[CANCER RESEARCH 35, 2109-2116, August 1975]

SUMMARY

Multiple molecular forms of glucose-6-phosphate dehydrogenase (G6PD) in normal, preneoplastic, and neoplasticmammary tissues were separated by polyacrylamide gelelectrophoresis and identified by specific staining for enzyme activity. Mammary tissue from lactating BALB/cmice showed considerable amounts (up to 50%) of aslower-migrating G6PD species, G6PD-III, which wasessentially absent from glands of pregnant mice, preneoplastic nodules, and mammary carcinomas. All tissuespossessed a faster-migrating species, G6PD-II, which accounted for up to 85% of the total G6PD in the glands ofpregnant mice. A third species, G6PD-I, migrating morerapidly than G6PD-II, was found in both abnormal tissues(preneoplastic and neoplastic) and accounted for up to 35%of the total enzymatic activity. G6PD-I was present inmoderate amounts (< 15%) in glands from pregnant miceand was essentially absent from the lactating gland (‘@@-5%).The addition of dithiothreitol did not alter the measurableG6PD activity but did increase the relative activity ofG6PD-II or G6PD-I, asjudged by the intensity ofthe bandson the gels. Mild oxidation (stirring overnight at 4°in air)resulted in a loss of G6PD activity, but preparations hadgreater amounts of G6PD-III; presence of dithiothreitolduring aeration partially prevented loss of G6PD activityand largely prevented the appearance of G6PD-III.Molecular-weight estimations with preparations from lactating mice yielded a value of 118,000 for G6PD-II and260,000 for G6PD-III, suggesting a monomer and dimer,respectively. The addition of nicotinamide adenine dinucleotide phosphate stabilized G6PD activity by preventingheat inactivation at 47°;nicotinamide adenine dinucleotidephosphate did not alter the pattern of species present. Thedata from heat inactivation studies suggest that G6PDIII (dimer) was the more stable species. The addition ofnicotinamide adenine dinucleotide phosphate to samplesafter oxidation in the absence of dithiothreitol (about 70%loss of activity) resulted in no change in patterns and inrecovery of full G6PD activity during heating at 47°. Apotential relationship between glutathione reductase activity and the pattern of G6PD species observed in thevarious tissues is noted.

I Research supported by National Cancer Institute Contract NOl

CB-33880 and by USPHS Grant CA-1736.2 To whom reprint requests should be addressed.

Received February 25, 1975; accepted May 2, 1975.

INTRODUCTION

G6PD,3 the 1st enzyme of the pentose phosphate pathway, plays an important role in the function of themammary gland. As the mammary gland differentiates tolactational stages, G6PD activity increases strikingly, anincrease that correlates with the NADPH-requiring pathways for lipid biosynthesis ( 1). Levy et a!. ( I2, 13) andNevaldine et al. (16, 17) purified G6PD from mammaryglands and have examined many properties of the enzyme,particularly its interactions with NADP@ and NAD@ andinhibition ofenzyme activity by steroids. They reported thatG6PD could be dissociated into 2 inactive subunits; activitywas restored by reassociation of the subunits to form amonomer4 with a molecular weight of@ I 30,000 daltons.Levy (12) proposed that the enzyme exists in 2 monomericforms, which are in rapid, mobile equilibrium with oneanother. Under certain conditions, dimerization of one ofthe monomeric forms can occur. Enzyme activity can thusreside in either a monomeric or dimeric structure, suggesting that multiple molecular forms of the enzyme may existin the mammary gland. Using polyacrylamide gel electrophoresis for protein separation, followed by specific stainingfor G6PD activity, Richards and Hilf (20) reported that 3forms of G6PD occurred in the rat mammary gland. Thebanding pattern was altered at different stages of glandulardifferentiation.

In a previous communication (7), we reported that thepattern of G6PD forms was different in preneoplastic5HAN and neoplastic tissues from BALB/c mice, from thosein normal, pregnant, and lactating mammary gland tissue.As a result, a more extensive study of the multiple

3 The abbreviations used are: G6PD, glucose-6-phosphate dehydrogen

ase (The terms used here for the different species of G6PD are: slowestmigrating, GSPD-III; intermediate migrating, G6PD-II; and fastestmigrating, G6PD-I. This designation is more in accord with the IUPACdefinitions than our earlier designation of G6PD-3, G6PD-2, andG6PD-l.); HAN, hyperplastic alveolar nodules; GR, glutathione reductase; DTT, dithiothreitol; GSSG, oxidized form of glutathione; GSH,reduced form of glutathione.

