Biochem. J. (1984) 222, 363-370 363Printed in Great Britain

Fructose is a good substrate for rat liver 'glucokinase' (hexokinase D)

Maria Luz CARDENAS,* Eliana RABAJILLE and Hermann NIEMEYERDepartamento de Biologia, Facultad de Ciencias Basicas y Farmaceuticas, Universidad de Chile, Casilla 653,

Santiago, Chile

(Received 23 February 1984/Accepted 14 May 1984)

Rat liver 'glucokinase' (hexokinase D) catalyses the phosphorylation of fructose witha maximal velocity about 2.5-fold higher than that for the phosphorylation of glucose.The saturation function is hyperbolic and the half-saturation concentration is about300mM. Fructose is a competitive inhibitor of the phosphorylation of glucose with aKi of 107 mm. Fructose protects hexokinase D against inactivation by 5,5'-dithiobis-(2-nitrobenzoic acid), and the apparent dissociation constants are about 300mM in thepresence of different concentrations of the inhibitor. The co-operativity of theenzyme in the phosphorylation of glucose can be abolished by addition of fructose tothe reaction medium. Fructose appears to be no better as a substrate for the othermammalian hexokinases than it is for hexokinase D. It is proposed that the name'glucokinase' ought to be reserved for enzymes that are truly specific for glucose, suchas those of micro-organisms and invertebrates, and that liver glucokinase must becalled hexokinase D (or hexokinase IV) within the classification EC 2.7.1.1.

Ever since the first work on yeast hexokinase(ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1)it has been known that this enzyme catalyses thephosphorylation of the C-6 position of glucose,mannose and fructose (Colowick & Kalckar, 1941,1943; Slein et al., 1950). Mammalian hexokinasesfrom brain and muscle exhibit the same broadsugar specificity (Ochoa, 1941; Sols & Crane,1954; Crane & Sols, 1955). Thus the discovery inrat and guinea-pig liver of an enzyme that seemedto be rather specific for glucose was of particularinterest (DiPietro etal., 1962; Walker, 1962, 1963;Vinluela et al., 1963). This enzyme exhibited,however, a rather high half-saturation point ofabout 10mM for glucose, and coexisted in the liverwith a hexokinase that more closely resembled theenzymes of brain and muscle, which have a Km ofabout 0.1 mm. The new enzyme was named gluco-kinase (ATP: D-glucose 6-phosphotransferase, EC2.7.1.2), in part as recognition of an earlierobservation by Slein et al. (1950), who had shownthat under certain conditions liver extracts wereable to phosphorylate glucose but not fructose, andhad postulated the existence of a glucokinase in theliver.

Rat liver hexokinases (including glucokinase)were later resolved into four isoenzymes that were

* Present address, and address for correspondence:Department of Biochemistry, University of Birming-ham, P.O. Box 363, Birmingham B15 2TT, U.K.

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named A, B, C and D (Gonzalez et al., 1964, 1967)or I, II, III and IV (Katzen et al., 1965). IsoenzymeD (IV) corresponded to glucokinase, because of itshigh Km for glucose and its low activity against100mM-fructose (Walker, 1963; Gonzalez et al.,1964, 1967; Salas et al., 1965). With the recognitionthat the saturation function of glucokinase withglucose is sigmoidal (Niemeyer et al., 1975a,b;Storer & Cornish-Bowden, 1976b), we becameinterested in the kinetic properties of the enzymewith several sugar substrates and on their effect ascompetitive inhibitors on the phosphorylation ofglucose (Niemeyer et al., 1978; Cardenas et al.,1979; Olavarria et al., 1982).

In the present paper we report data for the phos-phorylation of fructose catalysed by glucokinaseand show that this isoenzyme is no more specificfor glucose than are the other mammalian hexo-kinases. Thus the name 'glucokinase' appears to bea misnomer and ought to be replaced by hexokin-ase D, which is the name that we use in the presentpaper. Portions of this work have been publishedin abstract form (Cardenas et al., 1981).

