Mitochondrial and cytosolic hexokinase from rat brain: One and the same enzyme?

112

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BB~ 21325

MITOCHONDRIAL SAME ENZYME?

A N D C ' f T O S O L I C H E X O K I N A S E F R O M RAT BRAIN: O N E A N D T H E

ERIK D SPRENGERS. ANK'~ H L KO[-NDERMa~N,mdGI).RARD EJ S~.~[. [)wilton o/ ~,led~tal Enz~mo/ogv. 4<,nh,mt~ tt,v~tt,d. P 0 l:~,,,. 1:,25¢L 351,# l~'l~eJu ¢ lhe ",'cthcthmd~

(Received February 5th. 1982) (Rev~.,ed manu,~.r~ptrecep.cd September 10th 1982~

Kt'l ~t~,r(/~ [le~t,],ma~t' ~llltl~t', .~t)hlblh_-tltlt)tl.

(R(I[ [~ltl/HI

Rat brain mitochondrial hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was solubUized b} treatment of the mitochondria with glucose 6-phosphate and partl) purified. The solubilized e n z ) l n e ~ a s compared with the cytosolic e n z y m e fraction. ]'he solubilized and cytosolic e n z y m e s were also compared ~ith the e n z y m e bound to the mitochondrial membrane. The following observations were made. !. There is no difference in electrophoretic mobility on celhdose-acetate between the cb tosolic and the solubilized euz} me. Both fractions are hexokinase i s o e n z y m e I. 2. There is no difference in kinetic parameters between the c~tosolie or solubilized e n z y m e s ( P < 0.001). For the cytosolic enz.~me K,, for glucose was 0.067 m M (S.E. = 0.024, n = 7); K,, for M g A T P ~- was 0.42 m M (S.E. = 0.13. n = 7) and Ki..,pp for glucose 1,6-diphosphate was 0.084 m M ( S . E . = 0.011, n = 5). For the solubilized e n z } m e K m for glucose ~ a s 0.071 m M (S.E. = 0.021, n = 6): K,,, for M g A T P z - was 0.38 m M (S.E. = 0.11, n = 6) and Ki.ap p for glucose 1,6-diphosphate was 0.074 m M (S.E. = 0.010, n = 5). H o w m e r when bound to the mitochondrial membrane, the enz3 me has higher affinities for its substrates and a l o ~ e r affini~ for the inhibitor glucose 1,6-diphosphate. For the mitochondrial fraction K m for glucose was 0.045 m M (S.E. = 0.013, n = 7); K m for M g A T P 2~ was 0.13 m M (S.E. = 0.02, n = 7) and Ki.,p p for glucose 1,6-diphosphate ~,as 0.33 m M (S.E. = 0.03, n = 5). 3. The c}tosoli¢ and solubilized e n z y m e could be (re)-bound to depleted mitochondria to the same extent and ~ith the same affini~. Limited proteolysis fully destro~ ed the e n z y m e ' s abilit} to bind to depleted milochondria. 4. Our data support the hypothesis that soluble- and solubilizable e n z y m e from rat brain are one and the same e n z y m e , and that there is a simple equilibrium between the e n z y m e in these two pools.

Introduction Hexokinase (ATP: p-hexose 6-pho,phc, transferase. E(' 2.7.1.1) from m a m m a h a n brain tis.,,ue i,, present in tx~o ,,ubcellular fraction.,,. Approx. 20% of the total he,~okina~e activit'~ is found Jn the c\tosolic fraction. The other 80c~ is b o u n d to the m~tochondrml outer mernbrar~e, apparentl 3 attm.hed to a specific receptor. A b o u t 50c~ of the b o u n d enzyme can .,,peciflcallv be bolubilized from the mltoctlondria by Ira,. concentration., of glucose 6-phosphate. When b o u n d to the mitochondrm. 03o4-4165 83 11000 0000, $03 00 , 1983 F.l.,exler Biomcdu.al Pre.,.,

the enzxme t.,, less inhibited b'~ pho~phorylated hexose~. So. b i n d i n g to the mitochondrla x~Otlld facdate anaerobic glycoly.si.,, [I -4]. Whether there i.~ a .,,mlple e q u i l l b r m m betx~een the cytosolic a n d b o u n d enzyme, or whether a inore f u n d a m e n t a l difference between the t ~ o fractions exists, has been a matter of dispute. A h h o u g h such properties as molecular x~mght. electrophoretic mobility and chromatographic behavlour are ldenhcal [5 7]. T h o l n p s o n and Bachelard [8] claim, on the ba.,,la of an enzyme kinetic study, that a f u n d a m e n t a l difference between the

