Chromatographic Resolution and Kinetic Characterization Of

Proc. NatL Acad. Sci. USA Vol. 80, pp. 8589, January 1983 Biochemistry

Chromatographic resolution and kinetic characterization of glucokinase from islets of Langerhans (hexoldnase/islet glucose metabolism /N-acetylglucosamine kdnase/Cibacron blue) MARTIN D. MEGLASSON, PAMELA TRUEHEART BURCH, DONNA K. BERNER, HABIBA NAJAFI, ALAN P. VOGIN, AND FRANZ M. MATSCHINSKY* Diabetes Research Center and Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Communicated by Robert E. Forster, October 4, 1982'

ABSTRACT Glucokinase (ATP:D-glucose 6-phosphotransfer- ofislets equals serum glucose and islet glucose 6-phosphate in- ase, EC 2.7.1.2) from rat islets of Langerhans was partially pu- creases in proportion to extracellular glucose (1, 13). Ninety rified by chromatography on DEAE-Cibacron blue F3GA agar- percent ofislet glucose utilization occurs with an apparent glu- ose. The enzyme eluted in two separate peaks. Sigmoidal rate cose affinity constant 11.1 mM (6). Also, mannoheptulose, an dependence was found with respect to glucose (Hill coefficient inhibitor of glucokinase (8), profoundly inhibits islet glucose = 1.5) for both enzyme fractions. K. values for glucose were 5.7 metabolism. (14). mM for the major fraction and 4.5 mM for the minor fraction. The chromatographic separation of glucokinase from other Neither fraction phosphorylated GlcNAc. A' GlcNAc kinase is the most direct (ATP. 2-acetamido-2-deoxy-D-glucose' 6-phosphotransferase; EC glucose phosphorylating enzymes approach 2.7.1.59)-enriched fraction; prepared by affinity chromatography to establish its presence in pancreatic islets. It is the purpose on Sepharose-N46-aminohexanoyl)-GlcNAc, had a K. of 25 ,EM of this paper to report the partial purification of glucokinase,. for GlcNAc. Islet tissue also contained hexokinase (ATP:D-hexose free of hexokinase and GlcNAc kinase contamination, from is- 6-phosphotransferase, EC 2.7.1.1) eluting in multiple peaks. The lets of Langerhans. results are consistent with the concept that glucokinase serves as the glucose sensor of pancreatic beta cells. MATERIALS AND METHODS Tissue Preparation., Pancreatic islets were prepared from fed Glucose phosphorylation is considered to be the rate-limiting male Wistar rats (Hilltop Laboratory Animals, Scottdale, PA) step in glucose utilization by pancreatic islets and may deter- weighing 225-300g as described (6). Islets were separated from mine the relationship between extracellular glucose concentra- the collagenase digest on a Ficoll gradient (15). By using this tion and initiation of insulin- secretion (1-4). Homogenates of method, 6,000-8,000 islets could be prepared each day. rodent pancreatic islets contain glucose 6-phosphotransferase Isolated islets were homogenized after being washed free of activity of both low and high affinities for glucose (1, 2, 5, 6). glucose, which was present at 5 mM throughout the isolation The high-affinity component, composed of one or more hexo- protocol. Homogenization was performed with 10 vol of ho- kinase isozymes (ATP:D-hexose 6-phosphotransferase, EC mogenizing buffer (20 mM K2HPO4, pH 7.8, containing 1 mM 2.7.1.1), is largely inhibited in the intact cell (2). However, the dithiothreitol, 1 mM EDTA, and 110 mM KCl) in a Kontes 18 low-affinity component appears to be fully active under phys- glass homogenizer by 20 strokes of a machine-driven Teflon iological conditions and appears to determine the rate ofglucose pestle (Bellco Glass homogenizer drive unit; set on 3). This and utilization by islets (2, 6, 7). This enzyme exhibits a Michaelis- all subsequent purification steps were performed