Biochem. J. (1972) 130, 63-69Printed in Great Britain

Activation of Brain Hexokinase by Magnesium Ions andby Magnesium Ion-Adenosine Triphosphate Complex

By DANIEL L. PURICH and HERBERT J. FROMMDepartment ofBiochemistry and Biophysics, Iowa State University, Ames, Iowa 50010, U.S.A.

(Received 14 February 1972)

1. An alternative explanation for the kinetic data obtained by Bachelard (1971) for thebrain hexokinase reaction is presented. 2. Apparently sigmoidal saturation curves forMgATP2- based upon Bachelard's (1971) studies can be corrected to hyperbolic curvesby use of a stability constant for MgATP2- complex formation. 3. A number of othereffects related to the concentration-dependent stability of the MgATP2- complex and tothe presence of the inhibitory free uncomplexed ATP4- concentration are also explainedin terms of a non-allosteric role for either Mg2+ or MgATP2- fully consistent with anumber of previous reports on this enzyme. 4. A brief discussion of the validity of Hillplots in studies of multisubstrate co-operative enzymes is presented. 5. A simple model ispresented that demonstrates how enzymes obeying Michaelis-Menten kinetics candemonstrate sigmoidal velocity responses if the true substrate of the reaction is themetal-substrate complex.

In a recent report Bachelard (1971) described theallosteric activation of brain hexokinase by Mg2+ andMgATP2-. He found that substrate-saturation curvesof brain hexokinase for MgATP2- were sigmoidal atsubsaturating concentrations of glucose when theMgt0taj/ATP,otaj ratio was unity. On the other hand,he observed that these saturation curves werestrictly hyperbolic in the presence of excess of Mg2+(i.e., Mg2+a1/ATPtota1=5). Hill plots of these dataindicated that the number of binding sites forMgATP2- varied from 1.05 to 1.8 depending upon theMg2+ concentration. In addition, the sigmoidicityand deviation from Michaelis-Menten kinetics at a

Mgt0taj/ATP,ttaj ratio of 1.0 became less pronouncedwith increasing glucose concentration. Finally, hefound that although substrate-saturation curves forglucose were hyperbolic when the Mg2o+ta1/ATPtotajratio was unity, reciprocal plots were slightly non-linear. Bachelard interpreted these results to reflecthomotropic co-operative binding of MgATP2- tobrain hexokinase and postulated that Mg2+ acts as atopologically distinct activator site at low Mg2o,1/ATPtot,a ratios, but acts as an inhibitor whenever thisratio exceeds five.Examination of Bachelard's (1971) data has led us

to an alternative interpretation fully in agreementwith his experimental results but in harmony withprevious studies in which no evidence for suchcomplex binding of MgATP2- was obtained (Copley& Fromm, 1967; Ning et al., 1969; Purich & Fromm,1971). Our interpretation, which does not requirepostulating co-operative MgATP2- binding or thepresence of a distinct activator site for Mg2+, con-siders the presence of large quantities of ATP4- that

Vol. 130

may arise whenever the Mg,'.`aI/ATP,ttaj ratio is nearunity. Further, it is also possible to rationalize theglucose effects on the sigmoidicity and deviation fromMichaelis-Menten kinetics at low Mg2+ concentra-tions. Changes in the Hill coefficient for MgATP2-binding at different Mg2+ai/ATPt0taj ratios areexplained, and a discussion of the applicability of theHill equation in velocity studies of multisubstrateco-operative enzyme systems is presented. Thesestudies serve to suggest that there is no allostericinvolvement of Mg2` or MgATP2- in the brain hexo-kinase reaction, but that the concentration of freeuncomplexed Mg2+ in cerebral tissue may play animportant role in the regulation of the phosphoryla-tion of glucose by adjusting the concentration ofMgATP2- and ATP4-.

