Activities of glucokinase and hexokinase in mammalian and avian livers.
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ABSTRACT:
SUBMITTER: Stanley JC
PROVIDER: S-EPMC1144479 | biostudies-other | 1984 Dec
REPOSITORIES: biostudies-other
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Project description:Conflicting data have been reported concerning the anomeric specificity of glucokinase. In the present study, liver hexokinase (Km for D-glucose 0.4 mM) displayed a higher affinity for but lower Vmax. with alpha- than with beta-D-glucose. The velocity of the reaction catalysed by liver glucokinase was higher with with beta- than with alpha-D-glucose, whatever the glucose concentration. The apparent Km of glucokinase was somewhat lower, however, with alpha- than with beta-D-glucose. Comparable results were obtained for the high-Km glucokinase-like enzymic activity present in normal pancreatic islets or insulin-producing tumoral cells. These results suggest that the anomeric specificity of glucokinase cannot account for the higher rate of glycolysis found in islets exposed to alpha- as distinct from beta-D-glucose.
| S-EPMC1152624 | biostudies-other
Project description:Glucokinase catalyzes the ATP-dependent phosphorylation of glucose, a chemical transformation that represents the rate-limiting step of glycolytic metabolism in the liver and pancreas. Glucokinase is a central regulator of glucose homeostasis as evidenced by its association with two disease states, maturity onset diabetes of the young (MODY) and persistent hyperinsulinemia of infancy (PHHI). Mammalian glucokinase is subject to homotropic allosteric regulation by glucose-the steady-state velocity of glucose-6-phosphate production is not hyperbolic, but instead displays a sigmoidal response to increasing glucose concentrations. The positive cooperativity displayed by glucokinase is intriguing since the enzyme functions as a monomer under physiological conditions and contains only a single binding site for glucose. Despite the existence of several models of kinetic cooperativity in monomeric enzymes, a consensus has yet to be reached regarding the mechanism of allosteric regulation in glucokinase. Experimental evidence collected over the last 45 years by a number of investigators supports a link between cooperativity and slow conformational reorganizations of the glucokinase scaffold. In this review, we summarize advances in our understanding of glucokinase allosteric regulation resulting from recent X-ray crystallographic, pre-equilibrium kinetic and high-resolution nuclear magnetic resonance investigations. We conclude with a brief discussion of unanswered questions regarding the mechanistic basis of kinetic cooperativity in mammalian glucokinase.
| S-EPMC3294010 | biostudies-literature
Project description:Premature neonates are submitted to an early-life oxidative stress from parenteral nutrition, which is vitamin C (VC) deficient and induces low endogenous levels of glutathione. The oxidative stress caused by these deficiencies may permanently affect liver glycolysis and lipogenesis. This study evaluates the short- and long-term effects of neonatal VC and cysteine deficient diets on redox and energy metabolism. Three-day-old Hartley guinea pigs from both sexes were given a regular or a deficient diet (VC, cysteine, or both) until week 1 of life. Half of the animals were sacrificed at this age, while the other half ate a complete diet until 12 weeks. Liver glutathione and the activity and protein levels of glucokinase, phosphofructokinase, and acetyl-CoA-carboxylase were measured. Statistics: factorial ANOVA (5% threshold). At 1 week, all deficient diets decreased glutathione and the protein levels of glucokinase and phosphofructokinase, while cysteine deficiency decreased acetyl-CoA-carboxylase levels. A similar enzyme level was observed in control animals at 12 weeks. At this age, VC deficiency decreased glutathione, while cysteine increased it. Acetyl-CoA-carboxylase protein levels were increased, which decreased its specific activity. Early-life VC and cysteine deficiencies induce neonatal oxidative stress and an adult-like metabolism, while predisposing to increased lipogenic rates during adulthood.
