTICENZYMESAT DIFFERENTHYDROGENION

CONCENTRATIONS

ENZYIIEGlucokinasePhosphoglucoisomeraseAldolaseTriosephosphate

isomeraseGlyceraldehydephosphatede-hydrogenase*Glyceraldehyde

phosphatede-hydrogenasefPhosphoglycerate

kinasePhosphoglyceratemutasePhosphoglyceratemutaseÂ§EnolasePyruvate

kinaseLactÃ¤tedehydrogenaseLactate

dehydrogenasea-Glycerolphosphatede-hydrogenaaePhosphoglucomutaseGlucose-6-phosphate

dehydrogenaseMalate

dehydrogenaseSOUBCEYeastYeastRabbit

skeletalmuscleRabbitskeletalmuscleRabbitskeletalmuscleRabbit

skeletalmuscleYeastRabbit

skeletalmuscleRabbitskeletalmuscleRabbitskeletalmuscleRabbitskeletalmuscleRabbitskeletalmuscleBovineheartmuscleRabbitskeletalmuscleRabbit

skeletalmuscleYeastPig

heart muscleACTIVITY

ATpa7.6(U/MGPROTEIN)1263558.23664444141542125132036510314364171354RELATIVE

ACTIVITYATpH6.8192687716832527695867072439.511246627.2516188847472701031171239486542113972717.71001001001001001001001001001001001001001001001001008.0118119901011021081038853539110313689661041008.41171339494107118998639438189172â€”381101158.81131329410082118997536387475202653611270

* Measured in the direction, GAP -> 1,3-DPGA.t Measured in the direction, 1,3-DPGA -Â»GAP.ÃŽMeasured in the direction, 3-PGA -> 2-PGA.Â§Measured in the direction, 2-PGA â€”Â»3-PGA. Phosphofructokinase in a crude homogenate of

human skeletal muscle showed an activity of 23 U/g at pH 7.6; the activity of this preparation atother pH values expressed as per cent of the activity at pH 7.6 was as follows: pH 6.8, 6; pH 7.2, 11;pH 7.3, 37; pH 7.4, 50; pH 7.5, 88; pH 7.6, 100; pH 8.0, 132; pH 8.4, 145; pH 8.8, 150; pH 9.1, 147.

tion, and a more detailed account of these studies will bepresented in another paper.

DISCUSSION

Since the prospective aim of this study is to investigatethe enzyme patterns in human tissues, the patternsobtained with rodent tissues represent model systems, andthe handling of the rodent tissues was designed to simulatethe anticipated handling of human tissues. Since it isimprobable that freshly excised human tissue will bereadily available, most of the studies reported in thispaper were made on tissues which had been stored in thefrozen state at â€”20Â°C. Fortunately, the glycolyticenzymesâ€”even the reputedly labile glyceraldehyde phosphate dehydrogenaseâ€”were found to be stable in thefrozen intact tissues.

There are limitations on the size of the pieces of intacttissue that can be stored safely at â€”20Â°C.which vary

somewhat between different organs and different species,but pieces of 100 mg. or over could be safely stored in allcases. The reason for the instability of enzymes in smallpieces, and even more markedly in homogenates, isprobably due to availability or activation of proteolyticenzymes that are released from injured cells.

Conditions that are most suitable for storage of tissueprior to cell culture proved to be totally unsuited as far as

preservation of glycolytic enzymes were concerned. Notonly did these enzymes leak from the small tissue piecesinto the freezing medium, but, since some of the enzymeswere inactivated in the freezing fluid, highly distortedenzyme patterns were obtained.

Although some studies were performed on mouse andhamster tissues, these data were not included in thispaper because for the most part the situation was similar.The only exception was the greater lability of glyceraldehyde phosphate dehydrogenase in mouse tissues, becausein the storage of small pieces of mouse tissues for 83 daysat â€”20Â°C. it was observed that the glyceraldehyde

phosphate dehydrogenase activities were only 10 per centof that found in the corresponding fresh tissue, eventhough the activities of all the other enzymes were similarto those observed in the fresh tissues. In contrast, fullglyceraldehyde phosphate dehydrogenase activity appeared to be maintained in human liver stored for 1 yearat â€”20Â°C. Selective instability of single enzymes for

known or unknown reasons is an ever present possibility,particularly if pathologically altered tissues are understudy. This is an additional reason why patterns ofrelated enzymes rather than single enzymes should bemeasured.

