MANJU RAY AND AMAR BHADURI From the Division of Biochemistry, Department of Pharmacy, Jadavpur University, Calcutta-? OOOSZ, India

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1 THI JOURNAL OF BIOLOGICAL CHEYI~TRY Vol. 250, No. 10, Issue of My 26, PP , 1976 Printed in U.S.A. Glctose&phosphte Dehydrogense PURIFICATION AND PARTIAL CHARACTERIZATION (Received for publiction, November 15, 1974) MANJU RAY AND AMAR BHADURI From the Division of Biochemistry, Deprtment of Phrmcy, Jdvpur University, Clcutt? OOOSZ, Indi SUMMARY A new enzyme, glctose6phosphte dehydrogense hs been purified bout 50fold from got liver. The enzyme cn be distinguished from the nonspecific hexose6phosphte dehydrogense nd glucose6phosphte dehydrogense by its high substrte specificity nd bsolute pyridine nucleotide requirement. In contrst to the hexose6phosphte dehydrogense, this enzyme is locted exclusively in the cytoplsmic frction of the cell. The enzyme is metlloprotein nd is highly sensitive to mercurils. The product of the rection is possibly ketoldose, phosphorylted t the primry lcoholic group. We hve recently reported the presence of new enzyme in mmmlin liver tht specificlly oxidizes glctose 6phosphte in the presence of NAD (1, 2), glctose 6phosphte hs not yet been shown s norml metbolite for ny tissue or orgnism. Inouye et l. (3) climed tht the compound ccumulted in red blood cells under glctosemic conditions but this hs been disputed by Ng (4). In 1966, Ohno et l. (5) nd Shw (6) detected the presence of dehydrogense in mmmlin liver which could oxidize both glucose gphosphte nd glctose gphosphte quite efficiently in the presence of both NAD nd NADP. This enzyme ws lter found to be identicl with the well known microsoml glucose dehydrogense (7). Becuse of the much lower K, vlues for both glctose Bphosphte nd glucose 6phosphte in comprison to glucose, glucose dehydrogense ws renmed s hexosegphosphte dehydrogense. Becuse both hexose6 phosphte dehydrogense nd glctose6phosphte dehydrogense could utilize glctose gphosphte s substrte in the presence of NAD, detiled study regrding the differences in property nd the seprte identity of the two enzymes ws in order. Along with the bove spect of the problem, this pper is lso concerned with generl chrcteriztion of the new enzyme, glctose6phosphte dehydrogense. MATERIALS AND METHODS All of the biochemicls used for this work were purchsed from Sigm Chemicl Co. The mixed bed resin, Rexyn 300 ws purchsed from Fisher Scientific Co. The sodium slt of glctose gphosphte ws ssyed for ny possible contmintion of lcohol with lcohol dehydrogense. The compound ws found to be virtully free of lcohol. This precution ws tken to void ny possible confusion tht might rise becuse of the presence of lcohol dehydrogense in our enzyme preprtions (8,9). Clcium phosphte gel ws prepred ccording to the method of Keilin nd Hrtree (10). The reducing vlue of the sugrs ws determined by the method of Nelson (11). Estimtion of inorgnic phosphte ws crried out following the method of Lowry nd Lopez (12). In purified enzyme frctions, protein ws determined by the method of Wrbur nd Christin (13). For some other smples the method of Lowry et l. (14) ws used. Glctose6phosphte dehydrogense ws routinely ssyed spectrophotometriclly. The usul ssy mixture contined in totl volume of 1 ml, 100 pmol of potssium phosphte buffer, ph 7.6, nd 0.5 cmol of NAD. The rection ws strted by the ddition of 1 pmol of gletose gphosphte. The rte remined liner for more thn 5 min nd could be very conveniently mesured by following the increse in bsorbnce t 340 nm. One unit of the enzyme ws defined s the mount of enzyme needed for the formtion of 1 wmol of NADH per min. The optimum ph of the enzyme ws found to be round 8.4, when the enzyme ws ssyed with glycylglycine buffer. But t this ph there ws often nonspecific increse in bsorbnce which complicted the ccurte determintion of the rte. For some experiments however, the phosphte buffer for the routine ssy ws replced either by 100 Mmol of glvcvlnlvcine buffer. uh 8.4. or bv elvcine buffer, ph 9.6;which ismentioned in the pproprite pl&& in the text. For the ssy of the nonspecific hexose6phosphte dehydrogense, glctose Bphosphte ws not normlly used s the substrte. The enzyme ws usully ssyed in totl volume of 1 ml, contining 100 pmol of potssium phosphte buffer, ph 7.4, nd 1 umol of NAD. The rection ws strted bv the ddition of 500 rmol of glucose nd the rte of increse in bsorbnce t 340 nm ws followed over period