at2-40C. The Menten constant ofabout 10 mM for glucose (1, 2, 5, 6), similar homogenate was centrifuged at 105,000 X g for 60 min. The to that reported for liver glucokinase (ATP:D-glucose 6-phos- supernatant fraction was either used immediately or stored at photransferase, EC 2.7.1.2) (8). It has been suggested that this -800C and pooled with the islet supernatant prepared the sub- enzyme in islets is glucokinase similar to that found.in liver (1, sequent day. 5). Chromatography. Sepharose-N-(6-aminohexanoyl)-GlcNAc Glucokinase has been claimed to be present also in a number was prepared by modification of the method of Rijksen and of other extrahepatic tissues (8). Recently, these claims have Staal (16). 6-Aminohexanoic acid-activated Sepharose 4B (Sigma), been disputed when those tissues examined were shown to con- stated by the supplier to have 30-42 tkmol of active ester per tain GlcNAc kinase (ATP:2-acetamido-2-deoxy-D-glucose 6- g of gel, was allowed to react with D-glucosamine at a ratio of phosphotransferase, EC 2.7.1.59) rather than glucokinase (9, 200 /imol of glucosamine per g. The ligand concentration was 10). The failure to observe glucokinase on electrophoretograms decreased by mixing the GlcNAc-coupled gel with an equal of rodent islets has been reported also (4). Like glucokinase, packed wet weight of Sepharose 4B (17). Cibacron blue F3GA GlcNAc kinase has been shown to phosphorylate glucose; how- agarose and DEAE-Cibacron blue F3GA agarose were supplied ever, the reported K, values are very high, being 210 mM (9), by Bio-Rad. The basic equilibration buffer for all columns was 370 mM (10), 410 mM, or 600 mM (11). Rodent islets are known 20 mM.K2HPO4, pH 7.8, with 1 mM EDTA and 1 mM di- to contain substantial amounts of GlcNAc kinase (12). There- thiothreitol. Supplements to this buffer and elution conditions fore, the proposed role of glucokinase in islet physiology has are described in Results. Elution of DEAE-Cibacron blue been questioned (9, 10). F3GA agarose columns was accomplished by using a complex Many observations suggest, however, a physiological role for gradient ofKCI and MgCl2 produced by an LKB Ultrograd gra- glucokinase or a similar enzyme in islets. Intracellular glucose dient maker. This gradient was verified by determining the Mg2' and K+ content of the column fractions with an ion ana- The publication costs ofthis article were defrayed in part by page charge lyzer (Beckman Select Ion 5000). payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 85 Downloaded by guest on September 24, 2021 86 Biochemistry: Meglasson et aL Proc. Natl.- Acad. Sci. USA 80 (1983)

Assay Methods. Glucose phosphorylation was determined 251 *12 C- by a fluorometric enzymatic assay method (6). The reaction 0 20- -10 volume was 100 Al and the reaction was conducted for 2 hr at - 30'C. The ATP concentration was 4.3 mM. Magnesium was 1 8 0 mM in excess ofthe ATP concentration after-the EDTA content cl1-15'5- 0' the added eluant had been corrected for. Phosphorylation of 0o 6 E of 10- sugars other than glucose was measured by a fluorometric en- . 4 C~ E zyme assay with pyruvate kinase and lactate delhydrogenase n5 0 o e- 2 (Boehringer Mannheim) as coupling enzymes (18). The reaction A, D a. components were 50 mM Tris-HCl (pH 8.2), 100 mM KC1, 2 0 mM MgCl2, 7.1mM 2-mercaptoethanol, 0.05mM NADH, 0.05 mM phosphoenolpyruvate, 0.6 mM ATP, pyruvate kinase from Elution Volume,ml rabbit muscle at 50 ug/ml, and lactate dehydrogenase from beef muscle at 10 ,ug/ml. The reaction was conducted in 100 35. 70 E ,ul for 2 hr at 300C and was terminated by addition of 1 ml of 0 0.05 M NaOH containing 0.5mM cysteine. The half-time ofthe C- 2 0 :,_ reaction was about 15 sec. Sample fluorescence was corrected I 50 D for tissue NADH oxidase and ATPase activity subtraction of E E by E o -. C

