Oct 4, 1982 - rified by chromatography on DEAE-Cibacron blue F3GA agar- ose. The enzyme ... results are consistent with the concept that glucokinase serves as the glucose sensor of ..... In contrast, isletglucokinase prepared on either.
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-phosphotransferase, EC 2.7.1.2) from rat islets of Langerhans was partially purified by chromatography on DEAE-Cibacron blue F3GA agarose. The enzyme eluted in two separate peaks. Sigmoidal rate dependence was found with respect to glucose (Hill coefficient = 1.5) for both enzyme fractions. K. values for glucose were 5.7 mM for the major fraction and 4.5 mM for the minor fraction. Neither fraction phosphorylated GlcNAc. A' GlcNAc kinase (ATP. 2-acetamido-2-deoxy-D-glucose' 6-phosphotransferase; EC 2.7.1.59)-enriched fraction; prepared by affinity chromatography on Sepharose-N46-aminohexanoyl)-GlcNAc, had a K. of 25 ,EM for GlcNAc. Islet tissue also contained hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) eluting in multiple peaks. The results are consistent with the concept that glucokinase serves as the glucose sensor of pancreatic beta cells.
of islets equals serum glucose and islet glucose 6-phosphate increases in proportion to extracellular glucose (1, 13). Ninety percent of islet glucose utilization occurs with an apparent glucose affinity constant 11.1 mM (6). Also, mannoheptulose, an inhibitor of glucokinase (8), profoundly inhibits islet glucose metabolism. (14). The chromatographic separation of glucokinase from other glucose phosphorylating enzymes is the most direct approach to establish its presence in pancreatic islets. It is the purpose of this paper to report the partial purification of glucokinase,. free of hexokinase and GlcNAc kinase contamination, from islets of Langerhans.
MATERIALS AND METHODS Tissue Preparation., Pancreatic islets were prepared from fed male Wistar rats (Hilltop Laboratory Animals, Scottdale, PA) weighing 225-300 g as described (6). Islets were separated from the collagenase digest on a Ficoll gradient (15). By using this method, 6,000-8,000 islets could be prepared each day. Isolated islets were homogenized after being washed free of glucose, which was present at 5 mM throughout the isolation protocol. Homogenization was performed with 10 vol of homogenizing buffer (20 mM K2HPO4, pH 7.8, containing 1 mM dithiothreitol, 1 mM EDTA, and 110 mM KCl) in a Kontes 18 glass homogenizer by 20 strokes of a machine-driven Teflon pestle (Bellco Glass homogenizer drive unit; set on 3). This and all subsequent purification steps were performed at2-40C. The homogenate was centrifuged at 105,000 X g for 60 min. The supernatant fraction was either used immediately or stored at -800C and pooled with the islet supernatant prepared the subsequent day. Chromatography. Sepharose-N-(6-aminohexanoyl)-GlcNAc was prepared by modification of the method of Rijksen and Staal (16). 6-Aminohexanoic acid-activated Sepharose 4B (Sigma), stated by the supplier to have 30-42 tkmol of active ester per g of gel, was allowed to react with D-glucosamine at a ratio of 200 /imol of glucosamine per g. The ligand concentration was decreased by mixing the GlcNAc-coupled gel with an equal packed wet weight of Sepharose 4B (17). Cibacron blue F3GA agarose and DEAE-Cibacron blue F3GA agarose were supplied by Bio-Rad. The basic equilibration buffer for all columns was 20 mM.K2HPO4, pH 7.8, with 1 mM EDTA and 1 mM dithiothreitol. Supplements to this buffer and elution conditions are described in Results. Elution of DEAE-Cibacron blue F3GA agarose columns was accomplished by using a complex gradient of KCI and MgCl2 produced by an LKB Ultrograd gradient maker. This gradient was verified by determining the Mg2' and K+ content of the column fractions with an ion analyzer (Beckman Select Ion 5000).
Glucose phosphorylation is considered to be the rate-limiting step in glucose utilization by pancreatic islets and may determine the relationship between extracellular glucose concentration and initiation of insulin- secretion (1-4). Homogenates of rodent pancreatic islets contain glucose 6-phosphotransferase activity of both low and high affinities for glucose (1, 2, 5, 6). The high-affinity component, composed of one or more hexokinase isozymes (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), is largely inhibited in the intact cell (2). However, the low-affinity component appears to be fully active under physiological conditions and appears to determine the rate of glucose utilization by islets (2, 6, 7). This enzyme exhibits a MichaelisMenten constant of about 10 mM for glucose (1, 2, 5, 6), similar to that reported for liver glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.2) (8). It has been suggested that this enzyme in islets is glucokinase similar to that found. in liver (1,
5).