113 two enzyme forms exists. Other authors believe that developmental changes in the s o l u b l e / p a r ticulate distribution in cerebellum, and cerebrum of rat and chicken is an indication of a more fundamental difference between the two enzyme form> [9]. Also differences in soluble/particulate distribution in different regions of the brain are used as an argument in favour of the latter h 5pothesis [10,11]. Limited proteolysis of the enzyme ~otall~ destroys its ability to bind to mitochondrial membranes [1]. So. soluble hexokinase might be a post-translational modification product or even an artefact of preparation. We investigated the similarities and differences between the cytosohc and b o u n d enzyme, in order to establish the relationship between soluble and mitochondrial hexokinase. Materials and Methods

Whole brain was taken from male Wistar rats and either used immediately or stored at - 8 0 ° C before use. Preparation of enzyme fractions. Unless otherwise indicated, all steps were performed at 4°C. Brain was homogenized in a Potter homogemzer, in a 10-fold excess of extraction buffer, containing 10 mM Tris-HCl (pH 7.4 at 20°C): 0.25 M sucrose: 1.0 mM E D T A and 1.0 mM dithiothre~tol. The homogenate was centrifuged for 10 rain at 800 x g to remove the cell debris. The 800 × g pellet was washed once with extraction buffer. The 800 x g supernatants were combined and centrifuged for 15 rain at 27000 × g. The 27000 x g >upernatant is referred to as the 'cytosolic fraction'. The 27 000 × g pellet was washed once with extraction buffer. The final pellet is referred to as "mitochondrial fraction'. Hexokinase was solubilized from the ' m i t o chondrial fraction" by incubation of the pellet m extraction buffer, containing 0.5 m M glucose 6phosphate, for 15 rain at 25°C. Subsequently the mixture was centrifuged for 15 min at 27000 x g. Th~s supernatant is referred to as the "solubilized enzyme'. The pellet was washed twice with extraction buffer to remove the glucose 6-phosphate. The final pellet is referred to as the "depleted mitochondria'. Cytosolic and solubilized enzymes

were further purified by batchwise DEAE-Sephadex chromatography, as described by Rijksen et al. [12]. The eluates were precipitated bx' dialysis against extraction buffer, containing 50 g / 1 0 0 ml ( N H 4 ) 2 S O 4. The precipitates were collected and frozen at - 8 0 ° C until use. (Re)-binding experiments were performed either wxth 'depleted mLtochondrla' or with the unstripped ~mitochondrial fraction'. Enzymes were dlalvsed before use against e x t r a c t i o n buffer. E n z y m e s and "depleted mitochondria" were mixed, then 10 mM MgCI= was added, and the m~xture was incubated for 15 minutes at 25°C. After incubation the mixture was centrifuged for 15 rain at 27000 × g to separate b o u n d and u n b o u n d enzyme. Enzyme activity was determined using the coupled glucose 6-phosphate dehydrogenase assay. A part of the mitochondrial hexokinase is not readily assayable, but becomes apparent only after treatment of the mitochondria by a variety of membrane disrupting techniques [13]. This activity l> called latent activity [14]. For the determination of the hexokinase activity before and after (re)-bmding all fractions were incubated for 20 rain at 0°C with 0.5% Triton X-100. The activity of u n b o u n d hexokmase is not at all affected by such treatment. For the determination of the kinetic constants of the soluble and bound enzyme fractions, Triton X-100 was, of course, omitted. Cellulose acetate electrophoresis was performed a> described previously [15]. Determination of K,,, for glucose and K,,, for M g A T P z was performed at 37°C in a buffer containing 33 mM Tris-HC1 (pH 8.0 at 20°C), 0.2 mM N A D P ~ and 1 I U / ' m l glucose-6-phosphate dehydrogenase, lk'lg~r~, was kept at a constant excess of 5.0 raM, using a K = 2.51 • 10 s M for the M g A T P z complex [16]. Reactions were started by the addition of enzyme. Glucose was in the range of 0.016 10.0 raM: M g A T P z was in the range of 0.05 5.0 raM. The possibility of the presence of substrate consuming side-reactions was ruled out by end-point analysis of the hexokinase reaction. The data were fitted into the model of the sequenhal r a n d o m blsubstrate reaction mechanism, using the S E Q U E N computer p r o g r a m m e described by Cleland [17]. K, ,,pp for glucose 1,6-diphosphate, defined as that concentration of this c o m p o u n d resulting in a 50% inhibition of the enzyme, x~as determined at 37°C