appropriate tissue and reagent blanks. .2 _ Sample protein content was determined by the method of x aC0 0 E 30 o Bohlen and coworkers (19). Islet homogenate DNA content was x at 0 Robins (20). 0U, 0 determined by the method of Kissane and ,_) 0 0 Kinetic Analysis. Kinetic constants were determined by o E a- a.O least-squares linear regression of Hanes-Woolf plots (ref. 21, 10 < p. 210). Deviation of glucokinase from Michaelis-Menten ki- z netics was evaluated by using the Hill plot (ref. 21, pp. 371- C) 375). The slope of this plot was determined at v = 0.5 Vm,. 3 4 5 Enzyme activity is expressed as units; one unit is equal to phos- Elution Volume, ml phorylation of 1 /Lmol min' of sugar at 30'C. Data are ex- means and their standard errors. FIG. 1. Chromatography of liver or islet supernatants on Sepha- pressed as sample rose-N-(6-aminohexanoyl)-GlcNAc columns. The columns were equilibrated with the basic buffer supplemented with 5% (vol/vol) glycerol. RESULTS The arrow indicates addition of 0.5 M KCl to the equilibration buffer. Enzymatic activity was evaluated with 100mM glucose (-), 0.5mM Glucose phosphorylation activity in islet supernatant was re- glucose (---), or 2 mM GlcNAc -) as substrate. Note that phos- solved into two kinetic components on a Hanes-Woolfplot (not phorylation of 0.5 mM glucose is indicated only where the activity shown). The high-affinity component had a Km value of 0.047 differed from that measured with 100 mM glucose. x-x, Protein ± 0.010 mM and a Vm_ of71 + 7 units per g of DNA (n = 9). content. (Upper) Rat liver supernatant containing 18.6 milliunits The low-affinity component, corrected for hexokinase activity, (mUnits) of glucokinase activity applied to a 0.7 x 0.6 cm column. had a Km of 10.40 ± 1.74 mM and a Vm. of 69 ± 3 units per (Lower) Supernatant from 7,250 islets containing 5.7 milliunits of glu- DNA = These values are generally similar to those cokinase and 4.9 milliunits of hexokinase was applied by batch incu- g of (n 9). bation for'30 min at 4rC. The slurry was loaded into a 0.6-cm column, previously reported by our laboratory for the 12,000 x g su- producing a bed height of 1.3 cm. Sixty-three percent of the applied pernatant ofsmall batches ofrat islets prepared by hand-picking glucokinase activity bound the affinity matrix during incubation. after collagenase digestion (6). Islets prepared by the latter method were larger (20 ng of DNA vs. 9.6 ng of DNA per islet) and contained 25% more glucokinase activity, expressed as a this fraction phosphorylated GlcNAc with an apparent Km of function of DNA content, than the islets in this study, which 0.025 mM. No evidence of deviation from Michaelis-Menten were prepared on Ficoll gradients. Preliminary studies per- kinetics was observed over the range 0.007-0.250 mM GlcNAc. formed with Ficoll-prepared islets demonstrated that super- Phosphorylation of 0.015 mM GlcNAc was not inhibited by 60 natant glucokinase activity was stable when stored for 6 hr at mM D-mannoheptulose. GlcNAc kinase activity equal to 75 ± 5 units per g ofDNA (n = 3) was recovered in the peak eluting 4°C or 24 hr at -80°C. with high KC1. GlcNAc kinase could not be determined in the Glucokinase from islets was partially purified by affinity crude supernatant fraction with our present assay; however, the chromatography on Sepharose-N-(6-aminohexanoyl)-GlcNAc value measured in the partially purified fraction appears to be (Fig. 1). For comparison, a similar experiment was performed at least as large as the GlcNAc kinase