ExperimentalMaterialsATP (disodium salt), NADP+, cx-D-glucose, f-

D-fructose, D-glucosamine, glucose-6-phosphatedehydrogenase (EC 1.1.1.49, type VII, from

M. L. C'ardenas, E. Rabajille and H. Niemeyer

baker's yeast), phosphoglucose isomerase (EC5.3.1.9, type III, from yeast), Tris, EDTA, dithio-threitol, 2-mercaptoethanol, 5,5'-dithiobis-(2-nitrobenzoic acid) and 1-(3-dimethylamino-propyl)-3-ethylcarbodi-imide were products fromSigma Chemical Co. (St. Louis, MO, U.S.A.).DEAE-cellulose was either from Sigma ChemicalCo. or from Whatman (Maidstone, Kent, U.K.)(DE-52). Sephadex G-100 and G-150 andCH-Sepharose 4B were obtained from PharmaciaFine Chemicals (Uppsala, Sweden). Sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-D-gluco-pyranose was prepared by coupling D-glucosaminewith CH-Sepharose 4B by means of 1-(3-dimethyl-aminopropyl)-3-ethylcarbodi-imide, under condi-tions recommended by Pharmacia Fine Chemi-cals. Other chemicals were the highest gradecommercially available.

Enzyme preparationHexokinase D was purified from the livers of

well-fed rats by the procedure described in C'ar-denas et al. (1978), which involves successivechromatographic separations on DEAE-cellulose,Sephadex G-100 or G-150 and Sepharose-N-(6 - aminohexanoyl) - 2 - amino - 2 - deoxy - D - gluco -pyranose. Most of the experiments were done withenzyme at the Sephadex step (specific activity 3-6units/mg of protein), but the same results wereobtained with enzyme of specific activity of 70units/mg of protein.

Enzyme assayHexokinase D activity with fructose as substrate

was assayed by measuring the production offructose 6-phosphate through the reduction ofNADP+ in the presence of an excess of glucose-6-phosphate dehydrogenase and phosphoglucoseisomerase. The increase in absorbance at 340nmwas monitored in a Gilford 2400 spectrophoto-meter, with circulating water at 30°C. The stan-dard reaction mixture contained (final concentra-tions), in a total volume of 0.5 ml: 80mM-Tris/HClbuffer, pH 8.0; 100mM-KCl; 6mM-MgCl2 or 1mM-MgCl2 over the ATP concentration as indicated;0.1 mM-EDTA; 2.5 mM-dithiothreitol; 5mM-ATP(or other concentrations as indicated); 0.5mM-NADP+; 0.7-0.8 unit of glucose-6-phosphatedehydrogenase/ml and 2 units of phosphoglucoseisomerase/ml. When glucose was the substrate theisomerase was omitted. The concentrations ofMg2+ used ensured that the MgATP2- concen-tration would be a high and nearly constantproportion (86%) of the total ATP concentration(Storer & Cornish-Bowden, 1976a). The reactionwas started by the addition of the sugar substrateunless otherwise indicated. When fructose was thesubstrate the omission of the isomerase from the

assay medium gave no detectable increase inabsorbance, at least during the first 10min ofincubation, and even at the highest fructose con-centration, indicating no contamination withglucose. When fructose was used as an inhibitor ofglucose phosphorylation blanks without ATP wereperformed at each glucose concentration andcorresponded to glucose dehydrogenase activity ofglucose-6-phosphate dehydrogenase. The absor-bance at 100mM-glucose was generally below 5% ofthe hexokinase activity, and at below 10mM-glucose was negligible. With fructose as the sub-strate similar blanks gave undetectable values,even at the highest concentration. One unit ofenzyme activity is defined as the amount ofenzyme required to catalyse the conversion of1 ymol of glucose/min at 30°C in standardconditions.

Enzyme inactivation by 5,5'-dithiobis-(2-nitrobenzoicacid)

Samples of the enzyme preparation were freedfrom dithiothreitol or 2-mercaptoethanol by usinga small DEAE-cellulose column. The enzyme waseluted with 20 mM-glycylglycine/NaOH buffer,pH8.0, containing 300mM-KCI, 5% (v/v) glyceroland 1 mM-EDTA. The procedure also concentratedthe preparation. About 50munits of the enzymewere treated with variable concentrations of 5,5'-dithiobis-(2-nitrobenzoic acid) at 30°C in 0.5 ml ofthe same glycylglycine buffer in the presence ofvarious fructose concentrations. Samples wereremoved for hexokinase assay at various timeintervals, with 100mM-glucose as substrate. Thissugar concentration quenched any possible effectof 5,5'-dithiobis-(2-nitrobenzoic acid) during theassay. 5,5'-Dithiobis-(2-nitrobenzoic acid) was pre-pared fresh for each experiment in 100mM-glycyl-glycine buffer, pH 8.0, containing 1 mM-EDTA.