114

in a buffer c o n t a i n i n g 33 m M T r i s - H C l (pH 7.15 at 37°C): 1.75 m M MgCI~: 0.35 m M A T P 4 • 7.0 m M glucose: 0.75 m M N A D P + a n d 1 IU.."ml g l u c o s e - 6 - p h o s p h a t e dehydrogenase. Gluco,,,e 1.6d i p h o s p h a t e was in the range of 0 . 0 - 1.0 raM. The kinetic constants of the different enzyme fracuon,, were c o m p a r e d , using the Student'.,, t-test prog r a m m e of a pocket calculator. Partial proteolysis by c h y m o t r y p s m was performed in e x t r a c t i o n buffer for 30 rain at 25°C. at a c h y m o t r y p s i n c o n c e n t r a t i o n s of 10 / , g / m l for the purified soluble enzyme and 1.0 p . g / m l for the purified solubilized enzyme. Chemicals. G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e from yeast, c h y m o t r y p s i n A a from bovine pancreas, ATP, N A D P * a n d glucose 1,6-diphosphate were o b t a i n e d from Boehrmger. M a n n h m m , F.R.G. All chemicals were of the highest purity available. Results In fresh rat brain approx. 18e/ of the total h e x o k m a s e activity t~ found in the cvto,,ohc fraction (18 + S.D. = 4, n = 9). Freezing of the brain tl.,,sue at - 8 0 ° C does not change this percentage, nor is there any. difference m the enzyme kinet,c b e h a v i o u r of the e n z y m e fractions p t m f i e d from either fresh or frozen t,ssue. Of the "mltochondrml fraction' a b o u t 50% can be .,,olubihzed by gluco.,,e 6 - p h o s p h a t e (0.5 raM) under ,sotomc condition~. The remaining activlt,, can only be solubfllzed by the c o m b i n e d action of m e m b r a n e &.,,ruptmg agents {Triton X-100: s o m c a u o n ) and glucose 6p h o s p h a t e (0.5 mM). This latter acti~it\ i~ app a r e n t l y e n t r a p p e d m ~ynaptosomes, "pinched-off" nerve endings, origininating d u r i n g the homogenization of brain tissue under ,.,,otomc conditzons [181. Purification of the enzymes from the two subcellular fractions results m a p r e p a r a t i o n with a specific activity of 0.5 I U / ' m g protein for the soluble enzyme, a n d a specific activity of 10 ILI/ ' mg p r o t e i n for the solubihzed form. The ,,leld of thi,, p a r t i a l purification is approx. 70~. In n o r m a l rat brain, only the t_~pe I h e x o k m a s e is present, as can be seen m Fig. 1. There ts no difference in electrophorettc m o b i h t v on cellulo.,,eacetate between the "c,,.tobohc" and ' s o l u b d i z e d ' enzyme. S u b f r a c t i o n s of hexokinase, as ha.,, been

Pig 1. Cellulo,,e at.elate ele,._tlophore,,i,, ol h e \ o k l n a ' , e F l o m lelt to right, h u m a n e r s t h r o c ~ t e h e x o k m a s e . ,,oluble rat br,un he\oklna,,,e. ~o[ublhzed rat br,lul h e \ o k l n d x e

r e p o r t e d for the ervthrocvte enzyme, are iaot found in brain tls.,,ue [15]. F,g. 2 shox~.,, the rebinding d a t a of "cvtosolic" and 'solubilized" h e x o k m a s e to "depleted n m o c h o n d n a ' . ' ( ' v t o s o h c hexoklna,,c" binds equall_~ well to d e p l e t e d m , t o c h o n d r i a a,, ",,olubihzed enzyme" doe~. The increase in h e x o k m a s e act,vtt\' in the pellet c o r r e s p o n d s well to the decrease in act,vtt\ in the ~oluble fraction after binding. ThL,, mean.,, that. tit saturating sub~trate c o n c e n t r a u o n s , the enzyme ,,, equally actmxe in the soluble form, as ~ h e n b o u n d to the m , t o c h o n d n a l outer m e m b r a n e . L,mited p r o t e o l s •,i.,, of rather "cvtosohc" or 'solubilized" h e x o k m a s e x~,th ctaymotr 5psln g,ve~ no decrease ,11 euz\l'la¢' act,xLtv at ,ill. but fully de.,,troxs the enz,,nae',, abfl,tv to brad to depleted pellets (data not sho~n).