4 We have adopted the nomenclature of Nevaldine et al. (16), in which

the minimum enzymatically active G6PD is referred to as the monomer,MW.@ 120,000;this is composed of2 enzymatically inactive subunits. Thelarger-molecular-weight species, M.W.@ 240,000, is referred to as thedimer.

5 The term normal is used here in contrast to abnormal; abnormal refers

to the preneoplastic HAN and to carcinomas.

AUGUST 1975 2109

Multiple Molecular Forms of Glucose-6-Phosphate Dehydrogenase inNormal, Preneoplastic, and Neoplastic Mammary Tissues of Mice'

Russell Hilf,2 Regina lckowicz, J. C. Bartley, and S. Abraham

Department of Biochemistry and University of Rochester Cancer Center. University of Rochester School of Medicine and Dentistry, Rochester, NewYork 14642 [R. H., R. I.], and Bruce Lyon Memorial Research Laboratory, Children's Hospital Medical Center of Northern California, Oakland,C'alifornia 94609 EJ. C. B., S. A.]

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R. Hilfet al.

molecular forms of this enzyme was undertaken in normaland abnormal tissues. The results presented here indicatethat (a) the physiological state of the host influences thepattern of G6PD forms, (b) enrichment of the sulfhydrylmilieu alters the patterns seen, and (c) NADP@ affordsconsiderable protection to G6PD against heat inactivation.A possible relationship between GR activity and the patterns of G6PD is proposed.

MATERIALS AND METHODS

HAN were transplanted into isologous mice using thetechnique developed by DeOme et al. (4). Animals weremated and tissues were obtained during the last trimester ofpregnancy (Days 15 to 18) or during postpartum lactation(Days 10 to 15); these represented tissues from pregnant orlactating hosts. Mammary glands from the inguinal region,HAN, and transplanted or spontaneous mammary adenocarcinomas were removed from BALB/c mice, weighed,and quick-frozen in Dry Ice. All tissues were shipped by airin Dry Ice and placed in a freezer at —80°unti 1 assayed.Similar G6PD activities and patterns were obtained onfresh tissues stored at —80°for 1 to 2 months; all determinations were carried out prior to this time.

G6PD Activity and Acrylamide Gel Electrophoresis.Frozen tissues were thawed and homogenates (10 to 33%)were made with ice-cold 0.05 M Tris buffer, pH 7.4. Thehomogenate was centrifuged at 20,000 x g for 20 mm at2—4°;supernatants were used for enzyme assays and for

gel electrophoresis. The total activity of G6PD was determined by following the change in absorbance at 340 nm dueto NADPH production. The conditions of the assayassured zero-order kinetics for at least 5 mm. The methodused was the double-substrate technique of Glock andMcLean (6) in which the blank contained 6-phosphogluconate; G6PD activity was calculated by difference. Oneunit of enzyme activity is defined as 1 ,ttmole NADPHproduced per mm per 100 mg tissue. Specific activity isreported as nmoles NADPH produced per mm per mgprotein.

Multiple molecular forms of G6PD were separated bypolyacrylamide gel electrophoresis, according to themethods described earlier (20), except that a 7% separatinggel was used. G6PD activity was localized on gels by specificstaining and the gels scanned (20). The area under each peakwas calculated by triangulation, and its percentage of thetotal was determined.

Effects of SulfhydrylReagentsand Oxidation. Variousamounts of DTT were added to the supernatant preparations, and the mixture was incubated at room temperaturefor 15 mm prior to enzyme assay or gel electrophoresis. Onthe basis of preliminary studies, a final DTT concentrationof 2 mr@@iwas found to be satisfactory. Some samples weresubjected to oxidation by stirring overnight in air at 4°,inthe presence or absence of 2 mM DTT, and then were usedfor enzyme assay and gel electrophoresis.

Effectsof NADP@on EnzymeActivitiesandElectrophoretic Pattern. The ability of NADP@ to protect the enzymefrom heat inactivation was examined. Supernatant prepara

tions from various tissues were incubated at 47° in theabsence or presence of 2 mM NADP@. Aliquots were removed at 15-mm intervals and were assayed for G6PDactivity and subjected to gel electrophoresis.