| S-EPMC8231532 | biostudies-literature
Project description:The release of glucokinase (hexokinase IV) from digitonin-permeabilized hepatocytes from rat, guinea pig or mouse liver is inhibited by physiological concentrations of Mg2+ (> 0.25 mM). Preincubation of hepatocytes with fructose increases glucokinase release during permeabilization in the presence of Mg2+ but decreases glucokinase release in the absence of Mg2+, suggesting that fructose causes translocation of glucokinase from the Mg(2+)-dependent site. Glucose (25 mM) and sorbitol (1 mM) also induce translocation of glucokinase from the Mg(2+)-dependent site in guinea-pig, as in rat hepatocytes, but glucose is less effective than fructose or sorbitol, and the concentrations of fructose and sorbitol that cause half-maximal activation (A50) are 3-fold and 20-fold higher, respectively, in guinea-pig than in rat hepatocytes (170 microM and 257 microM, compared with 61 microM and 13 microM). Dihydroxyacetone and glycerol have no effect on fructose-induced or sorbitol-induced translocation in guinea-pig hepatocytes, in contrast with the potentiation and inhibition, respectively, by these substrates in rat hepatocytes. Some, but not all, of the differences between rat and guinea-pig hepatocytes could be due to the more reduced cytoplasmic NADH/NAD+ redox state in guinea-pig cells. The activity of low-Km hexokinases accounts for 30% of total hexokinase activity (low-Km hexokinases + glucokinase) in guinea-pig hepatocytes. Of the low-Km hexokinase activity, approx. 30% is released in the presence of Mg2+, 9% shows Mg(2+)-dependent binding and 60% shows Mg(2+)-independent binding. There was no substrate-induced translocation of low-Km hexokinase activity, indicating that translocation is specific for hexokinase IV.
| S-EPMC1137623 | biostudies-other
Project description:Fungi contain several hexokinases, which are involved either in sugar phosphorylation or in carbon source sensing. Glucose and fructose phosphorylations appear to rely exclusively on glucokinase and hexokinase. Here, we characterized the catalytic glucokinase and hexokinase from the opportunistic human pathogen Aspergillus fumigatus and showed that both enzymes display different biochemical properties and play different roles during growth and development. Glucokinase efficiently activates glucose and mannose but activates fructose only to a minor extent. Hexokinase showed a high efficiency for fructose activation but also activated glucose and mannose. Transcript and activity determinations revealed high levels of glucokinase in resting conidia, whereas hexokinase was associated mainly with the mycelium. Consequentially, a glucokinase mutant showed delayed germination at low glucose concentrations, whereas colony growth was not overly affected. The deletion of hexokinase had only a minor impact on germination but reduced colony growth, especially on sugar-containing media. Transcript determinations from infected mouse lungs revealed the expression of both genes, indicating a contribution to virulence. Interestingly, a double-deletion mutant showed impaired growth not only on sugars but also on nonfermentable nutrients, and growth on gluconeogenic carbon sources was strongly suppressed in the presence of glucose. Furthermore, the glkA hxkA deletion affected cell wall integrity, implying that both enzymes contribute to the cell wall composition. Additionally, the absence of either enzyme deregulated carbon catabolite repression since mutants displayed an induction of isocitrate lyase activity during growth on glucose-ethanol medium. Therefore, both enzymes seem to be required for balancing carbon flux in A. fumigatus and are indispensable for growth under all nutritional conditions.
| S-EPMC2901669 | biostudies-literature
Project description:Congenital hyperinsulinism causes persistent hypoglycemia in neonates and infants. Most often, uncontrolled insulin secretion (IS) results from a lack of functional K(ATP) channels in all ?-cells or only in ?-cells within a resectable focal lesion. In more rare cases, without K(ATP) channel mutations, hyperfunctional islets are confined within few lobules, whereas hypofunctional islets are present throughout the pancreas. They also can be cured by selective partial pancreatectomy; however, unlike those with a K(ATP) focal lesion, they show clinical sensitivity to diazoxide. Here, we characterized in vitro IS by fragments of pathological and adjacent normal pancreas from six such cases. Responses of normal pancreas were unremarkable. In pathological region, IS was elevated at 1 mmol/L and was further increased by 15 mmol/L glucose. Diazoxide suppressed IS and tolbutamide antagonized the inhibition. The most conspicuous anomaly was a large stimulation of IS by 1 mmol/L glucose. In five of six cases, immunohistochemistry revealed undue presence of low-K(m) hexokinase-I in ?-cells of hyperfunctional islets only. In one case, an activating mutation of glucokinase (I211F) was found in pathological islets only. Both abnormalities, attributed to somatic genetic events, may account for inappropriate IS at low glucose levels by a subset of ?-cells. They represent a novel cause of focal congenital hyperinsulinism.