115

sites are also available in the "mitochondrial fraclon': only half of the binding sites are occupied by enzyme. Rebound hexokinase, either to "depleted mitochondria" or to "mitochondrial fraction' could quantitatively be re-solubilized by glucose 6-phosphate (0.5 mM). This indicates that rebinding is a glucose &phosphate-dependent process. and not ju.st aspecific absorbuon to mltochondrial membranes. The kinetic behaviour of the "cvtosolic' and "solubllized enzymes' is identical ( P < 0.001: Table I). The data fit v, ell m the rapid equilibrium random bireactant kinetic model, as is indicated by the low values of sigma (the square root of the residual least square), produced by the SEQUEN programme of Cleland [17]. "Mitochondrial fraction" and "depleted mitochondria' also show identical kinetic behaviour ( P < 0.001. Table I). However. the "bound' enzyme ('mitochondrlal fraction' and 'depleted mitochondria') showed a 3-tlimes hligher affinity for MgATP 2 and a 1.5times higher affinity for glucose than the "soluble" enzvme ('cytosolic enzyme' and "solubilized enzyme') ( P < 0.002: Table I). Moreover the "bound' enzyme s h o w e d a four times higher K, ,pt, for glucose 1.&&phosphate than the "soluble" enzyme ( P < 0 . 0 0 1 : T a b l e l ) . It is noteworthy that the difference m K, MgATP 2 /; K m MgATP 2 between the "cytosolic' and ",~olubilized enzyme" is not statistically significant: 0.1 > P > 0.05 (the value K, MgATP2 ./K,,, MgATP z determines whether the intersection point of the set of Lineweaver-Burk plots hes above, on, or below the x-axis [19]).

L

(tU)

;

o.~t

o

C3 i

--c---.4--

Fig. 2 R e b i n d i n g plot of soluble ~O O ) and .solubdlzed 10 O ) h e x o k m a s e to depleted m~tochondrla F r o m the quanlilt,, of depleted m l t o c h o n d n a used m this e x p e r i m e n t 0.1 I l l h e x o k m a s e ( H K ) had been solubdlzed

'Depleted mitochondria" have more binding sites available than can be expected as a rebult of >olubilization of hexokmase. In the experiment shown in Fig. 2, a quantit~ of depleted pellet has been used, from which only 0.1 IU hexoklnase had been solubilized. To this quantity 0.25 IU of hexokinase could be (re)-bound. Addition of hexokina~e to unstripped "n-fitochondrial fraction' lead.,, to an increase of activity' of the pellets by ahmost a factor 2. This means that excess binding

TABLE I KINETIC P-~RAMETERS OF SEVERAL FRACTIONS OF HEXOKIN¢SE Value:, are average,, of fl%e to se,,en indepelldent delicrmlnallon~, lihe ,~tanddrd errors IS E ) of t~.hlch ,ire indlcdted m bracket,, Sample

K., for glucose (raM)

K

for

Mg~Tp2 mM)

( K, Mg~TP2 k , . MgATP-"

)

, ,,pp fOl"

gluco,e 1.6dlphosphate

(raM) I Soluble h e x o k m a s e 11. S o l u h h z e d he\oklna:,c 111 M t t o c h o n d n a l fracuon IV Depleted nuto~,hondrm

0 0 6 7 (_+0.024) 0071 t + 0 0 2 1 ) 0045(±0013} 0 043 ( + 0 016)

042 ~ ± 0 13) 038 ( ± 0 I1) 0 13 (+0(12) 0 14 ( + 0 0 3 )

0 84 ( + 0 44) 1 49 ( _+0 28) I 04 I + 0 38) 1 31) I ±(174)