GRAssay.Theassayusedwasessentiallythatdescribedby Racker (18). The reaction mixture contained: GSSG, 26mM; NADPH, 0.2 mM; supernatant, 0. 1 ml; and triethanol

amine buffer, 0.1 M, containing bovine serum albumin (10mg/mi), pH 7.6, to a final volume of 3.0 ml. The presence ofEDTA (1.5 mM) did not alter GR activity. The oxidation ofNADPH was followed at 340 nm under conditions thatyielded zero-order kinetics for at least 5 mm; a unit ofactivity represents 1 @moleNADPH oxidized per mm per100 mg tissue. Specific activity is reported as nmolesNADPH oxidized per mm per mg protein.

Chemicals. Trizma base and triethylanolamine werepurchased from Sigma Chemical Co. (St. Louis, Mo.);6-phosphogluconate, trisodium salt, glucose 6-phosphate,disodium salt, NADP, and NADPH were purchased fromGrand Island Biological Co. (Grand Island, N. Y.); glutathione was purchased from Boehringer Mannheim (NewYork, N. Y.), and acrylamide and bisacrylamide for gelelectrophoresis were purchased from Ortec (Oak Ridge,Tenn.). The standards employed for molecular-weight determinations were purchased as a kit from Schwarz/Mann(Orangeburg, N. Y.). All other chemicals used were ofreagent grade quality.

RESULTS

G6PD Patternsin Normal, Preneoplastic,andNeoplasticMammary Tissue. For normal mammary gland, we chosethe gland from the late pregnant mouse (last trimester),since it contains proportionately more epithelial cells thanthe gland from the virgin mouse. Confirming our earlierfindings (7), the pattern seen in the lactating mammarygland was distinct from the other tissues examined (Table1); as much as 40 to 50% of the G6PD activity was found ina slower-migrating species (G6PD-III). This form was notfound in significant quantity in any other tissue examined.The G6PD pattern in mammary tissue from virgin orpregnant mice or in HAN and tumors from virgin mice wascharacterized by 2 faster-migrating forms (Table I). InHAN, there was a small amount of G6PD-III (<5%) andthe presence of 2 faster-migrating species, G6PD-II andG6PD-I. The fastest-migrating species (G6PD-I) represented about 25 to 35% of the total activity in HAN and intumors. G6PD-II, the species migrating with an intermediate rate, represented the major component in these abnormal tissues (usually 60% or more) and frequently appearedas a doublet or triplet. G6PD-II was the predominant formin the gland from late pregnant mice. Thus, preneoplasticand neoplastic tissues demonstrate a greater amount ofG6PD-I than does the normal gland.

An estimation of molecular weight was made on samplesof lactating mammary glands by the method of Zwann (24).The standards used were: bovine serum albumin, alcoholdehydrogenase, catalase, and apoferritin. From these data,we estimated that G6PD-III in the lactating mammary

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PhysiologicalG6PD%

totalforms―FreshOxidizedTissuestateform―Fresh+

DTTOxidized+DTTMammaryglandPregnantIII

III0

85150

831778

2200

7228MammaryglandLactatingIII

III41

5454

91588

12051

445HANVirginIII

III4

64324

306660

231717

4736TumorVirginIII

III6

60345

326376

23118

757

G6PD Forms in Mammary Glands and Tumors

Table I

G6PD multiple molecularforms (influence of DTT and oxidation)

C G6PD-lll, slowest-migrating form; G6PD-II, form migrating with intermediate mobility;

G6PD-I, fastest-migrating form.0 Calculated as given in “Materials and Methods.― Numbers represent averages of 3 to 9 separate

determinations. Numbers for oxidized (overnight stirring) and oxidized in presence of 2 mM DTT areaverage of 2 to 3 separate experiments. The standard error was 10 to 15% of the mean.

gland had a molecular weight of @@.@260,000,whereas thefaster-migrating G6PD-II species had a molecular weightof―@.-ll8,000.

Effects of Sulfhydryl Reagents on G6PD Patterns. At 2.0mM DTT, we could find no slower-moving species (G6PD

II) in supernatant preparations from glands of lactatingmice. The effects of DTT were identical when added eitherprior to or after homogenization. When added to theelectrophoresis buffer, no change in patterns occurred, butbackground staining increased. Examination of preparations from HAN exposed to 2.0 m@iDTT indicated that thepredominant species was now the faster-migrating form,G6PD-I, rather than G6PD-II seen previously (Table 1).G6PD-I, which originally represented 30 to 35% of totalactivity, increased to 60 to 65% after treatment with DTT.The small amount of G6PD-III originally observed in thistissue was no longer visible after treatment. Similar resultswere obtained with preparations from carcinomas (Table 1).