activity in rat islet ho- with supernatant from rat liver. Liver supernatant was diluted mogenates determined byAshcroft, using a radiometric method with 3 vol ofthe column equilibration buffer to decrease the KC1 (12). concentration and added directly to the column. The more di- Islet glucokinase activity can be dissociated from GlcNAc lute supernatant prepared from islets was bound to the affinity kinase and hexokinase activity by chromatography on a column matrix by batch incubation, then the gel was loaded into a small of Cibacron blue F3GA agarose (Fig. 2). The binding of glu- chromatography column for elution. The bulk ofthe protein and cokinase to this triazine dye was dependent on the presence of most ofthe hexokinase eluted first. After addition of0.5 M KCl magnesium in the buffer and elution was accomplished by its to the equilibration buffer, more hexose phosphorylating activ- omission. Hexokinase was eluted from the column by inclusion ity eluted. With glucose as substrate, approximately 8% could of 0.5 M KC1 in the buffer. GlcNAc kinase activity was not de- be attributed to hexokinase (Km for glucose = 0.050 mM), tected in any fraction eluting from this column and hexokinases whereas the remaining glucose phosphorylating activity had an were found early and late in the chromatograms. apparent Km for glucose of 12 mM and evidenced marked coop- The magnesium-dependent binding of glucokinase, as well erativity with respect to glucose. GlcNAc kinase that eluted in as total protein, was pH dependent, a characteristic oftriazine Downloaded by guest on September 24, 2021 Biochemistry: Meglasson et aL Proc. Nati. Acad. Sci. USA 80 (1983) 87

ey C- .20 E E -5p +E+ 0 0 +o, +' C- -C 0 0E a 0>1 ,E94 RD D ._ go E -CD00 -y E.0 0 &.E e31 t E 0 u . Elutionr Volume, ml 3.4 &D 2

16 C - ._20. 0 0 _ -12 'I- - %. E +- E 00 3 - - - C o 0 1. ~-C X CF CL - a. 0 to .2 C. I' C c._ .4 0C -F0 E 0.E Ct0 E -.

o c o 8 16 24 32 40 48 56 64- Elution. Volume, ml 36 48 FIG. 2. Cibacron blue F3GA agarose chromatography of liver and Fraction brain or islet supernatants. The columns were equilibrated with the basic equilibration buffer, supplemented with 11 MM MgCI2. At point FIG. 3. Chromatography of supernatants of rat liver or islets on A, MgCl2 was omitted and, at pointB, 0.6 M KC1 was added. The flow DEAE-Cibacron blue F3GA agarose. The columns were equilibrated rate was 20 ml/hr. Enzymatic activities and protein were recorded as with the basic buffer supplemented with MgCI2 as indicated. Super- described for Fig. 1. (Upper) Mixture of rat liverandbrain supernatant natant samples were diluted with 3 vol of equilibration buffer and 0.01 containing 11.2 milliunits of glucokinase and 10.3 milliunits of hexo- vol of 1 M MgCl2 before loading. Elution was accomplished by the KCl kinase. Column dimensions were 0.9 x 4.6 cm. (Lower) Supernatant and MgCl2 gradients as indicated (--). The flow rate was 2 ml/hr. from 7,500 islets containing 4.3 milliunits of glucokinase and 4.9 mil- Enzymatic activity and eluate protein (- --) were determined in liunits of were x cm. hexokinase. Column dimensions 0.9 3.9 every fraction, 0.45 ml each. Enzymatic activity is indicated as in Fig. 1. (Upper) Supernatant from rat liver homogenized in homogenization dye-protein association (22). The buffer pH of 7.8 used in these buffer with 1 mM phenylmethylsulfonyl fluoride and 0.2 mM leupep- tin. Elution buffer was supplemented with 0.1 mM phenylmethylsul- experiments resulted in 53% recovery ofislet glucokinase with fonyl fluoride. Enzyme activity applied was 31.9 milliunits of gluco- approximately 100-fold purification. Another characteristic of kinase and 5.1 milliunits