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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 equil- ibrated 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).
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