| S-EPMC3636634 | biostudies-literature
Project description:The glucokinase (GCK) gene was one of the first candidate genes to be identified as a human "diabetes gene". Subsequently, important advances were made in understanding the impact of GCK in the regulation of glucose metabolism. Structure elucidation by crystallography provided insight into the kinetic properties of GCK. Protein interaction partners of GCK were discovered. Gene expression studies revealed new facets of the tissue distribution of GCK, including in the brain, and its regulation by insulin in the liver. Metabolic control analysis coupled to gene overexpression and knockout experiments highlighted the unique impact of GCK as a regulator of glucose metabolism. Human GCK mutants were studied biochemically to understand disease mechanisms. Drug development programs identified small molecule activators of GCK as potential antidiabetics. These advances are summarized here, with the aim of offering an integrated view of the role of GCK in the molecular physiology and medicine of glucose homeostasis.
| S-EPMC2780631 | biostudies-other
Project description:The enzyme-catalyzed phosphorylation of glucose to glucose-6-phosphate is a reaction central to the metabolism of all life. ADP-dependent glucokinase (ADPGK) catalyzes glucose-6-phosphate production, utilizing ADP as a phosphoryl donor in contrast to the more well characterized ATP-requiring hexokinases. ADPGK is found in Archaea and metazoa; in Archaea, ADPGK participates in a glycolytic role, but a function in most eukaryotic cell types remains unknown. We have determined structures of the eukaryotic ADPGK revealing a ribokinase-like tertiary fold similar to archaeal orthologues but with significant differences in some secondary structural elements. Both the unliganded and the AMP-bound ADPGK structures are in the "open" conformation. The structures reveal the presence of a disulfide bond between conserved cysteines that is positioned at the nucleotide-binding loop of eukaryotic ADPGK. The AMP-bound ADPGK structure defines the nucleotide-binding site with one of the disulfide bond cysteines coordinating the AMP with its main chain atoms, a nucleotide-binding motif that appears unique to eukaryotic ADPGKs. Key amino acids at the active site are structurally conserved between mammalian and archaeal ADPGK, and site-directed mutagenesis has confirmed residues essential for enzymatic activity. ADPGK is substrate inhibited by high glucose concentration and shows high specificity for glucose, with no activity for other sugars, as determined by NMR spectroscopy, including 2-deoxyglucose, the glucose analogue used for tumor detection by positron emission tomography.
| S-EPMC4759153 | biostudies-literature
Project description:Rat liver 'glucokinase' (hexokinase D) catalyses the phosphorylation of fructose with a 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 about 300 mM. Fructose is a competitive inhibitor of the phosphorylation of glucose with a Ki of 107 mM. Fructose protects hexokinase D against inactivation by 5,5'-dithiobis-(2-nitrobenzoic acid), and the apparent dissociation constants are about 300 mM in the presence of different concentrations of the inhibitor. The co-operativity of the enzyme in the phosphorylation of glucose can be abolished by addition of fructose to the reaction medium. Fructose appears to be no better as a substrate for the other mammalian 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, such as those of micro-organisms and invertebrates, and that liver glucokinase must be called hexokinase D (or hexokinase IV) within the classification EC 2.7.1.1.
| S-EPMC1144187 | biostudies-other
Project description:1. Methods of homogenizing suspensions of washed mammalian spermatozoa were studied. The most useful methods were those using sonication and those using a French press. 2. Hexokinase, phosphofructokinase, glucose phosphate isomerase and adenosine triphosphatase activities in ram, bull and boar spermatozoa were investigated by using these two homogenization methods. Glucose phosphate isomerase, representative of soluble cytoplasmic material, was very readily extracted and remained entirely in the supernatant after centrifugation at 145000g for 60min. In contrast, the other three activities were less easily extracted and were sedimented in various proportions under the described conditions of centrifugation. 3. Attempts to obtain subcellular fractions from sperm homogenates by ;classical' methods failed, owing apparently to the inhomogeneity of subcellular particles in the homogenates. It is concluded that, after removal of sperm heads, the only meaningful fractionation is a separation of spermatozoal material which sediments at 145000g during 60min from that which does not. 4. The stabilities of hexokinase and phosphofructokinase activities in bull, boar and ram sperm homogenates were investigated. Hexokinases showed very little dependence on the various environments tested, whereas the optimum conditions for phosphofructokinase stability were: a minimum of sonication, the presence of phosphate ions and of a thiol-group protectant, and a pH7.5. Activities of hexokinase, phosphofructokinase and glucose phosphate isomerase per sperm cell were compared with published data on rates of fructolysis by spermatozoa; the potential catalytic activities were shown to be considerably in excess of these rates. However, phosphofructokinase may be the rate-limiting enzyme of glycolysis in vivo in bull and ram spermatozoa.
| S-EPMC1177250 | biostudies-other