Hexokinase isoenzymes of RIN-m5F insulinoma cells. Expression of glucokinase gene in insulin-producing cells.

We have analysed the pattern of expression of the hexokinase isoenzyme group in RIN-m5F insulinoma cells. Three hexokinase forms were resolved by DEAE-cellulose chromatography. The most abundant isoenzyme co-eluted with hexokinase type II from rat adipose tissue and displayed a Km for glucose of 0.15 mM, similar to the adipose-tissue enzyme. Hexokinase type II was in large part associated with a particulate subcellular fraction in RIN-m5F cells. The two other hexokinases separated by ion-exchange chromatography were an enzyme similar to hexokinase type I from brain and glucokinase (or hexokinase type IV). The latter isoenzyme was identified as the liver-type glucokinase by the following properties: co-elution with hepatic glucokinase from DEAE-cellulose and DEAE-Sephadex; sigmoid saturation kinetics with glucose with half-maximal velocity at 5.6 mM and Hill coefficient (h) of 1.54; suppression of enzyme activity by antibodies raised against rat liver glucokinase; apparent Mr of 56,500 and pI of 5.6, as shown by immunoblotting after one- and two-dimensional gel electrophoresis; peptide map identical with that of hepatic glucokinase after proteolysis with chymotrypsin and papain. These data indicate that the gene coding for hepatic glucokinase is expressed in RIN-m5F cells, a finding consistent with indirect evidence for the presence of glucokinase in the beta-cell of the islet of Langerhans. On the other hand, the overall pattern of hexokinases is distinctly different in RIN-m5F cells and islets of Langerhans, since hexokinase type II appears to be lacking in islets. Alteration in hexokinase expression after tumoral transformation has been reported in other systems.     

Primary structure of the calcium ion-transporting adenosine triphosphatase of rabbit skeletal sarcoplasmic reticulum. Soluble peptides from the alpha-chymotryptic digest of the carboxymethylated protein.

1. Procedures for the extensive purification in high yield of N-acetyl-D-glucosamine kinase from rat liver and kidney are described. The separation of this enzyme from hepatic glucokinase depended primarily on their differing behaviour on an affinity column of Sepharose--N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucopyranose. 2. This N-acetyl-D-glucosamine kinase also catalyses the phosphorylation of N-acetyl-D-mannosamine and, at a lower rate, several other sugar analogues, including D-glucose. 3. A comparison of the behaviour of the enzyme during gel filtration and electrophoresis in sodium dodecyl sulphate/polyacrylamide gels suggests that N-acetyl-D-glucosamine kinase is a symmetrical dimer of mol.wt. 80000.     

Contributions of glucokinase and phosphofructokinase-2/fructose bisphosphatase-2 to the elevated glycolysis in hepatocytes from Zucker fa/fa rats

The insulin-resistant Zucker fa/fa rat has elevated hepatic glycolysis and activities of glucokinase and phosphofructokinase-2/fructose bisphosphatase-2 (PFK2). The latter catalyzes the formation and degradation of fructose-2,6-bisphosphate (fructose-2,6-P(2)) and is a glucokinase-binding protein. The contributions of glucokinase and PFK2 to the elevated glycolysis in fa/fa hepatocytes were determined by overexpressing these enzymes individually or in combination. Metabolic control analysis was used to determine enzyme coefficients on glycolysis and metabolite concentrations. Glucokinase had a high control coefficient on glycolysis in all hormonal conditions tested, whereas PFK2 had significant control only in the presence of glucagon, which phosphorylates PFK2 and suppresses glycolysis. Despite the high control strength of glucokinase, the elevated glycolysis in fa/fa hepatocytes could not be explained by the elevated glucokinase activity alone. In hepatocytes from fa/fa rats, glucokinase translocation between the nucleus and the cytoplasm was refractory to glucose but responsive to glucagon. Expression of a kinase-active PFK2 variant reversed the glucagon effect on glucokinase translocation and glucose phosphorylation, confirming the role for PFK2 in sequestering glucokinase in the cytoplasm. Glucokinase had a high control on glucose-6-phosphate content; however, like PFK2, it had a relative modest effect on the fructose-2,6-P(2) content. However, combined overexpression of glucokinase and PFK2 had a synergistic effect on fructose-2,6-P(2) levels, suggesting that interaction of these enzymes may be a prerequisite for formation of fructose-2,6-P(2). Cumulatively, this study provides support for coordinate roles for glucokinase and PFK2 in the elevated hepatic glycolysis in fa/fa rats.     