The effects of oxidation on the G6PD patterns wereexamined in samples from mammary glands, HAN, andtumors (Table 1). For HAN, oxidation caused the appearance of the slowest-migrating species G6PD-III, a smallamount of G6PD-II, and no detectable G6PD-I. WhenDTT waspresentduringoxidation,theG6PDpatternwassimilar to that seen in fresh preparations treated with DTT.Thus, oxidation enhanced the formation of G6PD-III,which was partially prevented by the presence of DTT,suggesting that —SH groups may be involved in theconversion of one form to the other. Similar results wereobtained with tumors and are summarized in Table 1. As aresult of oxidation, a loss of 70 to 80% of the G6PD activitywas observed in all tissues; however, in the presence of DTT,the loss in total activity was only 40 to 50%. Loss of activitythrough oxidation did not alter the enhanced formation ofG6PD-III.

Possible Relationship of G6PD and GR. We consideredthat GSH in the cell might influence the G6PD patternsobtained from fresh tissue preparations. Glutathione can bereduced by GR, an enzyme found in the cytoplasm, utilizingNADPH as a cofactor. Tissue preparations were measuredfor GR activity, and the results are presented in Table 2,along with the activities and patterns of G6PD. The resultsindicate that GR activity was similar in normal mammaryglands, regardless of the physiological state of the host, butwas significantly higher in HAN and in carcinomas. Theabnormal tissues showed that G6PD patterns contained thegreatest amount of G6PD-I. Thus, the activity of GR mayinfluence the pattern of G6PD species seen.

Effect of NADP@ on G6PD Forms and Heat Inactivation.It has been shown that NADP@ acts to stabilize G6PDduring purification (10, 14). When 2 mM NADP@ was addedto either the tissuesupernatantpreparationor the electrophoresis buffer, no significant alteration in the G6PDpattern of normal or abnormal tissues was seen.

Studies were performed to ascertain the effect of NADP@on the inactivation of G6PD by heating at 47°. Withcytosol preparations from lactating mammary gland(Chart I), the addition of 2 mrvi NADP@ afforded considerable protection against inactivation. In the presenceof NADP@, no decreasein G6PD activity was observed,whereas complete loss of G6PD activity occurred in itsabsence. To determine whether concentration of enzymeplayed a role in the rate of heat inactivation, variousdilutions of the supernatant fractions were tested (Chart 2).The protection by NADP@ was consistently observedwhereas, in the absence of this cofactor, the rate of enzymeinactivation was related to the amount of enzyme activity inthe initial preparation. Such inactivation was not due simplyto dilution of total protein; the addition of albumin to acomparable total protein content did not alter the rate of

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TissuePhysiological stateSpecific

activity―%totalactivit?

in G6PDspeciesG6PDGRIII

IIIMammary

glandVirgin105 ±108 ±15 905MammaryglandPregnant126±1911 ±30 8515Mammary

glandLactating297 ±229 ±I40 555HANVirgin100±1234±556530TumorVirgin64±730±6560 35

FO 30 60 FO 30 60

Table2Relationship of G6PD and GR activity and G6PD species

a Reported as nmoles of pyridine cofactor reduced or oxidized per mm per mg protein. Data

presented as mean ±S.E., derived from assays on 8 to 31 different tissue samples.b See Table 1 for terminology of G6PD species. Percentages were calculated as described in

“Materials and Methods.―

0 60 120

TIME (MINUTES)Chart 2. Effect of overnight oxidation on heat inactivation of G6PD

from either lactating mammary gland (left) or from mammary gland ofpregnant mice (right). Initial enzyme activities (F, fresh), with or withoutaddition of 2 mM DTT, are shown as •,A, and U. One portion ofthe freshsupernatant preparation was oxidized overnight without DTT; another wasoxidized overnight with DTT. The fraction oxidized in the absence of DTTwas further divided into 3 parts. Nothing was added to the 1st, DTT wasadded 15 mm prior to heating the 2nd, and DTT + NADP@ was added 15mm prior to heating the 3rd. At selected times during the heating periods, aportion was removed from each for G6PD assay and for gel electrophoresis.

phoresis (Chart 2). Samples oxidized oyernight withoutDTT had 0.23 unit of G6PD activity remaining and lostabout 40% of this activity after 1 hr of exposure to 47°.In contrast, those samples oxidized overnight in the presence of DTT (0.43 unit G6PD activity) showed no measurable G6PD activity after 45 mm at 47°. Samples, oxidized in the absence of DTT but then incubated for 15 mmwith DTT prior to heat inactivation, demonstrated a complete loss of G6PD activity within 60 mm. Thus, the addition of DTT in this manner did not protect against heatinactivation. The patterns of G6PD demonstrate the heat