of 5 min. Control blnks without glucose were simultneously run. Assy of the enzyme t ph vlues over 9 often posed some problems becuse of rpid increse in bsorbnce even in the bsence of the substrte. This ws lso noted by Beutler nd Morrison (7). In such cses, the enzyme could be conveniently ssyed in the presence of NADP becuse the nonspecific increse in bsorbnce ws much smller when NADP ws used insted of NAD. Lctic dehydrogense nd glucose6phosphte dehydrogense were ssyed following the stndrd methods described in Refs. 15 nd 16. Glyoxlse I ws ssyed ccording to the method of Rcker (17). RESULTS Pur$ction of EnzymeSince our preliminry communiction on this enzyme (l), the purifiction procedure hs been somewht modified nd extended. The enzyme being highly sensitive to the presence of ethylenediminetetrcette, it ws omitted from the extrction medium. Unless otherwise stted ll of the opertions were crried out t 56. A typicl purifiction procedure with got liver proceeded s follows. Twelve grms of freshly frozen got liver were tken into 36 ml of 0.02 M sodium phosphte buffer, ph 7.4. The liver ws homogenized in Wring Blendor 3595

2 3596 for 60 s. The homogenized suspension ws centrifuged t 12,000 s expected, lso showed ctivity when ssyed with glctose X g for 30 min. The superntnt ws now treted with solution 6phosphte s substrte nd NAD or NADP s the hydrogen of protmine sulfte (2% w/v) until the volume of dded prot cceptor. The rte of oxidtion of glctose 6phosphte however mine sulfte ws 15% of the originl volume of the superntnt. ws considerbly slower thn in cse of glucose indicting tht in After stnding for 10 min the precipitte ws centrifuged t got liver t lest glucose is much more efficiently oxidized thn 12,000 x g. The protmine sulftetreted superntnt ws now the hexose 6phosphtes by this enzyme. In contrst to this subjected to frctiontion with the incresing mmonium sulfte enzyme frction, the frction contining glctose6phosphte concentrtion. Solid, recrystllized mmonium sulfte ws dehydrogense ctivity filed to ctlyze oxidtion of glucose slowly dded to the superntnt with constnt stirring until the under different conditions of ph nd glucose concentrtions. mount of the mmonium sulfte present, s determined t 0, Substrte Spec$GtyThe purified enzyme from the got liver ws 35% of its sturting concentrtion. The ccumulted obtined fter the second mmonium sulfte tretment ws found precipitte ws rejected nd to the superntnt nother quntity to be completely free of glucose6phosphte dehydrogense nd of mmonium sulfte ws slowly dded until 55% sturtion hexose6phosphte dehydrogense. The enzyme ws found to be ws obtined. The centrifuged precipitte ws collected nd bsolutely specific both for glctose 6phosphte nd for NAD. redissolved in 5 ml of 0.02 M sodium phosphte buffer, ph 7.4. Our previous report tht NADP could prtilly replce NAD (1) The solution ws dilyzed for 6 hours with one chnge ginst ws shown not to be correct with this purified enzyme. A lrge the sme buffer. Two milliliters of the dilyzed solution contin number of sugrs nd sugr phosphtes including glucose, ing pproximtely 40 mg/ml of protein ws then treted with glctose, fructose, mnnose, glucose 6phosphte, mnnose 6 clcium phosphte gel. Two milliliters of the gel (16 mg dry phosphte, glucosmine 6phosphte, glucose lphosphte, weight per ml of solution) ws lightly centrifuged nd the glctose lphosphte, nd fructose gphosphte were tested both centrifuged gel ws dded to 2 ml of the dilyzed enzyme solution. t ph 7.4 nd 9.6 nd with NAD nd NADP s the potentil The gel ws mixed with stirring for 3 min nd then centrifuged electron cceptors. No significnt increse in bsorbnce t 340 t 2,000 x g. The enzyme ws not bsorbed on the gel nd ws nm ws detected with ny of these compounds s the substrte. further purified on DEAEcellulose column. The cler difference between glctose6phosphte dehydro Corse mesh DEAEcellulose, purchsed from Sigm Co. ws gense nd the nonspecific hexose6phosphte dehydrogense thoroughly wshed. About 2 g of wshed DEAEcellulose ws ws lso evident when the enzyme preprtions were obtined used to prepre column of bout 22 cm in height with dimeter from rt liver. The two enzymes from the rt liver could be of 1.6 cm. The column ws first equilibrted with 0.02 M sodium seprted by bsiclly following the sme procedure s tht phosphte buffer, ph 7.4 One milliliter of enzyme solution contining 0.35 unit of glctose6phosphte dehydrogense TABLE I ctivity nd 20 mg of protein ws now bsorbed on the