Effect of retinoic acid on glucokinase activity and gene expression in neonatal and adult cultured hepatocytes.

It has been shown that all-trans retinoic acid induces prematurely hepatic glucokinase mRNA in ten days-old neonatal rat hepatocytes, however, this effect could be related to the capacity of the retinoid to promote a more differentiated state of the hepatocyte. In this report we demonstrate that physiological concentrations of all-trans retinoic acid stimulate glucokinase activity in both mature fully differentiated hepatocytes and at the onset of the induction of the enzyme in 15 to 17 days-old neonatal hepatocytes. The effects produced by the retinoid were similar both in magnitude and in time, to those elicited by insulin, a well-known stimulator of hepatic glucokinase expression. No additive effect was observed when insulin and retinoic acid were tested together. Using the branched DNA assay, a sensitive signal amplification technique, we detected relative increases in glucokinase mRNA levels of about 70% at 3 and 24 h after the treatment with 10(-6) M all-trans retinoic acid, in both neonatal and adult hepatocytes. These data show that retinoic acid exerts a stimulatory effect on hepatic glucokinase independent of the hepatocyte stage of maturity and suggest a physiological role of retinoic acid on glucose metabolism. The action of retinoic acid on hepatic glucokinase might explain previous observations on the relationship between vitamin A status and liver glycogen synthesis. These findings may serve as basis for further investigations on the biological functions of retinoic acid derivatives on hepatic glucose metabolism.     

The amino acid sequence of rat liver glucokinase deduced from cloned cDNA

Rat liver glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified to homogeneity, cleaved, and subjected to amino acid sequence analysis. Forty-five percent of the protein sequence was obtained, and this information was used to design oligonucleotide probes to screen a rat liver cDNA library. A 1601-base pair cDNA (GK1) contained an open reading frame that encoded the amino acid sequences found in the peptides used to generate the oligonucleotide probes. A second cDNA was subsequently identified (GK.Z2), which is 2346 base pairs long and corresponds to nearly the entire glucokinase mRNA. Blot transfer analysis of hepatic RNA showed that glucokinase mRNA exists as a single species of about 2400 nucleotides. Four hours of insulin treatment of diabetic rats resulted in a 30-fold induction of this mRNA. GK.Z2 has a long open reading frame which, with the known partial peptide sequence, allowed us to deduce the primary structure of glucokinase. The enzyme is composed of 465 amino acids and has a mass of 51,924 daltons. Glucokinase has 53 and 33% amino acid sequence identities with the carboxyl-terminal domains of rat brain hexokinase I and yeast hexokinase, respectively. If conservative amino acid replacements are also considered, glucokinase is similar to these two enzymes at 75 and 63% of positions, respectively. The putative glucose- and ATP-binding domains of glucokinase were identified, and these regions appear to be highly conserved in the hexokinase family of enzymes.     