Protein determinationProtein concentration was calculated from the

absorbance at 260 and 280nm by the procedure ofWarburg & Christian (Dawson et al., 1972).

Results

Saturation functionIn contrast with the behaviour with glucose, the

saturation function of hexokinase D with fructosewas hyperbolic [h (Hill coefficient) = 1.01, as isshown in Fig. 1. The apparent Km values, atpH 8.0, were between 276 and 380mM (me-dian = 325mM), measured with several differentenzyme preparations with specific activities in therange 2-70 units/mg of protein. The Km for thereactive tautomer of fructose is only about one-quarter of these values if it is considered that in

1984

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Fructose: a good substrate for 'glucokinase' (hexokinase D)

6

IC1 ~~~~~~4

2 -

02

0 0.009 0.018

v/lFruII I I I I

0 300 600 900[Fructose] (mM)

Fig. 1. Hyperbolic kinetics of hexokinase D with fructoseExperimental conditions were as described in theExperimental section. The straight line seen in theEadie-Hofstee plot shown in the inset indicates thatthe enzyme obeys Michaelis-Menten kinetics(h = 1.0). Km = 276mM.

aqueous solution, at 25-30°C, only about 25% ofthe fructose is in the furanose form (Gottschalk,1943a,b; Que & Gray, 1974), which is the only oneable to act as substrate of hexokinases (Slein et al.,1950). As with glucose (Cardenas et al., 1979),decreasing the pH from 8.0 to 7.5 did not modifythe Hill coefficient with fructose, but increased thehalf-saturation concentration by about 30%(420mM at pH7.5). Most previous studies of thesaturation function of hexokinase D with fructosegave very high values of Km (>800mM), whichwere in some cases even higher than the concentra-tion range used in the experiments (Salas et al.,1965; Parry & Walker, 1966; Grossman et al.,1974). An exception was the report by Gonzalez etal. (1967), which gave a low Km value, similar to theone with glucose. In general, all these resultsintroduced doubts on the real capacity of theenzyme for phosphorylating fructose. We do nothave a satisfactory explanation for the. disagree-ment between the previous studies and the presentresults.

Relative maximal velocities with glucose andfructoseIn spite of the higher value of half-saturation

concentration with fructose than with glucose, theapparent maximal velocities with fructose (ob-tained by double-reciprocal plots, Hanes-Woolfplots or Eadie-Hofstee plots) were always 2.1-2.8-fold higher than the ones with glucose under thesame conditions. The apparent maximal velocitieswith glucose were obtained by utilizing a computer

2

0

0 1.5 3.0l/[ATPl (mM-')

Fig. 2. Double-reciprocal plots with the concentrations ofsugar substrate and ATP varied at constant ratio

The phosphorylation of glucose (0) or of fructose(-) was measured in parallel experiments done withthe same preparation of enzyme and in equalamount. Variable concentrations of ATP and sugarsubstrates were used, but the ratios of sugar andATP concentrations were held constant as follows:[glucose]/[ATP]= 10; [fructose]/[ATP] = 200. Thetotal Mg2+ concentration exceeded the total ATPconcentration by I.OmM. The abscissa correspondsto the reciprocal value of the total ATP concentra-tion. The parabolic curves are expected in this typeof experiment where two substrate concentrationsare simultaneously varied at a constant ratio, if theenzyme presents a ternary-complex mechanism.

program with h, K0.5 and Vmax. varied to yield thebest statistical fit of the equation

V = Vmaxj[S]/(K0 5h+ [S]h)to the data or by graphical extrapolation. Avariation of no more than 5% was obtained by thetwo methods. With the purpose of establishing amore rigorous comparison between the maximalvelocities for glucose and fructose, initial velocitieswere measured in parallel experiments with thesame enzyme solution with variation of the con-centration of the hexose and ATP at a constantratio, as suggested by Gulbinsky & Cleland (1968).The ratio [glucose]/[ATP] was 10:1, and the ratio[fructose]/[ATP] was 200:1. With both hexoses aparabolic graph was obtained when I/v was plottedagainst /[ATP] (Fig. 2), as expected for a ternary-complex (sequential) kinetic mechanism (Gul-binsky & Cleland, 1968). (In the case of glucose thenon-Michaelian kinetics do not allow this conclu-sion to be drawn.) Extrapolation gave different

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365

M. L. C'ardenas, E. Rabajille and H. Niemeyer

intercepts on the ordinate for glucose and fructose,which indicated a maximal velocity 2.6-fold higherwith fructose than with glucose, in excellent agree-ment with previous results, as commented above.This determination contrasts with experiments ofthe same design performed with 2-deoxyglucose,which showed that the maximal velocity with thissubstrate was only 73% of the value with glucose(Monasterio, 1980).