column Purijiction of glctose6phosphte dehydrogense from got liver nd the column ws then eluted with incresing concentrtions of sodium phosphte buffer of the sme ph. The rte of flow ws step Totl Totl Yield Specific rctivity protein ctivity mintined t 15 drops/min nd 2.5 ml of the eluted smple ws unitdmg collected in ech tube. The column ws eluted with 40 ml of ech units w protein x 10 of 0.02 M, 0.04 M, 0.07 M, nd 0.1 M of sodium phosphte buffer, ph 7.4. The enzyme which ws unbsorbed in the column Crude usully cme out between tubes 7 to 10. Two to three tubes Protmine sulfte Ammonium sulfte contining bout 80% of the totl eluted ctivity were now Clcium phosphte gel combined nd the enzyme ws reprecipitted with the ddition DEAEcellulose column of mmonium sulfte. The enzyme which precipitted between Second mmonium sulfte to 60% of sturting mmonium sulfte concentrtion ws redissolved in 1 ml of 0.02 M sodium phosphte buffer, ph 7.4. When kept frozen t 10 the enzyme ws found to be stble for 06 3 to 4 dys. Becuse the spectrophotometric ssy could not be.e used on the crude extrct, the yield nd purifiction t this stge 05,E is expressed in terms of the totl ctivity present in the superx ntnt frction fter protmine sulfte tretment. The finl yield 04 5 over the protmine sulfte frction ws usully between 50 to c 55% nd the enzyme ws purified bout 30fold over this frc 03: m tion. The respective yield nd purifiction during the vrious G stges of purifiction re presented in Tble I, 02 Seprtion from Hexose&phosphte DehydrogenseDuring the purifiction of the enzyme, the hexose6phosphte de 01 hydrogense ctivity ws lso ssyed in the vrious frctions..? More thn 60% of the totl ctivity of this enzyme ws precipi t tted between 0 to 35 % of the sturting concentrtion of IO mmonium sulfte. The remining ctivity ws ultimtely Tube completely seprted from the glctose6phosphte dehydro FIG. 1. Seprtion of glctose6phosphte dehydrogense gense on the DEAEcellulose column (Fig. 1). HexoseB nd hexose6phosphte dehydrogense on DEAEcellulose column. OO indictes the elution pttern of the proteins phosphte dehydrogense which ws mesured in terms of the from the column. OO, denotes the ctivity of glctose6 glucose dehydrogense ctivity ws eluted with 0.07 M sodium phosphte dehydrogense in vrious tubes. The ctivity of hexosephosphte buffer between tubes 38 to 42. This enzyme frction, gphosphte dehydrogense is indicted BH.

3 developed for the got liver. Ten grms of freshly excised rt liver were homogenized in 30 ml of 0.02 M potssium phosphte buffer, ph 7.4. The suspension ws centrifuged t 12,000 x g for 30 min. The crude homogente ws treted with protmine sulfte exctly in the sme mnner s described for got liver. The superntnt obtined fter the protmine sulfte tretment ws seprted into two frctions of 0 to 40% nd 40 to 55y0 of sturting concentrtion of mmonium sulfte. The precipittes obtined from both these frctions fter centrifugtion were seprtely redissolved in 5 ml ech of 0.02 M sodium phosphte buffer ph 7.4. Hexose6phosphte dehydrogense ws precipitted completely in the 0 to 40% frction wheres glctose 6phosphte dehydrogense ws obtined lmost exclusively in the 40 to 55% frction. The difference in substrte specificity between these two frctions t two different ph vlues nd using both NAD nd NADP s the potentil oxidnts is shown in Tble II. The substrte specificity of the 0 to 40% frction followed very closely the substrte specificity obtined for the hexose6phosphte dehydrogense in rt liver by Beutler nd Morrison (7). In contrst, the other frction contining glctosegphosphte dehydrogense could oxidize glctose 6phosphte lone only in presence of NAD, thus clerly indicting the seprte identity of glctose6phosphte dehydrogense from the nonspecific hexose6phosphte dehydrogense in the rt liver system. ph OptimumGlctose6phosphte dehydrogense ws found to be ctive over wide rnge of ph. The enzyme could be conveniently ssyed t ph 7.4 in 0.1 M sodium phosphte buffer or round ph 9.2 in glycinesodium hydroxide buffer. Using glycylglycine buffer of vrying ph nd sturting concentrtion of the substrte, the optimum ph of the enzyme ws determined to be 8.4 (Fig. 2). When glycylglycine buffer ws replced by Trishydrochloric cid buffer, the rte ws found to be considerbly lower though the nture of the ph curve remined bsiclly the sme. Determintion of &The Michelis constnt for glctose 6 phosphte ws determined for the enzyme t ph 7.4 with phosphte buffer nd t ph 8.4 with glycylglycine buffer. The