0.1iS4( ± 0 01 I ) l) 074 I + 0 0101 033 1+t)03) ()31 ( + l ) l ) 3 )

116

Discussion H e x o k i n a s e in rat brain is found in two subcellular fractions. A p p r o x . 20% of the total activity is found in the 'cytosolic fraction'. The other 80% is found in association with the " m i t o c h o n d r i a l fraction'. Of this latter fraction, a b o u t 50% can be solubilized by glucose & p h o s p h a t e (0.5 mM). The r e m a i n i n g activity can be solubilized by a comb i n e d action of m e m b r a n e - d i s r u p t i n g agents (0.5% T r i t o n X-100 or sonication) a n d glucose 6-phosphate. Apparently', this latter activity ,s e n t r a p p e d m s y n a p t o s o m e s . 'pinched-off" nerve endings. or,ginating d u r m g the h o m o g e n i z a t i o n of brain tissue under isotomc c o n d i t i o n s [18]. T r e a t m e n t of the "mitochondria[ fraction' with Triton X-100 (0.5%) alone, also solubilizes approx. 50% of the b o u n d activity. This may suggest that such a percentage of the hexokinase of the m i t o c h o n d r m l fraction is in fact soluble enzyme, e n t r a p p e d m s y n a p t o s o m e s . However, kinetic behaviot, r of " m l t o c h o n d r i a l fraction' and "depleted pellet" is identical, showing that all or at lea~t most of the m i t o c h o n d r i a l activ,tv is " b o u n d ' hexokmase. Solubilization of this activity bv Triton X-100 (0.5%,) could be due to e x t r a c t , o n of the hexok,nase-binding protein, conjugated ~lth hexokmase, from the t m t o c h o n d r i a l outer membrane. This is s u p p o r t e d by the observation that T r i t o n X-100-.,,olubfllzed h e x o k m a s e has a nluch lower affinity for glucose 1.6-diphosphate than glucose 6 - p h o s p h a t e :,olubdlzed enzyme. Treatment of the Triton X-100 solubilized enzyme ~ l t h glucoae & p h o s p h a t e increases its affinity for glucose 1.6-&sphosphate to the le,,el of gluco.,e 6p h o s p h a t e - s o l u b d l z e d enzyme (E.D. Sprengers. u n p u b h s h e d data). Extract,on of the hexokina..,eb i n d i n g protein from the m i t o c h o n d n a l outer m e m b r a n e b \ non-Joint detergents, has been observed by Feigner et al. [2]. The relationship between the "cytosohc" a n d " m i t o c h o n d r m l ' hexok m a s e has been a m a t t e r of &~pute. Some author,, beheve that a simple e q u i l i b r i u m exists between these two enzynle forms. Others claim a more f u n d a m e n t a l difference between the enz\ me.., from these two subcellular fractions (for review .,,ee Ref. 3). M a i n l y three lines of evidence are followed to pro,,e the existence of a distract cy topla.,,matlc and m i t o c h o n d r m l enzyme. In the first place, changes