The baseline for expressing enzyme activities hasalways been a difficult problem. Any number of reason-




able baselines can be chosen if activities in a single organof a given species are to be compared, but there is noreasonable choice if comparisons between different organsor a given tissue and its malignant counterpart are to bemade. For example, cellularity, as measured by DNAcontent or cell count, does not necessarily correlate withmetabolic activity if liver and muscle are compared.Similarly, protein content has no correlation in comparingmetabolic potentialsâ€”for example, between liver andconnective or adipose tissue. It is, of course, equallytrue that wet weight is not correlated to metabolic function, but it has the decided advantage of ease and precisionof measurement.

Comparison of enzyme activities with data in theliterature always poses a series of problems. Enzymeactivities, being reaction rates, are extremely dependenton the conditions under which the measurements aremade. Consequently, direct comparisons of enzymeactivities are valid only if the conditions employed wereidentical. Unfortunately, this appears to be an unattainable ideal and only approximate comparisons canbe made with studies done under nearly identical conditions. In recent years a number of studies have appearedwith which some of the data discussed in this paper maybe compared, since they were obtained by similar methodsâ€”though not necessarily in the same species or the sametissues. Wherever a direct comparison can be made withthe studies of BÃ¼cher'sgroup (8, 9, 20-23), with those ofSchmidt et al. (29-30, 31) and to some extent with those ofAebi's group (14), the values reported here are in reasonably

quantitative agreement. These workers all employedtriethanolamine buffer containing EDTA at pH's between

7.5 and 7.6. Comparisons can probably be made withthe enzyme activities reported by Wu and Racker (39),even though these investigators employed trishydroxy-aminomethane as the buffer, for in a limited study ofseveral glycolytic enzymes it was observed that theenzyme activities were similar in trishydroxyamino-methane and triethanolamine buffers. Where the sameenzymes were reported the data are also in good agreementwith the studies of Fitch and Chaikoff (15, 16), who usedglycylglycine buffer of pH 7.6. In all these instancescationic buffers had been used which influence enzymeactivities much less than anionic buffers. It is doubtfulthat valid comparisons can be made with studies whereanionic buffers had been employed.

All the tissues discussed in this paper were obtainedfrom well fed, apparently healthy animals. Examinationof the enzyme activities condensed in Tables 5 and 6 showthat in any given tissue glucokinase, phosphofructokinase,glucose-6-phosphate dehydrogenase, fructose diphospha-tase, and a-glycerolphosphate dehydrogenase, which mediate entrance into or diversion from the main glycolyticpathway, display greater variability in activity than mostof the other enzymes. Since these enzymes representpossible sites of metabolic control, the observed variabilities may reflect the physiological state of the animal,and a careful study of this aspect is being done.

The existence of several enzymes in multiple formsâ€”isozymesâ€”is well documented. Since the individualisozymes can have different requirements of optimal

substrate concentration, the possibility exists that some ofthe observed variations in activities may be due to differences in isozyme content in different organs and underdifferent physiological conditions. This perhaps mightexplain the rather large variations observed with lactatedehydrogenase. The strikingly small variations observedin the activities of glyceraldehyde phosphate dehydrogenase, on the other hand, may be an indication that thisenzyme exists primarily as a single entity.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the skilled technicalassistance of Mrs. Adele Wells.

REFERENCES

1. AISENBEBG,A. C. The Glycolysis and Respiration of Tumors.New York: Academic Press, 1961.

2. BEISENHERZ, G.; BOLTZE, H. J.; BÃ›CHER,TH.; CZOK, R.;GARBADE, K-H.; MEYER-ARENDT, E.; UND PFLEIDERER, G.Diphosphofructose-Aldolase, Phosphoglyceraldehyde-Dehy-drogenase, Milchsaure-Dehydrogenase, Glycero-phosphateDehydrogenase und Pyruvate-Kinase aus Kaninchenmuskulatur in einem Arbeitsgang. Z. Naturforsch., 8b:555-77,1953.

3. BOXER, G. E., ANDSHONK,C. E. Low Levels of Soluble DPN-linked a-Glycerophosphate Dehydrogenase in Tumors. CancerRes., 20:85-91, 1960.

4. BROWN, D. H. Action of Phosphoglucomutase on o-Glucos-amine-6-Phosphate. J. Biol. Chem., 204:877-89, 1953.

5. CHRISTIE, G. S., ANDJUDAH, J. D. Intracellular Distributionof Enzymes. Proc. Royal Soc., B141:420-33, 1953.

6. CORI, G. T.; SLEIN, M. W.; AND CORI, C. F. CrystallineD-Glyceraldehyde-3-Phosphate Dehydrogenase from RabbitMuscle. J. Biol. Chem., 173:605-18, 1948.

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1964;24:709-721. Cancer Res Carl E. Shonk and George E. Boxer Determination of Glycolytic EnzymesEnzyme Patterns in Human Tissues. I. Methods for the

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