substrte sturtion curve followed the usul MichelisMenten kinetics t both the ph vlues. The K, when plotted ccording to the method of Linewever nd Burk cme out to be 1.5 mm t ph 7.4 when the concentrtion of NAD ws kept t fixed 3597 level of 1 mm (Fig. 3). No significnt vrition in the vlue of K,,, ws observed with the chnge of ph. At fixed concentrtion of glctose Bphosphte (1 mm), the Ii, for NAD ws clculted to be 0.6 mm t ph 7.4 (Fig. 4). Locliztion of EnzymeFor the determintion of the locliztion of the enzyme, freshly excised got liver ws homogenized nd frctionted ccording,to modified method of Schneider nd Hogeboom (18). The excised liver ws first cut into smll pieces. About 2 g of the liver were suspended in 14 ml of 0.25 M sucrose contining 0.02 M sodium phosphte buffer, ph 7.4. The cells were homogenized in PotterElvehjem type glss homogenizer with glss pestle. The opertion ws repeted four times, so tht totl of 8 g of liver were homogenized in totl volume of bout 60 ml. The nucler frction consisting of nucler mteril, whole cells, nd cell debris ws obtined by centrifuging the homogente for 20 min t 600 x g. For wshing, the precipitted mss w6 resuspended in 5 ml of the sme medium nd x 7.r IO E \ u L o. 5 : PH FIG. 2. Glctose6phosphte dehydrogense ctivity s function of ph. Enzyme ctivities were determined in 0.1 M glyeylglycine buffer of vrying ph contining 2.8 mm of glctose 6phosphte, 1 mr of NAD, nd requisite mount of glctose6 phosphte dehydrogense. TABLE II Differences between glctose6phosphte dehydrogense nd hexosebphosphte dehydrogense from rt liver in reltion to specificity towrds substrtes The method for obtining the two frctions re described in the text. Activity (ba/min) X 10 N 0 x Substrte Glctose 6phosphte., Glctose 6phosphte., Glctose gphosphte. Glctose 6phosphte. Glucose... Glucose... C0lKeKP trtion?nm CCXllZplle PH 3lctose 6phoslexose6 phte hosphte dehydro dehydrogense gense NAD NAD NADP NADP NAD NAD ' I I I I L I/Gl6P(mM) FIG. 3. Determintion of pprent Michelis constnt for glctose Bphosphte. Ech ssy mixture contined in totl volume of 1 ml, 166 pmol of sodium phosphte buffer, ph 7.4, 1 mol of NAD, requisite mount of purified glctose6phosphte dehydrogense, nd vrying mounts of glctose 6phosphte. Reciprocl of the rte ws plotted ginst the reciprocl of glctose gphosphte concentrtion.

4 3598 : IO no \ C I I I I 05 I 0 I 5 20 I/NAD(mM) x10 FIG. 4. Determintion of pprent Michelis constnt for NAD. Ech ssy mixture contined in totl volume of 1 ml, 100 pmol of sodium phosphte buffer, ph 7.4, 1 pmol of glctose 6phosphte, requisite mount of purified glctose6phosphte dehydrogense nd vrying mounts of NAD. Reciprocl of the rte ws plotted ginst the reciprocl of NAD concentrtion. recentrifuged t the sme speed. The superntnt, fter the seprtion of the nucler frction, ws centrifuged t 1000 x g for 20 min nd the precipitte collected t this stge ws rejected. The superntnt of 1000 x g ws then centrifuged for 10 min t 9000 x g. The precipitte collected t this stge ws wshed twice in the sme medium nd t the sme speed. The wshed frction ws designted s the mitochondril frction. The superntnt of QOOO x g ws further centrifuged t 20,000 x g for 20 min to remove ny unwnted mitochondri still remining in the superntnt. The superntnt from this tretment ws centrifuged t 100,000 x g for 40 min. The pellet collected t this stge ws wshed once in the sme medium nd ws presumed to contin the microsoml frction. The superntnt fter 100,000 x g centrifugtion ws designted s the soluble superntnt. The soluble superntnt ws frctionted with mmonium sulfte in two portions. Both of the precipittes obtined with 0 to 38% nd 38 to 55% sturtion of mmonium sulfte were redissolved in 3 ml of 0.02 M sodium phosphte buffer, ph 7.4. The wshed nucler, mitochondril, nd microsoml frctions were finlly disrupted by freezing nd thwing four times in 5 ml of 0.02 M sodium phosphte buffer contining 0.5% sodium cholte. The disrupted nucler nd mitochondril frctions were finlly centrifuged t 40,000 x g for 20 min. The microsoml frction ws centrifuged t 100,000 x g for 20 min. Both glctose6phosphte dehydrogense nd the hexose8phosphte dehydrogense were ssyed in the cler superntnts of the vrious frctions. The hexosegphosphte dehydrogense ctivity ws ssyed with glucose (500 mm) nd NAD (1 mm) s substrtes t ph 7.4. The results of such n experiment re shown in Tble III. Glctose6phosphte dehydrogense ppers to be lmost exclusively cytoplsmic enzyme s lmost 90% of the totl