of hexokinase. Column dimensions were 0.6 elution of bound protein from triazine dye affinity columns is x 7.2 cm. (Lower) Supernatant from 12,000 islets. Refer to Table 2 for the broad elution peak ofglucokinase, which resulted in a glu- additional information. Column dimensions 0.6 x 7.2 cm. cokinase fraction too dilute to allow kinetic characterization. Attempts to concentrate the glucokinase activity by using small tants were prepared from freshly killed rats and were imme- Sepharose-N-(6-aminohexanoyl)-GlcNAc or DEAE-Sephadex diately subjected to chromatography without storage. columns resulted in very low recovery of glucokinase activity. Purification ofglucokinase from rat islets also resulted in two The classical ion-exchange method for separation of hexoki- fractions ofglucokinase activity (Fig. 3). Although both fractions nase isozymes was modified by the use of a mixed function col- were invariably present when either freshly prepared or pooled umn of DEAE-Cibacron blue F3GA agarose. The use of this and partially stored supernatant was used, the relative activities material allowed complete separation ofglucokinase from hexo- of fractions IV and V varied (Table 1). Phosphorylation of kinase isozymes. Additionally, GlcNAc kinase, which elutes GlcNAc was not detected with either fraction IV or fraction V. with glucokinase on anion-exchange chromatography (9), was Hexokinase activity was not detected in the glucokinase frac- removed by binding to Cibacron blue. Typical chromatograms tions. Hexokinase activity eluted as three fractions. Hexokinase of supernatants prepared from islets or liver are illustrated in eluted with the major protein peak (designated Ia) and imme- Fig. 3. The columns were eluted with a complex gradient of diately subsequent to this fraction (designated Ib). Also, hexoki- potassium and magnesium as shown. Hexokinase from liver su- nase eluted as a fraction similar to fraction II observed with pernatant eluted as three fractions (designated I, II, and III). Fraction I eluted with the major protein peak and was appar- Table 1. Recovery of islet glucokinase from DEAE-Cibacron blue. ently unretarded by the column. Fractions II and III were F3GA agarose columns eluted by a shallow KC1 gradient. Fraction III is noteworthy in Recovered glucokinase, that inhibition by glucose is apparent. Low-affinity glucose phosphorylation activity, apparently glucokinase, eluted from Glucokinase Total % of total recovered supernatants ofrat livers as two fractions (designated IV and V). applied, glucokinase Fraction Fraction milliunits % IV Fraction IV was eluted by a KCl gradient in the presence of recovered, V MgCl2. Fraction V was eluted by decreasing the buffer MgCl2 4.1 46.1 68 32 concentration while increasing the KCl concentration. Liver 8.3 55.4 86 14 glucokinase activity invariably appeared as two distinct fractions 6.4 56.1 81 19 even in the presence of protease inhibitors. All liver superna- 6.7 66.4 89 11 Downloaded by guest on September 24, 2021 88 Biochemistry: Meglasson et aL Proc. Nad Acad. Sci. USA 80 (1983) Table 2. Purification of pancreatic islet glucokinase by chromatography on DEAX-Cibacron blue F3GA agarose Enzymatic Specific activity, Protein, activity, Purification, Yield, Description milliunits mg units/g fold % Supernatant Hexokinase 8.1 2.71 3.0 1 Glucokinase 6.7 2.5 1 Column fractions Ia (2-7) 0.58 1.047 .0.6 0.2 lb (8-15) .1.66 0.181 9.2 3.1 68.5 II (22-38) 3.31 0.126 26.3 8.8 IV (56-71) 3.98 0.059 67.5 27.0 V (75-80) 0.47 0.036 13.1 5.2 J 66.4