-

E0E

C.

Ei

E0crA

0

0

r.

r.;:2

E

Kinetic mechanism of hexokinase D with fructoseData from initial-velocity studies could be

described by a ternary-complex mechanism, asshown by the Hanes-Woolf plots of Fig. 3.Secondary plots were linear (insets in Fig. 3), thusconfirming the hyperbolic nature of the saturationfunction of hexokinase D with fructose and withMgATP at different concentrations of the fixed

Fig. 3. Initial-velocity studies of hexokinase D with fructose as substrateATP total concentrations were 0.3, 0.6, 0.9 and 1.2mM, with total Mg2+ concentration exceeding the total ATPconcentration by 1.0mM. The corresponding concentrations ofMgATP were calculated (Storer & Cornish-Bowden,1976a) to be 0.26, 0.52, 0.77 and 1.03mM respectively. The fructose concentrations were 200, 400, 750 and 900mM.Inset: secondary plots of slopes (0) and intercepts (-) of the Hanes-Woolf representations. (a) Fixed ATP concen-trations and varied fructose concentrations. (b) Fixed fructose concentrations and varied ATP concentrations. Inboth cases the total ATP concentration has been plotted.

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Fructose: a good substrate for 'glucokinase' (hexokinase D)

substrate. The equation for this type of behaviouris as follows (Cleland, 1963):

V = V. [AI[B]/(Kia Kb + [A]Kb+ [B]Ka + [A][B])where [A] is the concentration of fructose and[B] the concentration of MgATP. The constantshave the following values as determined from thedata of Fig. 3: Ka = 174mM, Kia = 1025mM andKb = 0.35mM, and KiaKb/Ka = 2.0mM. The linear-ity of the plots and the clear intersection of theprimary plots in the third quadrant agree with thebehaviour observed with 2-deoxyglucose as sub-strate (Monasterio, 1980).

Protection against inactivation by 5,5'-dithiobis-(2-nitrobenzoic acid)

5,5'-Dithiobis-(2-nitrobenzoic acid) inactivateshexokinase D, the inactivation being reversed bydithiothreitol and blocked by glucose (Monasterio,1980; Monasterio et al., 1981; Niemeyer et al.,1981). A reversible enzyme-5,5'-dithiobis-(2-nitro-benzoic acid) complex is formed previous to thechemical modification (Monasterio et al., 1981).When fructose was used as a protector of theinactivation of hexokinase D by 5,5'-dithiobis-(2-nitrobenzoic acid) it was as effective as glucose,blocking enzyme inactivation almost completely

15

10 1

5

0 150 300 450[Fructosel (mM)

Fig. 4. Protection byfructose ofhexokinase D inactivationby 5,5'-dithiobis-(2-nitrobenzoic acid)

The inactivations were performed with 20/4M-5,5'-dithiobis-(2-nitrobenzoic acid) in the absence or inthe presence of different concentrations of fructosein a total volume of 0.5ml as described in theExperimental section. At zero time 50Ol of hexo-kinase D (37munits) was added and 20ul sampleswere removed every 1 min, during 6min, for enzymeassay. Spontaneous inactivation without 5,5'-dithio-bis-(2-nitrobenzoic acid), with and without fructose,was negligible. kinactivation is the observed rateconstant of inactivation and corresponds to theslope of a plot of log(residual activity) against timeat the various fructose concentrations. Apparentdissociation constant = 290mM-fructose.

when added at zero time at concentrations aboveKm. The apparent dissociation constant estimatedby plotting the reciprocal of the observed rateconstant of inactivation versus ligand concentra-tions was about 300mM, which agrees with theapparent Km value (Fig. 4). The effect of fructoseon hexokinase D inactivation by 5,5'-dithiobis-(2-nitrobenzoic acid) resembles the mixed pattern ofenzyme inhibition, as the dissociation constant ofthe enzyme-fructose complex is virtually indepen-dent of the concentration of 5,5'-dithiobis-(2-nitro-benzoic acid) (results not shown). Similar behav-iour has been observed with glucose as a protector(Monasterio et al., 1981).