ctivity is present in the soluble superntnt frction. The locliztion of the hexose6phosphte dehydrogense is generlly consistent with the erlier report of the locliztion of this enzyme (7). Metl Ion InvolvementDuring the purifiction of glctose6 phosphte dehydrogense, we observed tht the presence of even low concentrtions of ethylenediminetetrcette rpidly destroyed the enzyme ctivity. On ddition of ethylenediminetetrcette t 5 mm concentrtion, the enzyme ws lmost TABLE Locliztion of glctose&phosphte dehydrogense nd hezose6 phosphte dehydrogense in subcellulr frctions from got liver Frction Nucler.. Mitochondril Microsoml. Soluble superntnt 35yo mmonium sul. fte % mmonium sulfte. III Glctosc6phosphte dehydrogense ;pecifi< ctivit: (fl*it/ m;&*x Totl ctivity units er cell If totl ctivity I Specific ctivity mit/ng x Hexose6phosphte dehydrogense 1 Totl i rctivity units Per cent of totl ctivity immeditely dectivted by bout 40%. When the enzyme ws preincubted for 10 min in presence of ethylenediminetetrcette t 10 mm concentrtion, the ctivity ws completely lost (Tble IV, A). To check if the inctivted enzyme could be rectivted in presence of specific metl ions, the inctivted enzyme ws precipitted with 70% sturtion of mmonium sulfte nd redissolved in 0.02 M glycinesodium hydroxide buffer, ph 8.4. The inctive enzyme ws then dilyzed ginst the sme buffer for 4 hours nd then tested for rectivtion with vrious metl ions. A lrge number of metl ions were used between 5 mm to 50 mm concentrtions but except for C.+ none of the metl ions were found to restore the ctivity even prtilly (Tble IV, B). C2+ ion t concentrtion of 10 mm restored bout onethird of the originl ctivity. Incresing the concentrtion of Ct+ however did not significntly further increse the ctivity of the inctivted enzyme. Addition of Znzf t 10 mm concentrtion led to some nonspecific precipittion of proteins. These were removed by centrifugtion. The clen superntnt ws found to be devoid of ny ctivity. Fresh ddition of Zn2+ (10 mm) in the ssy medium filed to restore ny enzyme ctivity. Involvement of Suljhydryl GroupThe enzyme ws lso found to be highly sensitive to the tretment of pchloromercuribenzote (Tble V). The enzyme could lso be inctivted with iodocette but the concentrtion of iodocette needed ws considerbly higher thn in the cse of pchloromercuribenzote. Incubtion of the pchloromercuribenzotetreted inctive enzyme with mercptoethnol for 10 min could fully restore the ctivity of the enzyme. Totl inctivtion t very low concentrtions of pchloromercuribenzote nd complete reversl of this effect with mercptoethnol suggest tht one or more specific sulfhydryl groups might be involved in the ctive site. Prtil Chrcteriztion of ProductsThe enzymtic rection ctlyzed by glctose6phosphte dehydrogense is not ccompnied by ny clevge of the ester linkge of glctose 6 phosphte. Thus, when the enzymtic rection ws llowed to proceed for over 30 min in glycine buffer, ph 8.8, until bout 25% of the initil glctose gphosphte ws converted to its oxidized product, ssy of n liquot t tht stge reveled tht the vlue of the totl orgnic phosphte remined constnt. Moreover, relese of no inorgnic phosphte could be detected in the incubtion medium. Formtion of 1:4 NADH during the course of this rection ws confirmed by reoxidizing completely nd quntittively the 1

5 3599 TABLE Involvement of metl ion for ctlytic ctivity of enzyme For this experiment, the enzyme ws prepred by replcing the usul sodium phosphte buffer with 0.02 M glycylglycine buffer, ph 7.4. The complete system for A contined in totl volume of 1 ml, 100 pmol of glycine buffer, ph 8.8,0.5 rmol of NAD, nd the requisite mount of the enzyme. The rection ws strted with the ddition of 1 firno1 of gletose 6phosphte. For experiments in B, 0.15 unit of the enzyme ws incubted for 10 min in totl volume of 1 ml contining 30 pmol of glycylglycine buffer, 7.4, nd 10 pm01 of ethylenediminetetrcette. The complete inctivtion of the enzyme ws checked by ssying n liquot. To remove excess ethylenediminetetrcette, the inctive enzyme ws then precipitted with 70y0 sturtion of mmonium sulfte nd the precipitte ws redissolved in 1 ml of 0.02 M glycylglycine buffer, ph 7.4. This process ws repeted once more. The complete (inctive) system for this set contined in totl volume of 1 ml, 100 pmol of glycine buffer, ph 8.8, 0.5 pmol of NAD, nd the requisite mount of the ethvlenediminetetrcettefree inctive enzyme. A control ws runsimultneously. Addition A. Complete... + Ethylenediminetetrcette (5 mm) + Ethylenediminetetrcette (10 mm) + Ethylenediminetetrcette (10 mm) IV Activity % After 10 min B. Control Complete (inctive) Mg + (10 mm) 0 + Mn*+ (10 mm). 