Results

Co-operative effects ofMgATP2

Bachelard (1971) recently explored the possibilitythat Mg2+ is involved in the hexokinase reaction in afashion distinct from its role as MgATP2- byexamining the initial reaction velocity dependence on[MgATP2-i at Mg2+,a/ATPt.ta1 ratios of 1.0 and 5.0.Whereas the velocity dependence in the presence ofexcess of Mg2+ appeared to be strictly hyperbolic,slightly sigmoidal responses were observed at thelower Mg2+ concentrations. Because all knownnucleoside di- and tri-phosphate-dependent phospho-transferase reactions require a bivalent metal ion tocombine with the nucleotide to form the activesubstrate (Cohn, 1968), we became interested in

63

D. L. PURICH AND H. J. FROMM

V

0.02

0 0.4 0.8

[MgATP2-] (mM,Fig. 1. MgATP2 saturation curves for brain hexo-

kinase at a Mg2o+,/ATPtot,l ratio of 1:1

Glucose concentrations were: o, 0.1 mM; *, 0.05mM;n, 0.035mM; and *, 0.025mM. The velocity measure-ments are those ofBachelard (1971) and theMgATP2-concentrations on the abscissa are corrected basedupon a stability constant of 73OOM-1, as describedin the text. Corrections were made on photographicenlargements of the original data presented byBachelard (1971) in Fig. l(b).

determining the percentage of ATPtotal that ischelated with Mg2+ under the conditions used byBachelard (1971). When the Mgt2+a/ATPtotal ratiowas 1.0, the percentage of ATPtotai present in solutionas MgATP2- varied with the extent of dilution. Forinstance, the concentration of Mg2o+aj and ATPtotaivaried from 0.05mM to 0.8mM in the experimentpresented in Fig. l(b) of Bachelard's (1971) report.If one assumes that the stability constant for the

MgATP2- complex in 0.15M-triethanolamine-HClbuffer (pH7.6) is 73OOM-1 (O'Sullivan & Perrin,1964; Bachelard, 1971), then the concentration ofMgATP2- will vary from 0.03mM to 0.71 mm. Thusconsiderable correction is necessary for the abscissavalues presented in Fig. l(b) of Bachelard's (1971)report. Since the correction is more substantial at thelower end of this concentration range, it appeared tous that such effects might well explain the sigmoidalcurvature that he observed. When such correctionswere made on photographic enlargmeents of Fig. 1(b)of Bachelard's (1971) paper, the rather normal hyper-bolic saturation curves shown in Fig. 1 were observed.For the sake of clarity, only the corrected curves arepresented.

Additional corrections are also possible for thedata presented by Bachelard (1971). For example, atpH7.6 approximately 18.3% of ATPto,at is present asthe protonated species, HATP3-, if one assumes thatHATP3- has a pKa of 6.95 (Smith & Alberty, 1956).The stability constant for the MgHATP'- complex isapproximately 31 M-l (Smith & Alberty, 1956), and itis quite unlikely that very much of the HATP3- ispresent as its Mg2+ ion complex. Further, ATP4- is apotent inhibitor of yeast and brain hexokinases(Hammes & Kochavi, 1962a,b; Ning et al., 1969;Bachelard & Goldfarb, 1969; Purich & Fromm,1971). Corrections for HATP3- and ATP4- were notmade as the deviations from hyperbolic velocityresponses observed by Bachelard (1971) appear to beaccountable in terms of the correction made in Fig. 1alone.The possibility that HATP3- inhibition can lead to

residual sigmoidicity remaining after the abovecorrection of Bachelard's (1971) data seems remote.As shown by Dalziel (1963) the presence of a com-petitive inhibitor as a constant fraction of the truesubstrate will still generate hyperbolic velocity versus[substrate] plots. At pH7.6 we have observed that,over a wide range of glucose saturation, velocityversus [ATP] plots remain hyperbolic (Ning et al.,1969). These measurements were made at 1.0mM-freeuncomplexed Mg2+, and the concentration andeffects of ATP4- are quite small. The observation ofhyperbolic responses under these conditions suggeststhat HATP3- is either a competitive inhibitor relativeto MgATP2- or not an inhibitor at all. If HATP3-acts as a competitive inhibitor, then the following rateexpression describes the velocity of the hexokinasereaction at limiting [Mg2+] in the presence of ATP4-and HATP3-:

Vmax. K4 / [ATPf4ree] [HATP3'\1+ 11+ + ~~

v [MgATP2] KATp4-free KHATP3-

+ K3 + K,K3 1+ [ATPfree]+ [HATP3-)[Glucose] [Glucose] [MgATP2-] KATP4-free KHATP3-

1972

AA

ACTIVATION OF BRAIN HEXOKINASE

Since at any pH the HATPt3-I/ATP4-aI ratio is equalto some constant (k), then [HATP3-]=k[MgATP2-]+k[ATPf4ree]. Substitution of this equality into theabove rate expression yields upon rearrangement:

that correction of Fig. 2(b) of Bachelard's (1971)paper requires large changes in the values plotted onboth the ordinate and the abscissa. The correctedplot is shown in Fig. 2 of the present paper.