Stoichiometry of slow binding of palmitoyl-CoA to liver glucokinase

The interaction of palmitoyl-CoA with porcine glucokinase was studied by the gel permeation technique. The finding that glucokinase "bound" up to 60 molecules was unexpected from the specific inhibition of rat glucokinase by long chain acyl-CoA (Tippett & Neet, J. Biol. Chem. (1982) 287, 12839-12845). Sephacryl S-200 gel filtration in the presence of palmitoyl-CoA demonstrated a protein peak without enzyme activity that was eluted earlier than the active enzyme peak, indicating a large molecular weight shift for the inactivated enzyme form and confirming a large number (greater than or equal to 30) of associated palmitoyl-CoA molecules. The binding was also verified by analyzing the absorption characteristics of the inactivated enzyme peak. In the presence of glycerol, the size of the inactivated peak greatly decreased, but the separation between the two peaks remained unchanged. Therefore, the amphiphile bound predominantly to the inactive enzyme and not to the active form, suggesting that the rapid inhibitory interactions between palmitoyl-CoA and glucokinase previously observed are specific. Parallel enzyme activity studies showed that in the time range of the column experiments (4-20 h), both the rat and pig enzyme were greatly inactivated (greater than 90%) in the presence of palmitoyl-CoA (15 microM) in the absence of glycerol. This slow inactivation is different from the immediate specific inhibition previously reported and depends on both enzyme and palmitoyl-CoA concentrations. The presence of up to 20% glycerol slowed this inactivation process. These results demonstrated that even below the critical micelle concentration, partial inactivation of glucokinase occurs in the presence of palmitoyl-CoA over a long period of time.     

Effect of dichlorvos on hepatic and pancreatic glucokinase activity and gene expression, and on insulin mRNA levels

Several studies have shown that organophosphate pesticides affect carbohydrate metabolism and produce hyperglycemia. It has been reported that exposure to the organophosphate pesticide dichlorvos affects glucose homeostasis and decreases liver glycogen content. Glucokinase (EC 2.7.1.1) is a tissue-specific enzyme expressed in liver and in pancreatic beta cells that plays a crucial role in glycogen synthesis and glucose homeostasis. In the present study we analyzed the effect of one or three days of dichlorvos administration [20 mg/kg body weight] on the activity and mRNA levels of hepatic and pancreatic glucokinase as well as on insulin mRNA abundance in the rat. We found that the pesticide affects pancreatic and hepatic glucokinase activity and expression differently. In the liver the pesticide decreased the enzyme activity; on the contrary glucokinase mRNA levels were increased. In contrast, pancreatic glucokinase activity as well as mRNA levels were not affected by the treatment. Insulin mRNA levels were not modified by dichlorvos administration. Our results suggest that the decreased activity of hepatic glucokinase may account for the adverse effects of dichlorvos on glucose metabolism.     

Bisphenol A Impairs Hepatic Glucose Sensing in C57BL/6 Male Mice

Aims/Hypothesis

Glucose sensing (eg. glucokinase activity) becomes impaired in the development of type 2 diabetes, the etiology of which is unclear. Estrogen can stimulate glucokinase activity, whereas the pervasive environmental pollutant bisphenol A (BPA) can inhibit estrogen action, hence we aimed to determine the effect of BPA on glucokinase activity directly.

Methods

To evaluate a potential acute effect on hepatic glucokinase activity, BPA in water (n = 5) vs. water alone (n = 5) was administered at the EPA’s purported “safe dose” (50 µg/kg) by gavage to lean 6-month old male C57BL/6 mice. Two hours later, animals were euthanized and hepatic glucokinase activity measured over glucose levels from 1–20 mmol/l in liver homogenate. To determine the effect of chronic BPA exposure on hepatic glucokinase activity, lean 6-month old male C57BL/6 mice were provided with water (n = 15) or water with 1.75 mM BPA (∼50 µg/kg/day; n = 14) for 2 weeks. Following the 2-week exposure, animals were euthanized and glucokinase activity measured as above.

Results

Hepatic glucokinase activity was signficantly suppressed after 2 hours in animals given an oral BPA bolus compared to those who received only water (p = 0.002–0.029 at glucose 5–20 mmol/l; overall treatment effect p<0.001). Exposure to BPA over 2 weeks also suppressed hepatic glucokinase activity in exposed vs. unexposed mice (overall treatment effect, p = 0.003). In both experiments, the Hill coefficient was higher and Vmax lower in mice treated with BPA.

Conclusions/Interpretation

Both acute and chronic exposure to BPA significantly impair hepatic glucokinase activity and function. These findings identify a potential mechanism for how BPA may increase risk for diabetes.     