0 + N+ (10 mr.q K+ (10 mm). 0 + Fe8+ (10 mm) Cu*+ (10 mrd). 0 + Zn + (10 mm) C*+ (5 mm) Cp+ (10 mbr) C*+ (50 mm) 35 + Cl+ (160 mm).. 42 NADH formed with pyruvte nd the lctic dehydrogense system (15). During the oxidtion of glctose gphosphte, the free ldehydic group remined unffected s ws evident by the incresed reducing vlue of the totl incubtion medium. The formtion of NADH over period of time closely corresponded with the increse in reducing vlue (Fig. 5). A control for this experiment ws simultneously run with the glucose6phosphte dehydrogense system which showed, s expected, progressive decrese in the reducing vlue with incresing time. Becuse the enzymtic oxidtion led to n increse in the reducing vlue, the formtion of n dditionl center of oxidtion should be ssumed. Further, becuse there ws no libertion of inorgnic phosphte, it my be presumed tht ketoldose phosphorylted t Cg position is the product of the rection. Confirmtion of the genertion of product hving the properties of reducing sugr contining keto group ws obtined by tretment of the product with lkline phosphtse nd subsequent chrcteriztion by pper chromtogrphy nd by color rections. For the chromtogrphic chrcteriztion of the product, the following procedure ws dopted unit of purified glctose6 phosphte dehydrogense ws dded to totl volume of 1 ml, contining 100 pmol of sodium phosphte buffer, ph 7.4, 2 pmol TABLE Involvement of sulfhydryl group for enzyme ctivity The complete system contined in totl volume of 1 ml, 100 pmol of sodium phosphte buffer, ph 7.4, 0.5 pm01 of NAD, nd the requisite mount of the enzyme. The rection ws strted with the ddition of 1 pm01 of glctose 6.phosphte. Additions Complete + pchloromercuribenzote (5 X UY3 mm) (2 x 10Z rnm) (5 x 10Z rnm) (5 X lo+ mm) + Mercptoethnol (10 mm) Complete + Iodocette V Activity % (lrnm) (2 rnm) 65 I Time ( mins 1 FIG. 5. Correspondence between NADH formtion nd incresed reducing vlue. For this experiment, the chnge in bsorbnce t 340 nm ws mesured in cuvette tht contined 100 pmol of sodium phosphte buffer, ph 7.4, 1 wmol of NAD, nd 1 pmol of glctose 6phosphte. The redings were tken t fixed intervls of time s is indicted in the figure. For the determintion of reducing vlue severl tubes were set up with the sme composition. The enzymtic rections were stopped in ech tube t different times with lkline copper sulfte solution nd put on wter bth. OC nd Rm indicte increse in bsorbnce t 340 nm nd bsorbnce t 540 nm. of glctose 6phosphte, nd 1 pmol of NAD. About 0.75 pmol of the product ws llowed to form s mesured by the increse in bsorbnce t 340 nm. Contents of five such tubes were pooled. Along with these incubtion mixtures, five control tubes were run which contined ll of the regents except NAD. Contents of these control tubes were collected together nd were subsequently treted in the sme mnner s the experimentl smple. After djusting the ph of the solution to 9, 30 units of lkline phosphtse were dded nd the rection ws llowed to proceed for 2 hours t 37. The protein ws removed by trichlorocetic cid precipittion nd centrifugtion. Mixed bed resin ws dded btchwise to the cler superntnt until the ph of the superntnt corresponded to the ph of the distilled wter. The solution ws evported to dryness under reduced pressure t room temperture. The dried mteril ws dissolved in 0.1 ml of wter. Ten microliters of the solution were now spotted on Wht,

6 3600 mn No. 1 pper. In seprte lnes the following were simultneously spotted, () 10 ~1 of the control smple, (5) 10 ~1 of mixture contining 15 pg ech of glucose, glctose, nd fructose, (c) ll of the reference sugrs individully in seprte lnes. Severl chromtogrms were developed with two solvent systems. Solvent A consisted of ethylcettepyridinewter in 8 :2: 1 proportions (v/v) nd Solvent B consisted of glcil cetic cidisopropyl lcoholwter in 3:l:l proportions (v/v). After 18 to 20 hours of descending chromtogrphy, chromtogrms were developed in ech cse with the following sprying regents, () lkline silver nitrte (19), (5) pminobenzoic cid (20), nd (c) npthoresorcinol (21). Assuming the distnce trvelled by glucose to be 0 (RoiJ, both the experimentl nd the control smple showed with both the solvents one spot which corresponded with known glctose. The experimentl smple however, showed fster moving