Vmax.1 kK4 K4 [ATPIf4re] [ATPfree]\+ K3 kK1 K1K3V KHATP3_ [MgATP2-] KATP4-free KHATP3- [Glucose] KHATP3J [Glucose] [MgATP2-]

1+ [ATPf4r-] ± [ATPfre]KATP4-fe KHATP3-free/

Although this expression appears complex, oneshould note that it predicts strictly hyperbolicvelocity versus [MgATP2-] plots. The absence ofquadratic terms in [MgATP2-] precludes anyhigher-order dependence of velocity upon[MgATP2-].Bachelard (1971) attempted to illustrate more

clearly the departure from Michaelis-Menten kineticsby comparing plots of [MgATP2-]/v versus[MgATP2-] at several Mg2+ concentraions. Again,in the presence of low [Mg2+] he found markedcurvature in these plots. If one corrects these data asdescribed in the legend to Fig. 1, the curvature ofsuch plots is far less pronounced. It should be noted

_.

.-

0.3

[MgATP2-] (mM)

Fig. 2. Plot of [MgATP2i/v versus [MgATP2i forbrain hexokinase at a Mgt2'.,ATPtot,l ratio of1:1

Glucose concentrations were: o, 0.1 mM; *, 0.05mM;C1, 0.035mM; and *, 0.025mM. The velocity measure-ments are those of Bachelard (1971) and have beencorrected on the ordinate and abscissa by multiplica-tion of a factor representing the fraction of ATPtotaias MgATP2-, calculated as described in the legend toFig. 1. Corrections were made on photographicenlargements of Fig. 2(b) of Bachelard's (1971)reported data.

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Effect ofglucose on MgATP2 binding

In his studies of the effects of Mg2+ and MgATP2-on the kinetic properties of brain hexokinase,Bachelard observed non-linear velocity versus[MgATP2-] plots became less pronounced atelevated glucose concentrations and was apparentlyabsent at saturating concentrations of glucose(Bachelard, 1971; Bachelard & Goldfarb, 1969). In acompanion experiment, Bachelard (1971) observedthat plots of [glucose]/v versus [glucose] were linearin the presence of excess of Mg2+, but slightly curvedwhen the Mg'+.1/ATPtotaj ratio was 1.0. WhereasBachelard (1971) interprets these results to suggestthat alterations in the conformation of the MgATP2--binding sites may affect the conformation of theglucose-binding site, a much simpler explanation canbe offered in terms of the kinetic reaction mechanismof brain hexokinase action.The kinetic mechanism appears to be of the

sequential random type for rat and bovine brainhexokinase, and it is generally assumed that allsubstrates bind rapidly relative to the interconversionof the productive ternary complex. These conclusionswere made on the basis of initial-rate studies in theabsence and the presence ofcompetitive inhibitors andproduct-inhibition studies (Fromm & Ning, 1968;Ning et al., 1969; Purich & Fromm, 1971), and havebeen independently confirmed by the use of similarapproaches (Bachelard et al., 1971). In addition, wehave found that the uncomplexed form of ATP actsas a linear competitive inhibitor with respect toMgATP2- for these mammalian enzymes (Ning etal., 1969; Purich & Fromm, 1971), whereas studiesfrom Bachelard's laboratory appear to indicate'mixed' inhibition (Bachelard & Goldfarb, 1969).The latter observation can be better understood ifoneconsiders the concentrations of ATP4- present in theexperiments of Bachelard and Goldfarb (1969)(assuming for the purpose of this analysis that all theuncomplexed ATP is ATP4-, although under theconditions used 20% of it would exist as HATP3-).They observed relatively small intercept changes inLineweaver-Burk-type plots (Lineweaver & Burk,1934) at the higher concentrations of ATP4- whichcorrespond to concentrations that are from 11.3- to

3

65

D. L. PURICH AND H. J. FROMM

E-MgATP2-K \ K3

E-ATP4=--E E-MgATP2 -glucose - ProductsK1

K\ 1111K4E-glucose

K11

E-glucose-ATP4'

Scheme 1.