Effect offructose on glucose phosphorylationFructose had been described as a competitive

inhibitor of 'glucokinase' with respect to glucose(Salas et al., 1965; Parry & Walker, 1966;Gonzalez et al., 1967). We re-examined itsbehaviour in the light of the kinetic co-operativityof the enzyme with glucose. Fig. 5 shows a Dixonplot drawn with data obtained at relatively highconcentrations of glucose (4-30mM) in order togive straight lines (M. L. C'ardenas, E. Rabajille &H. Niemeyer, unpublished work). The competitivecharacter of fructose with respect to glucose isshown by the set of parallel lines of the Cornish-Bowden graph of the inset (Cornish-Bowden,1974). The inhibition constant was estimated as107 mM, a value that is not in close agreement withthe K, value of 300mM obtained from the pro-tection studies. This discrepancy may be related tothe complexity of the kinetics of hexokinase D;thus we have found that the inhibitory capacity ofcompetitive inhibitors such as mannose and N-acetylglucosamine at low glucose concentrationsdecreases as the glucose concentration decreases(M. L. Cardenas, E. Rabajille & H. Niemeyer,unpublished work). Our K, value is lower than theone reported by Parry & Walker (1966) (350mM),but this discrepancy is not very surprising, as theyalso obtained a very high value of Km (about 2M),as already mentioned above.The presence of fructose in the assay mixture

abolished the co-operativity of the saturationfunction for glucose. Thus with 300mM-fructose astraight line is obtained in a double-reciprocal plot(Fig. 6), with a corresponding Hill coefficient of1.0. The effect of different fructose concentrationon the Hill coefficient and the half-saturationconcentration of glucose as substrate is shown inFig. 7. A similar effect on the saturation functionwith glucose is observed when mannose, 2-deoxy-glucose or N-acetylglucosamine is used as inhibi-tor (Cardenas et al., 1979, and unpublished work).In the present work the Hill plot and the Hillcoefficient have been used only to ascertain the

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co

-I

M. L. C'ardenas, E. Rabajille and H. Niemeyer

IFructose l (mM)

Fig. 5. Dixon plots of the inhibition by fructose of glucose phosphorylation by hexokinase DThe phosphorylation of glucose was measured at several fixed concentrations of this substrate, namely 4 (O), 8 (s),15 (@) and 30mM (e), and various fructose concentrations (40-900mM) as indicated. The reaction was initiated bythe addition of ATP (5mM, final concentration). K*= 107mM-fructose. Inset: Parallel Cornish-Bowden plotsindicate that any uncompetitive component of the inhibition was negligible.

4

0 0.5 1

1/[Glucosel (mM-')

Fig. 6. Double-reciprocal plot of the phosphorylation ofglucose in the absence and presence offructose

The phosphorylation of variable concentrations ofglucose (1-200mM) by hexokinase D (3-6munits)was measured in the absence (M) or in the presence(0) of 300mM-fructose. The reaction was initiatedby the addition of ATP (5mM, final concentration).*, KO.5 = 3.6mM-glucose, h = 1.6; 0, KO.5 =13.6mM-glucose, h = 1.0. The kinetic parameterswere determined as indicated in Fig. 7 legend.

M1.4 L 1io0

1.2 _ 5

1.0 -J11 _O0 100 200 300

[Fructose] (mM)

Fig. 7. Effect offructose on the kineticparameters KO.5 andh of the saturation function of hexokinase D with glucoseThe phosphorylation of glucose (range 1-200mM) by4munits of enzyme was measured at differentfructose concentrations, as indicated in the Figure.The reaction was initiated by the addition of ATP(5mM, final concentration). The kinetic parameterswere obtained by using a computer program with h,KO.5 and Vmax. varied to yield the best statistical fit ofthe equation

V = Vmax[Sp/((Ko.5h+ [SP)to the data. Similar values of h and KO.5 (variation ofless than 5%) are obtained from a Hill plot in whicha Vmax. value calculated as described above orestimated graphically was used.

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Fructose: a good substrate for 'glucokinase' (hexokinase D)

Table 1. Kinetic parameters for hexokinase isoenzymes with glucose and fructose as substrates at pH 7.5

YGIc

Isoenzyme (mM)Hexokinase A* 0.044Hexokinase B* 0.130Hexokinase C* 0.020Hexokinase D 7.5t

* From Niemeyer et al. (1975b).t From Niemeyer et al. (1975a).I Present work.