in the ratio c y t o a o l i c / m l t o c h o n d n a l hexokinase d u r m g d e v e l o p m e n t of rat c e r e b r u m and cerebellurn arc taken a~ indicative for the existence of distinct c y t o p l a s m i c a n d m i t o c h o n d r i a l forms of the enzyme, each follo,,ving its own characterb, uc p a t t e r n during d e v e l o p m e n t [9]. Secondly, difference in the ratio cytosolic/' m i t o c h o n d r i a l h e x o k m a s e m different regions of the brain, i.e. a relat,vely high c o n t e n t of ~oluble hexokina~e in white matter, and a relatively 1o~ content of thi.s enz,,me form in grey' inatter, is put foreward as an a r g u m e n t in favour of the hypothesis of the existence of two different enzx'mes [10,11]. However. the ratio c y t o s o l i c / m i t o c h o n d r l a l hexokinase ,s not only "in v,tro" but al~o "in rico" subject to c o n s i d e r a b l e changes due to external factors: in xitro the c o n c e n t r a t i o n s of Mg :+ . glucose 6-pho.,phate and ATP determine the ~oluble," nfitochondrial h e x o k m a s e ratio, in vwo the metabolic state of the cell [20.21]. So the d e v e l o p m e n t a l and r e g i o n a l difference.,, m the .,,oluble, m i t o c h o n d r m l hexokma.,,e ratio, are a p o o r argument to prove the exL,,tence of two d~.,,tinct enzymes. The third and most , m p o r t a n t a r g u m e n t in fa,,our of this latter hypothesis were the enzvmekinet,c differences bet~veen the "cvtosolic" and ",,olubilized enzyme' reported by T h o m p s o n and Bachelard. for the o x - b r a m enz 3 me [8]. However. x~,th the rat brain enzyme ~ e oht a m e d different re~ult~. We purified and dial'~zed the enz,,mes from the two subcellular fractions m the ~,arne wax, and their k m e u c b e h a v i o u r with re~pect t o the sub~trates M g A T P : and glucose arm to the inhlb,tor glucose 1.6-d,pho,,phate v.a.,, fully Mentical (Table I). Our d a t a are con,,lstent with the c o m m o n ~ e w that the enzyme operates by a .,,equenual reaction mechauism [22.23]. When b o u n d to the m i t o c h o n d r i a l outer m e m b r a n e the e n z w n e ~hows a higher affinity for its substrate~ and a lox~,er affimt,, for the inhibitor glucose 1.6d i p h o a p h a t e {and p r o b a b l y other p h o s p h o r y l a t e d hexoses a~ well). Such a p h e n o m e n o n has been observed for the enzyme of several other .,pecm.,, [24 26]. O u r kinetic con.~tant.,, agree well ~ l t h tho~e reported prevlou.,,ly for the ..,oluble and b o u n d e n z y m e fractions from rat brain enzxme [3,7.27]. T h e d i f f e r e n c e betx~een the xalue., of K, M g A T P z / K , , M g A T P : ( = K, gluco~,e.'K,,, g[t,-

117

cose) of the "cytosolic' and 'solubilized enzymes' is not statistically significant (0.1 > P > 0.05) due to the large standard errors in these constants. This is noteworthy because for the ox-brain enzymes the difference in K, MgATP-" / K m MgATP 2 between the "cytosolic' and 'solubdized enzyme" was one of the arguments in favour of the hypothesis that the 'cytosolic" and "solubilized enzymes' were not identical [8]. Glucose 1,6-diphosphate is able to solubihze the bound enzyme from the mitochondrial outer-membrane, just as glucose 6-phosphate. So, solubihzation might interfere with the determination of K,,app for glucose 1.6-disphosphate. How.'ever. in the reaction mixture we keep [Mgi-~] at 1.4 raM. which pushes the eqmlibrium towards the bound form. Moreover ~,,e start the reaction by the addition of enzyme and measure initial rates. This abolishes the effects of solubihzation by glucose 1,6-diphosphate. So our kinetic data are consistent with the hypothesis that soluble and solubilized hexokinase i~ one and the same enzyme. In favour of th~s hypothes~s we add a new argument. (Re)binding to depleted mitochodria is accomplished to the same extent and with the same affinity by 5oluble hexokinase as by solubilized enzyme. This strongly suggests that the soluble and solubd~zed enzyme are ,,imply in equilibrium with each other. Lmaited proteolysis of hexokinase - either soluble or solubilized - fully destroys its (re)-binding capacity, although enzyme activity ts not affected. So. soluble hexokinase found in brain in not a proteolyzed form of the bindable enzyme. Remarkabl,,. the soluble hexokinase type I from h,,er and ervthrocvteb full,,' lack the ability to bmd to depleted mitochondria (data not shown). Our data support the hypothesis that hexokmase from rat brain is a true anabiquitous enzyme: the distrubution between the soluble and membranebound forms is influenced by metabolites (glucose 6-phosphate) and has m turn influence on the metabolic state of the cell (the membrane-bound enzyme is more active). Indeed, m a recent paper Kurokawa et al. [28] showed that the bound hexokmase ~s almost twice as effective for glucose 6-phosphate production as the unbound enzyme. Bustamante and Pederson [29] proposed that a form of hexokinase with a propensit5 for mitochondrial binding, plays a key role in the high

anaerobic glycolysis of hepatoma cells. They showed that only dedifferentiated tumor cell lines with a h~gh growth rate and elevated glycolysis had detectable mitochondrial hexokinase activity.

Acknowledgement Mrs. E i . Huisman-Backer Dirks is thanked for secretarml assistance.

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