17.5-fold higher than the dissociation constant forthis inhibitor (Bachelard & Goldfarb, 1969). At suchhigh concentrations of the free uncomplexed ATP, itis possible that these small intercept changes couldresult from a weak non-specific binding not related tothe mode of binding at lower inhibitor concentrationswhere their results appear to suggest competitiveinhibition. If we assume that ATP4- is indeed acompetitive inhibitior of the brain enzyme, thenScheme 1 outlines the various interactions ofsubstrates and ATP4- with this phosphotransferase.The rate expression for the above kinetic mechanismcan be expressed in the following form:

Vmax.1+4 + K +KA3K 1 +K 1

+-I 1+- - lAL KJ B AB LKi

where v, Vmax., A, B and I represent initial-reactionvelocity, maximal velocity, [MgATP2-], [glucose],and [ATP4-], respectively. The various K's aredissociation constants defining the equilibria des-cribed in Scheme 1. If K1. K1l or K1 6 K4, increasingthe glucose concentration will make the AB term inthe denominator smaller and thereby lessen theinhibition by ATP4-. At the Mg2+a/ATP,o1aj ratio of1, there is a relatively high concentration of un-complexed ATP4- at the most dilute [ATP,otal], andthe amount becomes less significant as the concentra-tion of ATPtotai increases. These considerationstherefore explain why one would observe an inter-dependence between the binding of MgATP2- andglucose at the lower Mg2+ concentration. In asimilar manner, one can understand why [glucose]/vversus [glucose] plots are slightly curved at sub-optimal amounts of the bivalent cation. Although webelieve that ATP4- inhibition is competitive, it maybe important to note that similar arguments couldalso be made if the inhibition were 'mixed'. The rateequation for ATP4- acting as a 'mixed' inhibitorwould contain an AB term in its denominator as ineqn. 1. Elevated concentrations of glucose wouldagain be expected to decrease the ATP4- inhibitionin the manner described for the effect of glucose on acompetitive inhibitor of MgATP2-.

Discussion

This report attempts to re-interpret Bachelard's(1971) data in terms of previous kinetic studies inwhich no evidence was obtained for an allosteric rolefor either Mg2+ or MgATP2-, and is similar to areport (Blair, 1969) which re-evaluated the role ofMg2+ and ATP4- in the pyruvate carboxylasereaction (Keech & Barritt, 1967). Our ma,jor con-tention is that Bachelard's (1971) kinetic data mustbe corrected to reflect the concentration-dependentstability of the MgATP2- complex wheneverMgtoa1]=[ATPtotai] and their respective concentra-tions are sufficiently low. If such corrections aremade, the velocity dependence upon the concentra-tion of MgATP2- becomes strictly hyperbolic. Inaddition, corrections of this sort and also for ATP4-inhibition appear to reconcile the non-hyperbolicglucose-saturation curves with all previous studies, inwhich strictly Michaelis-Menten isotherms wereobserved. Although these corrections serve to clarifythe action of Mg2+ and MgATP2- in the brain hexo-kinase reaction, it may be of value to briefly sum-marize several additional arguments against anallosteric action by these compounds.On the basis of Hill plots, Bachelard (1971) has

stated that the number of binding sites for MgATP2-on brain hexokinase is 1.8 at a Mg2aJfATPto1a,ratio of unity, and 1.0 in the presence of excess ofMg2+. Here again, corrections for the large per-centage of total ATP that exists as the uncomplexedATP4- and for the inhibitory action of this speciessuggest that the Hill coefficient at high and low[Mg2+] is near unity (i.e., n < 1.25). In this regard, weshould also like to point out some possible limitationsin the use of Hill plots in kinetic studies of multi-substrate co-operative enzyme systems. The originalformulation of the Hill plot was in terms of ligandbinding to a protein assuming that the concentrationsof all protein-ligand complexes having less than nmolecules of bound ligand are negligibly small (Hill,1913). This was later put in terms of velocity on theassumption that vIVmax., the fractional attainment ofmaximal initial velocity, for a one-substrate enzymicreaction is strictly proportional to 7, the fractional

1972

66

ACTIVATION OF BRAIN HEXOKINASE

saturation of substrate-binding sites (Atkinson et al.,1965). Unfortunately, this equation can be ratherindiscriminately applied to studies of multisubstrateco-operative enzymes where the above proportion-ality may or may not hold depending upon the con-centration of the other substrate(s) involved in thereaction. For example, by analogy with the rapid-equilibrium random mechanism described earlier,the dependence of vlVmax. on the concentration ofsubstrate A is also related to the concentration ofsubstrate B, as indicated by the following relation-ship:

(EA)+(EAB) v (1+K3/B) (2)Eo Vmax.

where the different enzyme forms and kinetic termsare as defined earlier, and Eo is the total enzyme con-centration. Clearly, Y=vIVmax. only when substrateB is saturating, and the apparent values for themaximal velocity, obtained by the usual extrapola-tion of Lineweaver-Burk plots, cannot be used inplace of the true Vmax.. Finally, it is not altogetherclear what n truly means; Atkinson (1966) has statedthat treatments considering the slope to be either an'interaction factor' or an indication of the number ofbinding sites may be equally in error without furtheranalysis. These considerations point to an obviousneed for the cautious application of the Hill equationin kinetic studies of multisubstrate co-operativeenzyme reactions.One additional point should be made regarding the

differences in the observed maximal velocity atMga2/ATPt0ta1 ratios of 1.0 and 5.0 in Bachelard's(1971) study of the velocity dependence of the hexo-kinase reaction upon [MgATP2-]. From Fig. 4 of hispaper, it seems clear that the excess of Mg2+ inhibitsthe enzyme and may account for the lower maximalvelocities at Mg2l /ATPt.ta1 ratio of 5.0.

It should be noted that whereas it is possible torationalize all of Bachelard's (1971) data in terms ofnormal Michaelis-Menten kinetics, it is not possibleto make similar arguments regarding a recent studysuggesting allosteric interactions in the yeast hexo-kinase reaction (Kosow & Rose, 1971). In the latterstudy, velocities were determined at pH6.9, wherethe kinetics with respect to ATP are complicated bythe presence of ATP4-, MgATP2-, HATP3-, andMgHATP1-. In addition, the stability constant forthe MgATP2- complex is only 15000M1- in thebuffer used in those studies (D. L. Purich & H. J.Fromm, unpublished work), and the buffer, itself,binds appreciable amounts of the Mg2aI (Good etal., 1966). It should be pointed out, however, that ourstudies of the forward and reverse reaction kinetics ofyeast hexokinase at pH6.5 in another buffer showedno deviation from normal kinetics in either initial-rate studies or isotope-exchange at equilibriumstudies (Fromm et al., 1964).VoL. 130

With nucleoside 5'-triphosphate-dependent trans-phosphorylases, we have found that the free un-complexed Mg2+ concentration must be maintainedat a fixed value in each reaction mixture. This pro-cedure ensures that a constant fraction of the totalnucleotide is in the form of the Mg2+-nrucleotidecomplex. Whereas this free Mg2+ concentration isfrequently around 1mM (Copley & Fromm, 1967;Rudolph & Fromm, 1969), the free [Mg2+] optimumdepends upon the enzyme under study and, to a largeextent, upon the concentration, pH, and type ofbuffer used in the rate experiments. It is thereforeadvantageous to evaluate the optimum free Mg2.concentration experimentally at the extremes of theconcentration ranges of substrates to be studied. It isgenerally insufficient to maintain the Mgtoa/ATPtotai ratio at 2.0, as has been done in many ratestudies. For such cases, the percentage of ATP4-existing in solution as MgATP2- will vary with theextent of dilution. If, for example, the Mg,ot/ATPtotai ratio is 2.0 and the stability constant for thiscomplex is taken to be 7300OM-1 (O'Sullivan &Perrin, 1964), then the ATPtotal concentration mustexceed 0.25mm or else the free uncomplexed ATP4-will exceed 5% of the total ATP concentration. Thepotential seriousness of such an approach can beillustrated by a kinetic study of rabbit musclephosphofructokinase (Uyeda, 1970). In that study,the ratio of total Mg2+ to total ITP was maintainedat 2.0, and the total ITP was varied in the concentra-tion range from 12 to 200^*M. If one assumes that thestability constant for the MgITP2- complex is20000M-1 for the Mg2+-nucleotide complex in trisbuffer (O'Sullivan & Perrin, 1964), simple calculationshows that the percentage of total ITP as MgITP2-varied from approximately 29 to 83%. This variationbecomes especially important if the free uncomplexednucleotide is inhibitory.- However, it is also in-advisable to utilize higher Mg2?aj/ATPtotal ratios asthe free uncomplexed Mg2+ is often inhibitory athigher concentrations. The above statements alsopertain to studies of nucleoside 5'-diphosphate-dependent transphosphorylases, but the free Mg2+concentration must be maintained at a higherconstant and non-inhibitory concentration.