liver. Two islet chromatograms were-atypical in that in one frac- from fraction IV displayed Michaelis-Menten kinetics with re- tion lb was absent and in the other fraction II did not occur. It spect to ATP, with a Km averaging 0.51 mM in the presence of is unclear whether- this variability resulted from the sample 40 mM glucose. D-Mannose at 100 mM was phosphorylated at loading procedure which involved dilution of the supernatant 77% ofthe rate of 100 mM D-glucose by fraction IV. Inhibition with the column equilibration buffer to diminish the superna- by D-mannoheptulose ofglucose phosphorylation by fraction IV tant KC1 concentration. This resulted in the sample being was evaluated by a method of Cornish-Bowden (23). D-man- loaded into the column at a slightly higher KC1 concentration noheptulose was a competitive inhibitor with a KY of 0.7 mM than at equilibrium. The use ofthe dilution method to diminish with respect to glucose. Phosphorylation of 10 mM glucose by the supernatant KCI concentration was chosen instead of an fraction V was inhibited by 90% by 10 mM mannoheptulose. equilibration step such as dialysis to preclude the loss of glucokinase that would have occurred. Total hexokinase recovery DISCUSSION was not altered in those experiments in which a.hexokinase fraction was apparently absent. The results ofa purification scheme Glucokinase prepared from islets ofLangerhans evidences sub- are illustrated in Table 2. stantial chromatographic and. kinetic similarity to. glucokinase Kinetic characterization of islet glucokinase fractions IV and from liver. Glucokinase from islets behaved identically to liver V indicated that glucose phosphorylation by both fractions glucokinase on three chromatography systems.. In one of these obeyed the Michaelis-Menten equation at glucose concentra- systems, Sepharose-N-(6-aminohexanoyl)-GlcNAc, glucokinase tions of 10 mM or greater (Fig. 4). Michaelis-Menten constants and GlcNAc kinase activity were eluted concomitantly. This ± finding is consistent with previous reports ofthe use-ofthis af- computed over this substrate range were 5.72. 0.33 mM (n finity chromatography system for purification ofboth these en- = 4) and 4.52 ± 0.56 mM (n = 3) for fractions IV and V, re- zymes (17, 24). In contrast, islet glucokinase prepared on either spectively. Glucose phosphorylation by both fractions markedly ofthe chromatography systems based on Cibacron blue was free deviated from the Michaelis-Menten equation at glucose con- of.contamination by hexokinase and GlcNAc kinase. The mag- centrations between 0.6 and 10 mM, resulting in nonlinear nesium-dependent affinity of glucokinase for Cibacron blue is Hanes-Woolf plots (Fig. 4). Hill coefficients determined from similar to that reported for yeast hexokinase (25). The failure replots of these data averaged 1.52 ± 0.12 (n = 4) and 1.51 of GIcNAc kinase-to elute from columns prepared with this ± 0.09 (n = 3) for fractions IV and V, respectively, clearly in- material may be related to the strongly hydrophobic nature of dicating sigmoidal kinetics with respect to glucose. Glucokinase this enzyme (24).

8 C .Fraction IV 0 6 ./, 244 A x 4 Fraction IV K2 .0 20 - 0 / x C x cm 0o 16 - XE D X --E E :L 8- oE 12 - Fraction V Wu D 0TX 6- . Cv o 8- U E x i Ix 4- & 4- 2 0 0 .40 80 120 0 10 20 Glucose, mM Glucose, mM Glucose, mM