(mM)3.13.01.2420t

70.523.160.056.0

VFru/VGIc1.11.21.32.4t

VFruK

VGIcKFru0.0160.0520.0220.043

degree of co-operativity without implication ofmechanism.

Discussion

Since the first reports there has been generalagreement in considering that hexokinase D(glucokinase) shows a very low activity withfructose by comparison with the other animalhexokinases (Vifiuela et al., 1963; Gonz'alez et al.,1964; Parry & Walker, 1966; Niemeyer et al.,1975b; Weinhouse, 1976). This idea was mainlya consequence of the relative velocities of phos-phorylation of fructose and glucose at a sugar con-centration of 100mM. At this concentration hexo-kinases A, B and C catalyse the phosphorylation offructose better than glucose (1.1-1.3-fold) (Nie-meyer et al., 1975b), but for hexokinase D the ratioof phosphorylation of fructose and glucose is onlyabout 0.2: 1. However, if instead of comparing therelative velocities at a single substrate concentra-tion we examine the quotient VFrUK-/5VGIcK .

(Table 1), we can see that the four hexokinasesshow similar specificity for fructose, and in all ofthem glucose is a more specific substrate thanfructose. For an enzyme obeying Michaelis-Men-ten kinetics the ratio kcaJKm kcatIKo5 is the bestmeasure of specificity (Fersht, 1977); although it isnot strictly correct for a co-operative enzyme suchas hexokinase D, it is clearly preferable to otherparameters. Table 1 shows that, not only doesglucokinase have a much higher KO5 for fructosethan for glucose, but this is also true for hexo-kinases A, B and C. On the other hand, hexokinaseD has a higher maximal velocity with fructose thanwith glucose.

Although in the past the relative capacity forphosphorylating fructose at a concentration of100mM might have been a good argument for usingthe name glucokinase and a separate EC number,this name is no longer valid. In fact, even withoutour present knowledge of the specificity of the liverisoenzyme, some authors have questioned whetherglucokinase is the proper name for it (Walker,1966; Niemeyer et al., 1975b; Ureta et al., 1979;

Pollard-Knight & Cornish-Bowden, 1982). Es-pecially, Ureta et al. (1979) have made an extensiveanalysis of the problem.The name glucokinase and the classification as

EC 2.7.1.2 strictly implies an enzyme that isspecific for glucose, which is inappropriate for theliver enzyme, since it can also catalyse the phos-phorylation of mannose, fructose and 2-deoxy-glucose, sugars that are also competitive inhibitorsof the phosphorylation of glucose. In these proper-ties the liver enzyme differs from the glucokinasesfrom Aerobacter aerogenes (Kamel et al., 1966),Brevibacteriumfuscum (Saito, 1965), Bacillus stearo-thermophilus (Hengartner & Zuber, 1973), Strepto-coccus mutans OMZ 70 (Porter et al., 1982),Dictyostelium discoideum (Baumann, 1969), Schisto-soma mansoni (Bueding & Mackinnon, 1955) andEchinococcus granulosus (Agosin & Aravena, 1959),which are truly specific for glucose, but resemblesmultiple-substrate hexokinases of yeast and mam-mals. In addition, the analysis of Table 1 indicatesthat there is really nothing special about the liverhexokinase D in relation to fructose specificity.Furthermore, it seems that neither hexokinase Dnor the other hexokinases are responsible for thephosphorylation of fructose in vivo, since thephosphorylation of fructose in the liver occurs inposition C-l by the action of fructokinase (Hers,1955). Thus, instead of glucokinase, the namehexokinase D (or hexokinase IV), within theclassification number of EC 2.7.1.1, would be moreappropriate and is the one used throughout thepresent paper. The name 'glucokinase' should bereserved more properly for those enzymes knownto be specific for glucose, such as those isolatedfrom several invertebrates and micro-organisms,as mentioned above.

We are grateful to Dr. T. Ureta and Dr. A. Cornish-Bowden for helpful discussions during the preparation ofthis manuscript. We are indebted to Professor S. V. Perryfor allowing us to use the facilities of the Department ofBiochemistry, University of Birmingham, during thepreparation of this manuscript. This study was supportedby grants from the Departamento de Desarrollo de laInvestigaci6n, Universidad de Chile, from the Multi-

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370 M. L. C'ardenas, E. Rabajille and H. Niemeyer

national Program of Biochemistry, Organization of theAmerican States, and from the Regional Program forgraduate training in Biological Sciences, P.N.U.D.-U.N.E.S.C.O. RLA 78/024.

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