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180

TIME (MINUTES)Chart I. Effect of NADP@ on G6PD inactivation by heating. Superna

tant preparations from lactating mammary glands were incubated at 47°,in the presence or absence of 2 mr@iNADP@ . Aliquots were taken for assay of G6PD activity and for acrylamide gel electrophoresis. Freshtissue fractions contained 0.4 unit G6PD per 72.5 @sgprotein per ml. Dilutions were made with 0.05 M Tris buffer, pH 7.4.

heat inactivation. The time required to reduce G6PDactivity by 50% was estimated as ‘@.-87mm, ‘@-69mm, and“@27mm for undiluted (0.4 unit G6PD, 72.6 @gprotein) and

diluted (0.2 unit, 36.3 zg protein, and 0. 13 unit, 24.2 @sgprotein) preparations, respectively.

Examination of G6PD activity in supernatant fractionsprepared from spontaneous mammary tumors and from aline of transplantable mammary carcinoma also showedthat the addition of NADP@ protected G6PD activityagainst heat inactivation. The rate of G6PD inactivationwas similar in both tumors; the time to reach 50% inactivation was -“-25mm in both cases.

Sensitivity of Different Forms of G6PD to Heat Inactivation. Preliminary results suggested that the slowermigrating G6PD-III species was less sensitive to heatinactivation. Supernatant preparations from lactatingglands, subjected to overnight oxidation with or without 2mM DTT, were heated at 47° and aliquots were removed

at various time intervals for G6PD assay and electro

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G6PD Forms in Mammary Glands and Tumors

induced disappearance of the faster-migrating band withtime and are illustrated in Fig. 1.

The effect ofthe addition ofboth NADP@ and DTT (eachat 2 mM) to preparations that had been oxidized overnightwas examined. After 60 mm at 47°, G6PD activity waselevated and returned to the level of activity found infresh tissue preparations prior to overnight oxidation.This apparent reactivation of G6PD was not simply dueto the availability of more NADP@ in the enzyme assay,since the addition of equivalent amounts of NADP@ (2 mMplus that level used in the assay) did not increase themeasurable G6PD activity.

Essentially identical results were obtained with mammaryglands from pregnant mice (Chart 2). Equal portions(activity) of supernatant preparations from fresh glands ofpregnant and lactating animals were mixed and thenexposed to heat, in the presence or absence ofNADP@. Lossof activity was seen in the absence of NADP@, withG6PD-III demonstrating greater heat stability; NADP@prevented loss of activity and maintained the G6PD patterns (Fig. I).

DISCUSSION

The results presented here confirm our earlier report (7),in that, during pregnancy, the gland contains virtually noG6PD-III. In addition, G6PD-I during lactation is severelydecreased. A progression appears to occur in pregnant andlactating conditions such that the pattern is shifted fromG6PD-I to G6PD-III. In this regard, the HAN and tumorsresemble more closely the glands from pregnant or virginanimals rather than those from lactating animals. Uponcloser examination of the 3 forms of this enzyme, datapresented here demonstrate additional subtle differences.HAN and tumors from virgin animals contain a significantly higher proportion of G6PD-I than do glands frompregnant mice. Tissue supernatant preparations from lactating mice exposed to DTT showed a shift from G6PD-III toG6PD-II. In supernatant preparations from HAN andtumors to which DTT was added, a conversion fromG6PD-II to G6PD-I could be demonstrated. In preparations of glands from pregnant mice, however, DTT did notcause a conversion of G6PD-II (the major species) toG6PD-I and, likewise, G6PD-II in preparations fromlactating mice was not influenced by DTT to migrate faster.Thus, tissues not containing G6PD-I in the absence of DTTdid not show this form in the presence of DTT. A failure todemonstrate an increase in G6PD-I in the presence of DTTmight be useful to distinguish normal from abnormalmammary tissues.