spot with both of the solvents which ws bsent in the cse of the control smple. The RGle vlue of this fster moving spot ws 2.0 nd 1.2 for Solvent A nd Solvent B, respectively. Under identicl conditions, the RGlc vlues for glctose nd fructose were found to be 0.81 nd 1.37 in Solvent A nd 0.93 nd 8 in Solvent 3. paminobenzoic cid nd npthoresorcinol re known to be generlly specific for reducing sugrs. Moreover, oxidtion of the sugr t Ci position would hve resulted in the dsorption of the product on the resin. Color rection tests specific for ketoses were lso crried out. For this, the product of the enzymtic rection ws formed in the sme wy s for chromtogrphic seprtion except tht 0.3 pmol of glctose 6phosphte ws tken in ech tube to ensure compiete conversion of glctose 6phosphte into the product. Control smples without NAD were lso incubted in this cse. After tretment with lkline phosphtse nd the resin, liquots from the experimentl nd control tubes, ech contining bout 25 to 40 pg of reducing sugr were subjected to cysteinecrbzole (22) nd resorcinol (23) color rections, Control rections were lso crried out for tubes contining seprtely, 100 pg of glctose, 50 I.cg of fructose, nd 100 pg of methylglyoxl. After 10 min of color development with cysteinecrbzole rection, only tubes contining fructose nd the product showed the typicl pink colors for the keto group. Methylglyoxl developed blue color wheres tubes contining glctose nd the control showed the color of the blnk. The bsorbnce t 560 nm with lcm light pth for fructose nd the product were nd 0.095, respectively. The nture of the spectrum for the two smples were lso identicl between 500 nd 600 nm. When similr color rections were crried out with resorcinol (23), glctose nd the control filed to develop ny color but the tubes contining fructose, the product, nd methylglyoxl developed pink colors. The bsorbnce t 520 nm for these tubes ws 0.26, 0.09, nd 0.04, respectively. In this cse, lso the nture of the spectrum for fructose nd the product were lmost identicl. The positive color tests with cysteinecrbzole nd resorcinol indicted the possible presence of keto group in the molecule. The ketoldose nture of the phosphorylted product rised the possibility tht this compound could serve s the substrte for the glyoxlse enzyme system. This ws tested with glyoxlse I lone, following the method of Rcker (17). About 0.2 pmol of the product ws first formed in totl volume of 1 ml contining 100 pmol of sodium phosphte buffer, ph 7.4, 0.4 pmol of glctose 6phosphte, excess glctose6phosphte dehydrogense, nd limiting mounts (0.2 pmol) of NAD. Twotenths milliliter of this liquot contining bout 0.04 pmol of the product ws now trnsferred to n ssy mixture of 1 ml contining 100 pmol of sodium phosphte buffer, ph 7.0, nd 3.5 pmol of glutthione. Absorbnce of this incubtion medium t 240 nm ws 30. On ddition of 2 ~1 of glyoxlse I, contining 2 units of ctivity, no increse in bsorbnce could be observed for 4 min. When 0.2 pmol of methylglyoxl ws however dded to the system, very rpid increse in bsorbnce ws noted. The product therefore pprently filed to rect with glutthione in the presence of glyoxlse I. Further, ddition of excess glyoxlse I nd glyoxlse II to the product in the presence of ctlytic mounts of glutthione filed to show ny decrese in the totl reducing vlue. Under identicl conditions, with methylglyoxl s the substrte, significnt decrese in the reducing vlue ws observed t the end of 30min incubtion period. DISCUSSION Glctose hs been shown to be uniformly ctbolized in lrge vriety of cell types through the Leloir pthwy described in Ref. 24. The only other route of glctose metbolism tht hs been chrcterized in detil, is the one opertive in Pseudomons scchrophi2i (25, 26). There is, however, evidence in literture tht suggests tht n lternte weker pthwy my lso be opertive in the mmmlin systems. Thus, certin glctosemic individuls were shown to hve the bility to metbolize considerble quntities of glctose (27, 28). Further, Segl et l. (29) reported tht some glctosemic individuls were ble to convert crbon 1 of intrvenously dministered glctose to crbon dioxide to quite lrge extent. Using [1YJglctose, these workers lso showed tht glctose is metbolized to significnt extent in glctosemic liver when compred to norml liver control (30). More recently, Petriccini et l. (31) hve shown tht glctosemic fibroblsts produce 14C02 from [114C]glctose, but this does not begin until fter 4 dys of incubtion. Rdioutogrphy