The identification of extrahepatic "glucokinase" as N-acetylglucosamine kinase

Several research groups have reported the presence of a high Km glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.2) in tissues other than adult liver. As shown in this report, protein fractions catalyzing glucose phosphorylation only at high substrate concentrations (100 mM) are indeed found in bovine spleen, rat kidney, human placenta, and newborn rat liver. However, the study of substrate specificities and Michaelis constant values showed that those fractions could be better described as N-acetylglucosamine kinase (ATP:acetamide-2-deoxy-D-glucose-6-phosphotransferase, EC 2.7.1.9) which, in addition to N-acetylglucosamine (Km = 0.066 mM), can also phosphorylate glucose although with very high Km values (370 mM). Furthermore, a homogeneous preparation from bovine spleen was able to phosphorylate both N-acetylglucosamine and glucose. An immune serum against bovine spleen N-acetylglucosamine kinase did not cross-react with purified hexokinases or with glucokinase from rat. However, it was able to remove the putative "glucokinases" from extracts of rat kidney, newborn rat liver, and one of two electrophoretic bands of liver "glucokinase." It is proposed that any report of extrahepatic glucokinase should explicity rule out N-acetylglucosamine kinase as the enzyme being described.     

Free fatty acids increase basal hepatic glucose production and induce hepatic insulin resistance at different sites

To investigate the sites of the free fatty acid (FFA) effects to increase basal hepatic glucose production and to impair hepatic insulin action, we performed 2-h and 7-h Intralipid + heparin (IH) and saline infusions in the basal fasting state and during hyperinsulinemic clamps in overnight-fasted rats. We measured endogenous glucose production (EGP), total glucose output (TGO, the flux through glucose-6-phosphatase), glucose cycling (GC, index of flux through glucokinase = TGO - EGP), hepatic glucose 6-phosphate (G-6-P) content, and hepatic glucose-6-phosphatase and glucokinase activities. Plasma FFA levels were elevated about threefold by IH. In the basal state, IH increased TGO, in vivo glucose-6-phosphatase activity (TGO/G-6-P), and EGP (P < 0.001). During the clamp compared with the basal experiments, 2-h insulin infusion increased GC and in vivo glucokinase activity (GC/TGO; P < 0.05) and suppressed EGP (P < 0.05) but failed to significantly affect TGO and in vivo glucose-6-phosphatase activity. IH decreased the ability of insulin to increase GC and in vivo glucokinase activity (P < 0.01), and at 7 h, it also decreased the ability of insulin to suppress EGP (P < 0.001). G-6-P content was comparable in all groups. In vivo glucose-6-phosphatase and glucokinase activities did not correspond to their in vitro activities as determined in liver tissue, suggesting that stable changes in enzyme activity were not responsible for the FFA effects. The data suggest that, in overnight-fasted rats, FFA increased basal EGP and induced hepatic insulin resistance at different sites. 1) FFA increased basal EGP through an increase in TGO and in vivo glucose-6-phosphatase activity, presumably due to a stimulatory allosteric effect of fatty acyl-CoA on glucose-6-phosphatase. 2) FFA induced hepatic insulin resistance (decreased the ability of insulin to suppress EGP) through an impairment of insulin's ability to increase GC and in vivo glucokinase activity, presumably due to an inhibitory allosteric effect of fatty acyl-CoA on glucokinase and/or an impairment in glucokinase translocation.     

Studies on the use of sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucopyranose for the large-scale purification of hepatic glucokinase.

The synthesis of N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucose is described and it was shown to be a competitive inhibitor (Ki, 0.75 mM) with respect to glucose of rat hepatic glucokinase (EC 2.7.1.2). After attachment to CNBr-activated Sepharose 4B, this derivative was able to remove glucokinase quantitatively from crude liver extracts and release it when the columns were developed with glucose, glucosamine, N-acetyl-glucosamine or KC1. Repeated exposure of the columns to liver extracts led to rapid loss in their effectiveness as affinity matrices because proteins other than glucokinase are bound to the columns. The nature of such protein binding and methods for the rejuvenation of "used" columns are discussed along with the effect of the mode of preparation of the Sepharose-ligand conjugate and the concentration of bound ligand on the purification of glucokinase. Glucose 6-phosphate dehydrogenase is cited as an example of both non-specific protein binding to the affinity column and of the importance of the control of ligand concentration in removing such non-specifically bound proteins. Some guidelines emerged that should be generally applicable to other systems, particularly those which involve affinity chromatography of enzymes that are present in tissue extracts in very low amounts and possess only a relatively low association constant for the immobilized ligand.     