The G6PD patterns of mouse tissues differed from thoseof rat tissues (20). In the rat mammary gland, differentiation leading to lactation was accompanied by an increase inthe fastest-migrating species; the lactating stage had essentially 1 G6PD species (migrating like G6PD-I describedhere). In the mouse, differentiation of the mammary glandwas accompanied by an appearance of a slow-migratingspecies, G6PD-III, which was not present in significantamounts in the other tissues examined. Mammary tumors

of rats showed 2 G6PD forms (2, 19), with the predominantform or forms migrating rapidly. Similar results were foundhere with mouse mammary tumors and preneoplasticlesions. Preliminary data on human breast carcinomas alsoindicated the presence of 2 rapidly migrating G6PD formsaccounting for most of the G6PD activity (8). Thus, one canclearly distinguish abnormal tissues froth glands in lactationin the mouse, but not in the rat.

Estimated molecular weights of G6PD-III and G6PD-IIin glands from lactating mice suggest that 1 species wasapproximately twice the weight of the other. The value of

I 18,000 daltons for G6PD-II agrees well with the value of120,000 daltons for purified G6PD from rat mammarygland (16). The effects of DTT and oxidation suggest a roleof —SH groups in monomer-dimer formation, with themonomer being favored in the reducing milieu. The involvement of —SH groups in these interconversions was alsosuggested by Schmukler (2 1) from studies with erythrocyteG6PD. On the other hand, Hizi and Yagil (9) and Taketaand Watanabe (22, 23) reported reconversion of faster- toslower-migrating forms after the addition of sulfhydrylreagents to G6PD from mouse and rat liver, respectively.The reasons for these differences are not apparent.

The G6PD patterns observed could have resulted fromalterations in cellular oxidation-reduction milieu, particularly GSH-GSSG levels. We determined the activity of GR,an enzyme capable of regulating intracellular GSH formation, and the results showed that GR activity was higher inabnormal tissues. If the elevated GR activity reflectedincreased GSH level, then preneoplastic and neoplastictissues could have a greater sulfhydryl milieu, which shouldfavor G6PD in its monomeric forms. During pregnancy andlactation, GR activities were similar, although G6PD-IIIwas a dominant form during lactation. It is obvious,therefore, that GR activity per se does not determine themolecular form of G6PD.

Recently, Eggleston and Krebs (5) proposed that GSSGexerts control of the pentose phosphate pathway by counteracting the NADPH inhibition of G6PD. This effect wasnot attributed to GR activity, which would decreaseNADPH. They reported that the effect of GSSG was notseen in the lactating mammary gland. However, duringlactation, when lipogenesis is at a peak, NADPH utilizationfor fatty acid synthesis is favored and, thus, availability ofreductive hydrogen to GR may be diminished. The availability of NADP@ has been proposed as one of the factorscontrolling the pentose phosphate pathway activity (15),although effects on enzyme forms, such as monomerreformation after inactivation, seem to be equally influenced by NADP@ and NADPH (3, 11). From the workreported here, it appears that NADP@ does not change theform of G6PD but merely stabilizes it.

Although the addition of NADP@ to mammary tissuepreparations did not alter the electrophoretic profiles, thecofactor clearly stabilized the enzyme forms and preventedinactivation by heat. Levy (12) proposed that NADP@ (orNADPH) favored 1 form of G6PD monomer in ratmammary glands. It was proposed that this form favorsdimer formation. The fact that G6PD-III increases propor

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R. Hilfet al.

tionately during lactation may be related to the tissuesincreased content ofNADP@ plus NADPH (15), a situationwhich Levy (12) suggests favors dimer formation. By usingoxidation to enrich the proportion of dimer (G6PD-III), wedemonstrated that this enzymatic form was more stable toheat inactivation.Enzymeinactivationcausedby oxidationwas completely reversed by the addition of NADP@. Ifinactivation was accompanied by dissociation to a subunit,recombination of the subunits must have occurred in thepresence of NADP@ , as evidenced by a complete restorationof G6PD activity. Oxidation of erythrocyte G6PD similarlydecreased activity, which was restored by the addition ofpyridine nucleotide (1 1). This inactivation-reactivation appears to be a general property of mammalian G6PD.Although DTT was able to partially protect against oxidation-induced enzyme inactivation, it did not protect againstheat inactivation. If the dimer form is indeed a more stableform in vivo, perhaps its presence during lactation reflects aneed for maintaining high functional levels of the enzyme.The fact that oxidation caused a relative increase inG6PD-III in all of the tissues studied indicates that theability for dimer formation is not lost in the abnormaltissues examined.

REFERENCES

I. Abraham, S., and Chaikoff, I. L. Glycolytic Pathways and Lipogenesisin Mammary Glands of Lactating and Nonlactating Normal Rats. J.Biol. Chem., 234: 2246-2253, 1959.