Finally, we should note here that it is not our intentto suggest that the kinetic studies of Bachelard (1971)describing the sigmoidal dependence of reactionvelocity upon [total ATP] may not be of functionalimportance. Indeed, in cases where the true substrateofan enzymic reaction is the metal-substrate complex,functionally important non-hyperbolic velocity res-ponses to changes in the total substrate concentrationmay occur. In addition, if the uncomplexed substrateacts as a competitive inhibitor relative to the metal-substrate complex, the deviation from normal hyper-bolic responses may be more pronounced. This canb-illustrated in the case of a simple one-substrate

67

68 D. L. PURICH AND H. J. FROMM

0.8

A

0.6 -B

C

~0.4-

0.2-

0 0.2 0.4 0.6 0.8 I.0[Substrate] (mM)

Fig. 3. Plot of Vl Vmax. versus the millimolar concelntra-tion of total substrate for a model one-substrate

enzyme

The concentrations of Sf,ee and the metal-S complexwere estimated assuming a stability constant of100OOm-0. The Km and K, for metal-S complex andSfrcc, respectively, were assumed to be 0.5mM. Theratio vl Vmax. was calculated from the rate expressionfor a simple one-substrate enzyme obeyingMichaelis-Menten kinetics. Curve A (v) representsthe case where total [substrate]= [metal-S complex];curve B (o) is where the velocity is strictly a functionof the metal-S complex concentration, as determinedby using the stability constant; curve C (A) is wherevelocity is dependent upon metal-S complex con-centration as in curve B, but also accounts forcompetitive inhibition by Sfree relative to the metal-Scomplex. The total metal ion to total substrate ratiowas maintained at 1.0. Other details are described inthe text.

model enzyme system for which it is assumed that theactive substrate species is the 1 :1 complex of substrateand metal ion. For example, the theoretical velocity-response curves presented in Fig. 3 show how thenormal hyperbolic dependence (curve A) can givesigmoidal substrate-saturation curves (curves B andC). For curve A, the stability constant for the complexis assumed to be sufficiently great that [Stota,] isequivalent to the concentration of the metal-substrate complex. Alternatively, one may assumethat the stability constant is lower but that there is asufficient concentration of free metal ion to force thesubstrate into combination with the metal ion. Theresponses in curves B and C were computed assuming

a stability constant of 100Om-1. For curve B it isassumed that the free uncomplexed substrate is non-inhibitory, whereas for curve C it is assumed that freesubstrate acts as a competitive inhibitor. The non-linear responses of hexokinase reaction velocities tototal ATP concentration may represent such anexample for a two-substrate enzymic reaction. Theseresponses may be of functional importance since it islikely that the non-particulate Mg2+ concentration incerebral cortical tissue is approximately 1.5 to3.5mM (Bachelard & Goldfarb, 1969), slightly lowerthan the ATP concentration in this tissue. Whereasone might expect that the amount of Mg2+ willremain constant, it is important to recognize that theamount of Mg2+ associated with the ATP pool is theimportant factor. Clearly, this will change withchanges in the ATP concentration, and the otherMg2+-metabolite complexes within the cell will act asthe source of this bivalent metal ion. Moreover, thereis growing evidence suggesting that the free un-complexed Mg2+ concentration in many tissues isbelow 0.4mM (Rose, 1968; England et al., 1967;Peck & Ray, 1971). These results suggest thatappreciable fractions of the total ATP pool withinthe cell may be present as the inhibitory free un-complexed ATP4-. The effect of suboptimal Mg2+concentrations and the presence of free ATP4- on theresponses of several glycolytic phosphotransferasesto the adenylate energy charge has been recentlyevaluated (Purich & Fromm, 1972). These experi-ments would suggest that the above factors may playmetabolically important roles.

This research was supported in part by Research GrantAM-1 1041 from the National Institutes of Health,United States Public Health Service, and Research GrantGB-33400 from the National Science Foundation.This is journal paper J-7269 of the Iowa Agricultureand Home Economics Experiment Station, Ames, Iowa50010, U.S.A., project 1666. A United States PublicHealth Service Predoctoral Fellowship to D. L. P. is alsogratefully acknowledged.

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ACTIVATION OF BRAIN HEXOKINASE 69

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