Glucokinase has been claimed to be present also in a number of other extrahepatic tissues (8). Recently, these claims have been disputed when those tissues examined were shown to contain GlcNAc kinase (ATP:2-acetamido-2-deoxy-D-glucose 6phosphotransferase, EC 2.7.1.59) rather than glucokinase (9, 10). The failure to observe glucokinase on electrophoretograms of rodent islets has been reported also (4). Like glucokinase, GlcNAc kinase has been shown to phosphorylate glucose; however, the reported K, values are very high, being 210 mM (9), 370 mM (10), 410 mM, or 600 mM (11). Rodent islets are known to contain substantial amounts of GlcNAc kinase (12). Therefore, the proposed role of glucokinase in islet physiology has been questioned (9, 10). Many observations suggest, however, a physiological role for glucokinase or a similar enzyme in islets. Intracellular glucose The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
*
85
To whom reprint requests should be addressed.
86
Proc. Natl.- Acad. Sci. USA 80 (1983)
Biochemistry: Meglasson et aL
Assay Methods. Glucose phosphorylation was determined by a fluorometric enzymatic assay method (6). The reaction 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 mM in excess ofthe ATP concentration after-the EDTA content of the added eluant had been corrected for. Phosphorylation of sugars other than glucose was measured by a fluorometric enzyme assay with pyruvate kinase and lactate delhydrogenase (Boehringer Mannheim) as coupling enzymes (18). The reaction components were 50 mM Tris-HCl (pH 8.2), 100 mM KC1, 2 mM MgCl2, 7.1 mM 2-mercaptoethanol, 0.05mM NADH, 0.05 mM phosphoenolpyruvate, 0.6 mM ATP, pyruvate kinase from rabbit muscle at 50 ug/ml, and lactate dehydrogenase from beef muscle at 10 ,ug/ml. The reaction was conducted in 100 ,ul for 2 hr at 300C and was terminated by addition of 1 ml of 0.05 M NaOH containing 0.5 mM cysteine. The half-time of the reaction was about 15 sec. Sample fluorescence was corrected for tissue NADH oxidase and ATPase activity by subtraction of appropriate tissue and reagent blanks. Sample protein content was determined by the method of Bohlen and coworkers (19). Islet homogenate DNA content was determined by the method of Kissane and Robins (20). Kinetic Analysis. Kinetic constants were determined by least-squares linear regression of Hanes-Woolf plots (ref. 21, p. 210). Deviation of glucokinase from Michaelis-Menten kinetics was evaluated by using the Hill plot (ref. 21, pp. 371375). The slope of this plot was determined at v = 0.5 Vm,. Enzyme activity is expressed as units; one unit is equal to phosphorylation of 1 /Lmol min' of sugar at 30'C. Data are expressed as sample means and their standard errors.
RESULTS Glucose phosphorylation activity in islet supernatant was resolved into two kinetic components on a Hanes-Woolf plot (not shown). The high-affinity component had a Km value of 0.047 ± 0.010 mM and a Vm_ of 71 + 7 units per g of DNA (n = 9). The low-affinity component, corrected for hexokinase activity, had a Km of 10.40 ± 1.74 mM and a Vm. of 69 ± 3 units per g of DNA (n = 9). These values are generally similar to those previously reported by our laboratory for the 12,000 x g supernatant ofsmall batches of rat islets prepared by hand-picking 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 function of DNA content, than the islets in this study, which were prepared on Ficoll gradients. Preliminary studies performed with Ficoll-prepared islets demonstrated that supernatant glucokinase activity was stable when stored for 6 hr at 4°C or 24 hr at -80°C. Glucokinase from islets was partially purified by affinity chromatography on Sepharose-N-(6-aminohexanoyl)-GlcNAc (Fig. 1). For comparison, a similar experiment was performed with supernatant from rat liver. Liver supernatant was diluted with 3 vol ofthe column equilibration buffer to decrease the KC1 concentration and added directly to the column. The more dilute supernatant prepared from islets was bound to the affinity matrix by batch incubation, then the gel was loaded into a small chromatography column for elution. The bulk ofthe protein and most of the hexokinase eluted first. After addition of 0.5 M KCl to the equilibration buffer, more hexose phosphorylating activity eluted. With glucose as substrate, approximately 8% could be attributed to hexokinase (Km for glucose = 0.050 mM), whereas the remaining glucose phosphorylating activity had an apparent Km for glucose of 12 mM and evidenced marked cooperativity with respect to glucose. GlcNAc kinase that eluted in
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