Radiometric oil well assay for glucokinase in microscopic structures

Glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.1) plays a pivotal role in hepatic glucose metabolism and serves as the glucose sensor in pancreatic islet beta-cells. Biochemical studies of this enzyme are complicated by the cellular heterogeneity of the liver and the pancreas and because the presence of hexokinases (ATP:D-hexose 6-phosphotransferases, EC 2.7.1.1) seriously interferes with currently available analytical procedures. A radiometric assay was designed to deal with these problems. It is based on the liberation of 3H2O from D-[2-3H(N)]glucose 6-phosphate, the product of the glucokinase reaction, using exogenous phosphoglucose isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9). Interference by hexokinases was largely eliminated by using glucose 6-phosphate as inhibitor and the sensitivity of the assay was greatly increased by using small volumes with the oil well procedure. The assay was sufficiently sensitive to detect about 1 pg of glucokinase. It thus allowed the application of quantitative histochemical procedures to the study of intralobular hepatic glucokinase profiles and the pancreatic beta-cell glucose sensor. The quantitative histochemical procedures were sufficiently sensitive and reliable for measuring important kinetic constants of glucokinase (i.e., the Km and the Hill number) in microscopic samples of tissue.     

The mechanism by which rat liver glucokinase is inhibited by the regulatory protein

The fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive protein that inhibits rat liver glucokinase [Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179-184] was purified close to homogeneity by a procedure involving poly(ethyleneglycol) precipitation, chromatography on anion-exchangers and hydroxylapatite, gel filtration and chromatography on Mono S, a cation exchanger. In the last chromatographic step, the regulatory protein coeluted with a 62 kDa peptide. From the elution volume of the gel-filtration column a molecular mass of 60 kDa was determined, allowing the conclusion that the regulator is a monomer. The decrease in the apparent affinity of glucokinase for glucose, which the regulator induced, disappeared upon separation of the two proteins. Furthermore, the regulator did not appear to catalyse the formation of a heat-stable or a trypsin-resistant inhibitor of glucokinase. Finally, the inhibition exerted by the regulatory protein reached a steady value 1-2 min after the addition of the regulator. These results indicate that the regulator does not act by causing a covalent modification of glucokinase nor by catalysing the formation of a low-molecular-mass inhibitor. Raising the concentration of glucokinase in the assay from 6 mU/ml to 120 mU/ml caused a 2.5-fold increase in the concentration of regulator required to half-maximally inhibit the enzyme. The apparent mass of glucokinase, as determined by centrifugation in isokinetic sucrose gradient, was 55 kDa, and this value was unaffected by the separate presence of fructose 6-phosphate or of the regulatory protein. However, the apparent mass of the enzyme increased to 105 kDa when glucokinase was centrifuged together with both fructose 6-phosphate and the regulatory protein, although not when fructose 1-phosphate was also present. Conversely, the presence of glucokinase increased the apparent molecular mass of the regulator in the presence of fructose 6-phosphate. From these results, it is concluded that the regulatory protein inhibits glucokinase by forming a complex with this enzyme in the presence of fructose 6-phosphate, and that fructose 1-phosphate antagonises this inhibition by preventing the formation of the complex.     

High-Yield Purification of Glucokinase from Rat Liver

A rapid and reliable method for the purification of rat liver glucokinase was developed. The procedure consists of DEAE-cellulose ion-exchange chromatography, Phenyl-Sepharose hydrophobic interaction chromatography, DEAE-Affi Gel Blue dye-ligand chromatography, and duplicate steps of glucosamine-Sepharose affinity chromatography. Glucokinase was purified to a specific activity of 290 units/mg protein in a yield of 55% in 6 days. The final enzyme preparations were completely homogeneous in most experiments as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The estimated molecular weight (51.000) and sigmoidal saturation function for glucose of purified glucokinase were in good agreement with published data.     