2. Cho-Chung, Y. S., and Berghoffer, B. The Role of Cyclic AMP inNeoplastic Cell Growth and Regression. II. Growth Arrest andGlucose-6-Phosphate Dehydrogenase Isoenzyme Shift by DibutyrylCyclic AMP. Biochem. Biophys. Res. Commun., 60: 528-534, 1974.

3. Chung, A. E., and Langdon, R. G. Human Erythrocyte Glucose-6-Phosphate Dehydrogenase. II. Enzyme-Coenzyme Interrelationship.J. Biol. Chem., 238: 2317-2324, 1963.

4. DeOme, K. B., Faulkin, L. J., Jr., Bern, H. A., and Blair, P. B.Development of Mammary Tumors from Hyperplastic AlveolarNodules Transplanted in Gland-free Mammary Fat Pads of FemaleC3H Mice. Cancer Res., 19: 515-520, 1959.

5. Eggleston, L. V., and Krebs, H. A. Regulation of the PentosePhosphate Cycle. Biochem. J., 138: 425-435, 1974.

6. Glock, G. E., and McLean, P. Further Studies on the Properties andAssay of Glucose-6-Phosphate Dehydrogenase of Rat Liver. Biochem.J., 55: 400-408, 1953.

7. Hilf, R., Rector, W. D., and Abraham, S. A Glucose-6-phosphateDehydrogenase Isoenzyme Characteristic of Preneoplastic and Neoplastic Mouse Mammary Tissue. J. NatI. Cancer Inst., 50: 1395-1398,1973.

8. Hilf, R., and Wittliff, J. L. Characterization of Human Breast Cancer

by Examination of Cytoplasmic Enzyme Activities and EstrogenReceptors. In: K. W. McKerns (ed), Hormones and Cancer, pp.103—130.New York: Academic Press, Inc., 1974.

9. Hizi, A., and Yagil, G. On the Mechanism of Glucose-6-PhosphateDehydrogenase Regulation in Mouse Liver. 2. Purification andProperties of the Mouse-Liver Enzyme. European J. Biochem., 45:201-209, 1974.

10. Kirkman, H. N. Glucose 6-Phosphate Dehydrogenase from HumanErythrocytes. I. Further Purification and Characterization. J. Biol.Chem.,237:2364-2370,1962.

I 1. Kirkman, H. N., and Hendrickson, E. M. Glucose 6-PhosphateDehydrogenase from Human Erythrocytes. II. Subactive State of theEnzyme from Normal Persons. J. Biol. Chem., 237: 2371-2376, 1962.

12. Levy, H. R. The Interaction of Mammary Glucose-6-PhosphateDehydrogenase with Pyridine Nucleotides and 3$-Hydroxyandrost-5-ene-17-one. J. Biol. Chem., 238: 775—784, 1963.

13. Levy, H. R., Raineri, R. R., and Nevaldine, B. H. On the Structureand Catalytic Function of Mammary Glucose-6-Phosphate Dehydrogenase. J. Biol. Chem., 241: 2181-2187, 1966.

14. Marks, P. A., Szeinberg, A., and Banks, J. Erythrocyte Glucose-6-Phosphate Dehydrogenase of Normal and Mutant Human Subjects.Properties of the Purified Enzymes. J. Biol. Chem., 236: 10-17, 1961.

15. McLean, P. Carbohydrate Metabolism of Mammary Tissue. II.Levels of Oxidized and Reduced Diphosphopyridine Nucleotide andTriphosphopyridine Nucleotide in the Rat Mammary Gland. Biochim.Biophys. Acta, 30: 316-324, 1958.

16. Nevaldine, B. H., Hyde, C. M., and Levy, H. R. MammaryGlucose-6-Phosphate Dehydrogenase. Molecular Weight Studies.Arch. Biochem. Biophys., 165: 398-406, 1974.

17. Nevaldine, B. H., and Levy, H. R. Reversible Dissociation andAssociation of M ammary Glucose-6-Phosphate Dehydrogenase. Biochem. Biophys. Res. Commun., 21: 28-33, 1965.

18. Racker, E. Glutathione Reductase. Methods Enzymol., 2: 722-725,1955.

19. Richards, A. H., and Hilf, R. Glucose-6-Phosphate and LactateDehydrogenase Isoenzymes in Rodent Mammary Carcinomas and theEffect ofOophorectomy. Biochim. Biophys. Acta, 232: 753-756, 1971.