FIG. 4. Glucose dependence of glucokinase reaction. Hanes-Woolf plots of islet glucokinase fractions IV and V are given in A and B, respectively. The relative velocity of glucose phosphorylation as a function of glucose concentration is indicated in C andD for fractions IV and V, respectively. The Michaelis-Menten kinetic functions appropriate for the constants computed from the linear portions of Hanes-Woolf plots are presented in C and D (---) for comparison with the observed data. The assay mixtures differ from that described by Trus et-al. (6) by the addition of eluant to the assay mixture. For fraction IV, the assay mixture contained 4.3 mM ATP, 8.6 mM Mg2+, 6.1 mM Pi, and 0.3 mM EDTA, and the pH was 7.7. For fraction V, the assay mixture contained 4.3 mM ATP, 5.7 mM Mg2+, 6.1 mM Pi, and 0.3 mM EDTA, and the pH was 7.7. Downloaded by guest on September 24, 2021 Biochemistry: Meglasson et aL Proc. Nati Acad. Sci. USA 80 (1983) 89 Islet glucokinase appeared as two clearly separate fractions islet cell population, show parallel, sigmoidal concentration when chromatographed by DEAE-Cibacron blue F3GA agar- dependency functions for glucose (13, 31). We observe glucoki- ose. Presently available information is insufficient to decide nase occurring in islets to exhibit similar glucose-dependent whether the two fractions are true glucokinase isoenzymes. cooperativity. This study thus firmly establishes the presence Similarly, two bands of low-affinity glucose 6-phosphotransfer- ofglucokinase in islet tissue and strongly supports the hypoth- ase activity have been reported on starch-gel electrophoreto- esis that the relationships among extracellular glucose concen- grams of liver (26, 27). Electrophoresis of liver has been re- tration, glycolytic flux in islets, and insulin release are con- ported to produce four "glucokinase" bands, which also could trolled by this enzyme serving as glucose sensor. be partially differentiated on anion-exchange chromatography (28). One fraction from this preparation was subsequently iden- This work was supported by National Institutes ofHealth Grants AM- tified as GlcNAc kinase (9), which may also be the slow band 22122, AM-19525, AM-07069, and AM-07409. identified earlier on starch gels. Kinetic characterization of the glucokinase fractions pre- 1. Matschinsky, F. M. & Ellerman, J. E. (1968)J. Biol Chem. 243, pared from islets indicates marked similarity with glucokinase 2730-2736. 2. Matschinsky, F. M., Trus, M., Burch, P., Berner, D., Ghosh, prepared from liver. Both fractions of islet glucokinase exhibit A., Zawalich, W. & Weill, V. (1980) in Diabetes 1979, Interna- similar sigmoidal kinetics with respect to glucose. The Hill coef- tional Congress Series, ed. Waldhausl, W. K. (Excerpta Medica, ficient values of 1.5 that were observed with both peaks are Amsterdam), No. 500, pp. 154-159. similar to the coefficients reported by Storer and Comish-Bow- 3. Ashcroft, S. J. H. (1981) in The Islets ofLangerhans, eds. Coop- den for purified liver glucokinase (29, 30). Also, the Michaelis- erstein, S. J. & Watkins, D. (Academic, New York), pp. 117-148. Menten constants determined from the linear portion of 4. Taljedal, I. B. (1981) Diabetologia 21, 1-17. Hanes-Woolfplots were similar, 5. Ashcroft, S. J. H. & Randle, P. J. (1970) Biochem. J. 119, 5-15. being 5.7 mM and 4.5 mM for 6. Trus, M. D., Zawalich, W. S., Burch, P. T., Berner, D. K., islet glucokinase fractions and 5 mM for glucokinase from liver Weill, V. A. & Matschinsky, F. M. (1981) Diabetes 30, 911-922. (29). These Km values are lower than those commonly reported 7. Burch, P. T., Trus, M. D., Berner, D. K., Leontire, A., Zawal- for partially pure liver glucokinase (8) or islet glucokinase ac- ich, K. C. & Matschinsky, F. M. (1981) Diabetes 30, 923-928. tivity in homogenates (1, 5, 6). The source of this difference is 8. Weinhouse, S. (1976) Curr. Top. CelL Regul 11, 1-50. unclear, although Storer and Cornish-Bowden have 9. Allen, M. B., Brockelbank, J. L. & Walker, D. G. (1980) Bio- argued it chim. Biophys. Acta 614, 357-366. is based on faulty interpretation of kinetic plots (29). The Km 10. Davagnino, J. & Ureta, T. (1980)J. Biol