High-yield purification of glucokinase from rat liver

A rapid and reliable method for the purification of rat liver glucokinase was developed. The procedure consists of DEAE-cellulose ion-exchange chromatography, Phenyl-Sepharose hydrophobic interaction chromatography, DEAE-Affi Gel Blue dye-ligand chromatography, and duplicate steps of glucosamine-Sepharose affinity chromatography. Glucokinase was purified to a specific activity of 290 units/mg protein in a yield of 55% in 6 days. The final enzyme preparations were completely homogeneous in most experiments as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The estimated molecular weight (51,000) and sigmoidal saturation function for glucose of purified glucokinase were in good agreement with published data.     

Modulation of human glucokinase intrinsic activity by SH reagents mirrors post-translational regulation of enzyme activity

The low-affinity glucose phosphorylating enzyme glucokinase plays a key role in the process of glucose recognition in pancreatic B-cells. To evaluate mechanisms of intrinsic regulation of enzyme activity human pancreatic B-cell and liver glucokinase and for comparison rat liver glucokinase were expressed in E. coli bacteria. A one-step purification procedure through metal chelate affinity chromatography revealed 58 kDa proteins with high specific activities in the range of 50 U/mg protein and K_m values around 8 mM for the substrate d-glucose with a preference for the α-anomer. There were no tissue specific differences, no species differences in the electrophoretic mobility, and no differences of the kinetic properties of these well conserved enzymes. The deletion of the 15 tissue-specific NH₂-terminal amino acids of the human glucokinase resulted in a catalytically active enzyme whose kinetic properties were not significantly different from those of the wild-type enzymes. The human and rat glucokinase isoforms were non-competitively inhibited by the sulfhydryl group reagents alloxan and ninhydrin with K_i values in the range of 1 μM. The inhibition of glucokinase enzyme activity was reversed by dithiothreitol with an EC₅₀ value of 9 μM for alloxan and of 60 μM for ninhydrin. d-Glucose provided protection against alloxan-induced inhibition of human and rat glucokinase isoenzymes with half-maximal effective concentrations between 11 and 16 mM. The enzyme inhibition by alloxan was accompanied by a change in the electrophoretic mobility with a second lower molecular 49 kDa glucokinase band which can be interpreted as a compact glucokinase molecule locked by disulfide bonds. Quantification of free sulfhydryl groups revealed an average number of 3.6 free sulfhydryl groups per enzyme molecule for the native human glucokinase isoforms. Alloxan decreased the average number of free sulfhydryl groups to 1.9 per enzyme molecule indicating that more than one SH side group is oxidized by this compound. The extraordinary sensitivity of the SH side groups of the glucokinase may be a possible mechanism of enzyme regulation by interconversion of stable (active) and unstable (inactive) conformations of the enzyme. In pancreatic B-cells the glucose-dependent increase of reduced pyridine nucleotides may stabilize the enzyme in the 58 kDa form and provide optimal conditions for glucose recognition and glucose-induced insulin secretion.     

Characterization of glucokinase regulatory protein-deficient mice

The glucokinase regulatory protein (GKRP) inhibits glucokinase competitively with respect to glucose by forming a protein-protein complex with this enzyme. The physiological role of GKRP in controlling hepatic glucokinase activity was addressed using gene targeting to disrupt GKRP gene expression. Heterozygote and homozygote knockout mice have a substantial decrease in hepatic glucokinase expression and enzymatic activity as measured at saturating glucose concentrations when compared with wild-type mice, with no change in basal blood glucose levels. Interestingly, when assayed under conditions to promote the association between glucokinase and GKRP, liver glucokinase activity in wild-type and null mice displayed comparable glucose phosphorylation capacities at physiological glucose concentrations (5 mM). Thus, despite reduced hepatic glucokinase expression levels in the null mice, glucokinase activity in the liver homogenates was maintained at nearly normal levels due to the absence of the inhibitory effects of GKRP. However, following a glucose tolerance test, the homozygote knockout mice show impaired glucose clearance, indicating that they cannot recruit sufficient glucokinase due to the absence of a nuclear reserve. These data suggest both a regulatory and a stabilizing role for GKRP in maintaining adequate glucokinase in the liver. Furthermore, this study provides evidence for the important role GKRP plays in acutely regulating of hepatic glucose metabolism.     