20. Richards, A. H., and HiIf, R. Influence of Pregnancy, Lactation andInvolution on Glucose-6-Phosphate Dehydrogenase and Lactate Dehydrogenase Isoenzymes in the Rat Mammary Gland. Endocrinology,91:287-295,1972.

21. Schmukler, M. The Heterogeneity and Molecular Transformations ofGlucose-6-Phosphate Dehydrogenase of the Rat. Biochim. Biophys.Acta, 214: 309-317, 1970.

22. Taketa, K., and Watanabe, A. Interconvertible Microheterogeneity ofGlucose-6-Phpsphate Dehydrogenase in Rat Liver. Biochim. Biophys.Acta, 235: 19-26, 1971.

23. Watanabe, A., Taketa, K., and Kosaka, K. Glutathione-IndependentInterconversion of Microheterogeneous Forms of Glucose-6-Phosphate Dehydrogenase in Rat Liver. J. Biochem., 72: 695-701, 1972.

24. Zwann, J. Estimation of Molecular Weights of Proteins by Polyacrylamide Gel Electrophoresis. Anal. Biochem., 21: 155—168, 1967.

2114 CANCER RESEARCH VOL.35

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G6PD Forms in Mammary Glands and Tumors

Fig. I. Composite illustration of acrylamide gel patterns of G6PD. A, fractions from lactating gland heated at 47°(1, 0 time; 2, 30 mm; 3, 45 mm; 4, 60mm). Note relative stability of G6PD-III. B, fractions from lactating gland to which 2 mM DTT was added 15 mm prior to heating at 47°(1, 0 time; 2,30 mm; 3, 45 mm; 4, 60 mm). Note lack of G6PD-III and disappearance of G6PD-II with time. C, fractions from lactating gland to which 2 mM DTT+ 2 mr@iNADP@ were added prior to heating at 47°(1, 0 time; 2, 30 mm; 3, 45 mm; 4, 60 mm). Note maintenance of activity in presence of NADP@.

D, fractions from lactating gland after overnight oxidation at 4° and then exposure to 47° (1, 0 time; 2, 15 mm; 3, 30 mm; 4, 45 mm; s, 60 mm; 6, 75 mm).

Note relative stability of G6PD-III. E, fractions from lactating gland after overnight oxidation at 4°in the presence of 2 mM DTT and then heatedat 47°(1, 0 time; 2, 15 mm; 3, 30 mm: 4, 45 mm; s, 60 mm; 6, 75 mm). Note disappearance ofactivity. F, fractions from lactating gland after overnightoxidation at 4°to which 2 mt@iDTT + 2 mM NADP@ were added 15 mm prior to heating at 47°.Gel was obtained from sample removed after 60 mmof heating. Note maintenance of G6PD activity. G, supernatant fraction from mammary glands of pregnant mice oxidized overnight at 4°and thenexposed to heating at 47°(1, 0 time; 2, 15 mm; 3, 30 mm; 4, 45 mm; 5, 60 mm); results are similar to those shown in A. H, supernatant fraction frommammary glands of pregnant mice oxidized overnight in the presence of 2 mM DTT and then exposed to heating at 47°(1, 0 time; 2, 15 mm; 3, 30 mm;4, 45 mm). Note disappearance of G6PD-II and prevention of G6PD-III formation by DTT. J, supernatant fraction from mammary glands oxidizedovernight at 4°to which 2 mt@iDTT and 2 mM NADP@ were added 15 mm prior to heating at 47°(1, 0 time; 2, 60 mm). Note apparent @ncreasein

G6PD activity, K, fractions from lactating and pregnant glands were mixed on the basis of equal G6PD activity and the mixture was heated at 47°(1,0 time; 2, 15 mm; 3, 30 mm; 4. 45 mm; 5, 60 mm; 6, 75 mm). Note relative stability ofG6PD-III. L, supernatant fractions from lactating and pregnantglands were mixed on the basis of equal G6PD activity, and 2 mM NADP@ was added 15 mm prior to heating at 47°(1, 0 time; 2, 15 mm; 3, 30 mm;4, 45 mm; 5, 60 mm; 6, 75 mm). Note stabilization of G6PD activity by NADP@. Migration direction is from top to bottom.

AUGUST 1975 2115

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2116 CANCER RESEARCH VOL. 35

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1975;35:2109-2116. Cancer Res   Russell Hilf, Regina Ickowicz, J. C. Bartley, et al.   Mammary Tissues of MiceDehydrogenase in Normal, Preneoplastic, and Neoplastic Multiple Molecular Forms of Glucose-6-Phosphate

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