Chem. 255, 2633-2636. values observed for ATP were similar for islet and liver glucoki- 11. Allen, M. B. & Walker, D. G. (1980) Biochem.J. 185, 577-582. nases (8, 29). Relative phosphorylation rates for glucose and 12. Ashcroft, S. J. H. (1978) FEBS Symp. 42 (Al), 227-236. mannose were consistent with previous observations with liver 13. Ashcroft, S. J. H., Hedeskov, C. J. & Randle, P. J. (1970) glucokinase (8) and the higher Km value observed for phos- Biochem.J. 118, 143-154. phorylation of mannose by islet homogenates (6). Mannohep- 14. Zawalich, W. S., Pagliara, A. S. & Matschinsky, F. M. (1977) tulose, a competitive Endocrinology 100, 1276-1283. inhibitor of liver glucokinase (8), also in- 15. Scharp, D. W., Kemp, C. B., Knight, M. J., Ballinger, W. F. hibited islet glucokinase, although with a lower Ki than previ- & Lacy, P. E. (1973) Transplantation 16, 686-689. ously reported (0.7 mM vs. 2.5 mM). 16. Rijksen, G. & Staal, G. E. J. (1976) Biochim. Biophys. Acta 445, Extrahepatic glucokinase-like activity has been suggested to 330-341. be due to GlcNAc kinase (9, 10). In fact, GlcNAc kinase was 17. Holroyde, M. J., Chesher, J. M. E., Trayer, I. P. & Walker, D. found in islet tissue, yet the glucose phosphorylation activity G. (1976) Biochem. J. 153, 351-361. 18. Lowry, 0. H. & Passonneau, J. V. (1972) A Flexible System of we report in glucokinase fractions from islets cannot be ex- Enzymatic Analysis (Academic, New York), pp. 148-149. plained by this enzyme. We observe GlcNAc to be phosphoryl- 19. B6hlen, P., Stein, S., Dairman, W. & Udenfriend, S. (1973) ated with a Km of25 AM by a GlcNAc kinase-enriched fraction Arch. Biochem. Biophys. 155, 213-220. from islets. This value is similar to that reported by Ashcroft for 20. Kissane, J. M. & Robins, S. E. (1958)J. Biol Chem. 233, 184- islet homogenates (12) but lower than the values of 40-66 ,M 188. reported for GlcNAc kinase purified from several tissues (9-11). 21. Segel, I. H. (1975) Enzyme Kinetics (Wiley, New York). 22. Dean, P. D. G. & Watson, D. H. (1979) J. Chromatogr. 165, The reported observations of glucose phosphorylation by 301-319. GlcNAc kinase with Km values exceeding 210 mM (9-11) are 23. Cornish-Bowden, A. (1974) Biochem. J. 137, 143-144. inconsistent with our kinetic data. Also inconsistent with our 24. Allen, M. B. & Walker, D. G. (1980) Biochem. J. 185, 565-575. observations are reports of Michaelis-Menten kinetics (9, 11) 25. Hughes, P., Lowe, C. R. & Sherwood, R. F. (1982) Biochim. or negative cooperativity (10) of this enzyme with glucose as Biophys. Acta 700, 90-100. 26. Pilkis, S. J. & Hansen, R. J. (1968) Biochim. Biophys. Acta 159, substrate. Finally, we observed no GlcNAc phosphorylating 181-191. activity in glucokinase prepared by DEAE-Cibacron blue F3GA 27. Shatton, J. B., Morris, H. P. & Weinhouse, S. (1969) Cancer Res. agarose chromatography. 29, 1161-1172. Glucokinase appears to play a crucial role in regulation ofislet 28. Allen, M. B. & Walker, D. G. (1976) Biochern. Soc. Trans. 4, glucose metabolism. Starvation, which is known to decrease 1057 (abstr.) liver glucokinase (8), results in a concomitant decrease of islet 29. Storer, A. C. & Cornish-Bowden, A. (1976) Biochem. J. 159, 7- glucose utilization and glucokinase 14. activity (7). Mannoheptulose 30. Storer, A. C. & Cornish-Bowden, A. (1977) Biochem.J. 165, 61- and glucosamine, inhibitors of liver glucokinase (8), are simi- 69. larly effective in islet tissue (13, 14). Glucose utilization by islets 31. Zawalich, W. S. & Matschinsky, F. M. (1977) Endocrinology 100, and insulin secretion by beta cells, which compose 75% of the 1-8. Downloaded by guest on September 24, 2021