of cultured, humn glctosemic, nd norml cells lso suggests the possible existence of n lternte pthwy for glctose metbolism (32). The existence of UDPglctose pyrophosphorylse discovered by Isselbcher (33) is not dequte enough to explin these dt s the rte of conversion of glctose lphosphte to UDPglctose by this enzyme is not very significnt (34). The possibility tht hexose6 phosphte dehydrogense might be involved in the supposed uxiliry pthwy ws initilly suggested by Beutler nd Morrison (7) but hs subsequently been ruled out by these uthors on the bsis of both physiologicl (35) nd biochemicl (36) experiments. Thus, the product of hexose6phosphte dehydrogense rection with glctose 6phosphte s substrte is 6phosphoglctonic cid which is dedend product of metbolism (36). As for glctose6phosphte dehydrogense, there is no bsolutely direct evidence t the moment to suggest tht this enzyme might be involved in n uxiliry pthwy of glctose metbolism. However, we hve observed tht UDPglucose or UDPglctose specificlly inhibits the enzyme t low concentrtions of the substrte (2). In ny cse, if the product is phosphorylted ketoldose nd not 6phosphoglctonic cid, the possibility cnnot be excluded tht the enzyme lies on n lternte pthwy for the ctbolism of glctose. We hve lredy pointed out tht the presence of glctose 6 phosphte s norml metbolite hs not yet been unequivoclly demonstrted (3,4). Glctose 6phosphte hs been shown to be formed from glctose lphosphte by phosphoglucomutse in in vitro systems by Posternk nd Resselet (37) nd lso by Lowry nd Pssonneu (38). The rte of formtion of glctose 6phosphte in such rtificil systems is however too smll to

7 3601 suggest ny possible metbolic role for the enzyme in synthesizing 18. glctose 6phosphte in cellulr systems. It is therefore not 19, unlikely tht specific enzyme my be present in mmmlin liver for phosphorylting glctose in the Cg position. Whether 20. glctose6phosphte dehydrogense long with other undetected enzymes leds to n lternte pth in mmmlin liver 21. remins to be estblished. 22. REFERENCES RAY, M., PAL, D. K., AND BHADURI, A. (1972) FEBS (Fed. Eur. Biochem. Sot.) Lett. 26, RAY, M., AND BHADURI, A. (1973) Ninth Znt. Congr. Biochem. 25. Abslr. p INOUYE, T., TANNENBAUM, M., AND HSIA, D. Y.Y. (1962) Nture 193, 67 NG, W. G. (1964) Ph.D. thesis, University of Southern Cliforni. Universitv Microfilm Incorported, Los Angeles OHNO, S.; MORRISON, M., AND BEUTL& E. (i966) Science 163, SHAW, C. R. (1966) Science 163, BEUTLER, E., AND MORRISON, M. (1967) J. Biol. Chem. 242, BEUTLF,R, E. (1967) Science 166, SHAW. C. R.. AND KOEN, A. L. (1965) J. Histochem. Cytoch,em. 13, KEILIN, D., AND HARTREE, E. F. (1938) Proc. Roy. Sot. London Biol. Sci. 124, 397 NELSON, N. (1944) J. Biol. Chem. 163, 375 LOWRY, 0. H., AND LOPEZ, J. A. (1946) J. Biol. Chem. 162,421 WARBURG, O., AND CHRISTIAN, W. (1940) Biochem ,384 Arch. Biochem. Biophys. 141, 155 LOWRY, 0. H., ROSEBROUGH, N. J.,FARR, A. L., AND RANDALL, 36. SRIVASTAVA, S. K., AND BEUTLER, E. (1969) J. Biol. Chem. R. J. (1951) J. Biol. Chem. 193, , 6377 KORNBERG, A. (1955) Methods Enzymol. 1, POSTERNAK, T., AND RESSELET, J. P. (1954) Helv. Chim. Act HORECKER, B. L., AND SMYRNIOTIS, P. Z. (1955) Methods 37, 246 Enzymol. 1, LOWRY, 0. H., AND PASSONNEAU, J. V. (1969) J. Biol. Chem. RACKER, E. (1951) J. Biol. Chem. 190, , SCHNEIDER, W. C., AND HOGEBOOM, G. H. (1950) J. Biol. Chem. 183, 123 TREVELYAN, W. E., PROCTER, D. P., AND HARRISON, J. S. (1950) Nture 166, 444 HEFTMANN, E. (1962) Chromtogrphy, Reinhold Publishing Corportion, p. 507 ISHERWOOD, F. A., AND JERMYN, M. A. (1951) Biochem. J. 48, 515 DISCHE, Z., AND BORENFREUND, E. (1951) J. Biol. Chem. 192, 583 ROE, J. H., EPSTEIN, J. H., AND GOLDSTEIN, N. P. (1949) J. Biol. Chem. 178, 839 KALCKAR, H. M. (1958) Physiol. Rev. 20, 111 DE LEY, J., AND DUODOROFF, M. (1957) J. Biol. Chem. 227, 745 ORNSTON, L. N. (1971) Bcteril. Rev. 36, 87 CUSWORTH, D. C., DENT, C. E., AND FLYNN, F. V. (1955) Arch. Dis. Childhood KOMROWER, G. M., SCHW~RZ, V., HOLZKL, A., AND GOLDBERG, L. (1956) Arch. Dis. Childhood 31,254 &GAL, S., BLAIR, A., AND TOPPER, Y. J. (1962) Science 136, 150 TOPPER, Y. J., LASTER, L., AND SEG.~L, S. (1962) Nture 196, PETRICCIANI, J. C., BINDER, M. K., MERRIL. C. R.. AND GEIER. M. R. (19?2) Science 176; HILL. H. Z.. AND PUCK. T. T. (1973) Science ISSE&CHE~, K. J. (1958) J. kol. khem. 232, 4i9 34. ABRAHAM, H. D., AND HOWELL, R. R. (1969) J. Biol. Chem. 244, MANDULA, B., SRJVASTAVA, S. K., AND BEUTLER, E. (1970)