Phosphorylation of glucose by a guanosine-5′-triphosphate (GTP)-dependent glucokinase in Fibrobacter succinogenes subsp. succinogenes S85

Cell extracts of Fibrobacter succinogenes subsp. succinogenes S85 phosphorylated glucose with a GTP-dependent glucokinase. The enzyme showed little activity with ATP (12% of that with GTP). Of other phosphate donors tested, only dGTP and ITP gave high glucokinase activities. Dialyzed extracts required Mg⁺² and K⁺ for maximal activity. In potassium phosphate buffer, glucokinase showed maximum activity at pH 7.5 with glucose-6-phosphate dehydrogenase as the coupling enzyme. In this assay, glucokinase was active with glucose (100%), 2-deoxy-d-glucose (40%), and mannose (20%). Partially purified glucokinase had a molecular weight of 82,000 and a pl of 4.82. Double-reciprocal plots of substrate concentration versus velocity were linear and the enzyme had apparent K_m values of 55 M for glucose and 72 M for GTP. Dialyzed cell extracts of Fibrobacter intestinalis C1A also contained a GTP-dependent glucokinase that showed little activity with ATP. Potassium also stimulated the activity of this enzyme. These results suggest that this unusual glucokinase may be characteristic of the genus Fibrobacter.Abbreviations CHES cyclohexylaminoethanesulfonic acid - GK glucokinase - PEP phosphoenolpyruvate Published with the approval of the Director of the North Dakota Agricultural Experiment Station as journal article no. 2186     

A novel glucokinase regulator in pancreatic beta cells: precursor of propionyl-CoA carboxylase beta subunit interacts with glucokinase and augments its activity

A glucokinase regulatory protein has been reported to exist in the liver, which suppresses enzyme activity in a complex with fructose 6-phosphate, whereas no corresponding protein has been found in pancreatic beta cells. To search for such a protein in pancreatic beta cells, we screened for a cDNA library of the HIT-T15 cell line with the cDNA of glucokinase from rat islet by the yeast two hybrid system. We detected a cDNA encoding the precursor of propionyl-CoA carboxylase beta subunit (pbetaPCCase), and glutathione S-transferase pull-down assay illustrated that pbetaPCCase interacted with recombinant rat islet glucokinase and with glucokinase in rat liver and islet extracts. Functional analysis indicated that pbetaPCCase decreased the K(m) value of recombinant islet glucokinase for glucose by 18% and increased V(max) value by 23%. We concluded that pbetaPCCase might be a novel activator of glucokinase in pancreatic beta cells.     

ADP-dependent glucokinase/phosphofructokinase, a novel bifunctional enzyme from the hyperthermophilic archaeon Methanococcus jannaschii

A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing. The gene encoded a protein of 462 amino acids with a molecular weight of 53,361. The deduced amino acid sequence of the gene showed 52 and 29% identities to the ADP-dependent phosphofructokinase and glucokinase from Pyrococcus furiosus, respectively. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. To our surprise, the enzyme showed high ADP-dependent activities for both glucokinase and phosphofructokinase. A native molecular mass was estimated to be 55 kDa, and this indicates the enzyme is monomeric. The reaction rate for the phosphorylation of D-glucose was almost 3 times that for D-fructose 6-phosphate. The K(m) values for D-fructose 6-phosphate and D-glucose were calculated to be 0.010 and 1.6 mm, respectively. The K(m) values for ADP were 0.032 and 0.63 mm when D-glucose and D-fructose 6-phosphate were used as a phosphoryl group acceptor, respectively. The gene encoding the enzyme is proposed to be an ancestral gene of an ADP-dependent phosphofructokinase and glucokinase. A gene duplication event might lead to the two enzymatic activities.