Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The molecular basis for the beta-cell dysfunction that characterizes non-insulin-dependent diabetes mellitus (NIDDM) is unknown. The Zucker diabetic fatty (ZDF) male rat is a rodent model of NIDDM with a predictable progression from the prediabetic to the diabetic state. We are using this model to study beta-cell function during the development of diabetes with the goal of identifying genes that play a key role in regulating insulin secretion and, thus, may be potential targets for therapeutic intervention aimed at preserving or improving beta-cell function. As a first step, we have characterized morphology, insulin secretion, and pattern of gene expression in islets from prediabetic and diabetic ZDF rats. The development of diabetes was associated with changes in islet morphology, and the islets of diabetic animals were markedly hypertrophic with multiple irregular projections into the surrounding exocrine pancreas. In addition, there were multiple defects in the normal pattern of insulin secretion. The islets of prediabetic ZDF rats secreted significantly more insulin at each glucose concentration tested and showed a leftward shift in the dose-response curve relating glucose concentration and insulin secretion. Islets of prediabetic animals also demonstrated defects in the normal oscillatory pattern of insulin secretion, indicating the presence of impairment of the normal feedback control between glucose and insulin secretion. The islets from diabetic animals showed further impairment in the ability to respond to a glucose stimulus. Changes in gene expression were also evident in islets from prediabetic and diabetic ZDF rats compared with age-matched control animals. In prediabetic animals, there was no change in insulin mRNA levels. However, there was a significant 30-70% reduction in the levels of a large number of other islet mRNAs including glucokinase, mitochondrial glycerol-3-phosphate dehydrogenase, voltage-dependent Ca2+ and K+ channels, Ca(2+)-ATPase, and transcription factor Islet-1 mRNAs. In addition, there was a 40-50% increase in the levels of glucose-6-phosphatase and 12-lipoxygenase mRNAs. There were further changes in gene expression in the islets from diabetic ZDF rats, including a decrease in insulin mRNA levels that was associated with reduced islet insulin levels. Our results indicate that multiple defects in beta-cell function can be detected in islets of prediabetic animals well before the development of hyperglycemia and suggest that changes in the normal pattern of gene expression contribute to the development of beta-cell dysfunction.
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PMID:Evolution of beta-cell dysfunction in the male Zucker diabetic fatty rat. 758 53

Glucokinase is distinguished from yeast hexokinase and low Km mammalian hexokinases by its low affinity for glucose and its cooperative behavior, even though glucose binding residues and catalytic residues are highly conserved in all of these forms of hexokinase. The roles of Ser-151 and Asn-166 as determinants of hexose affinity and cooperative behavior of human glucokinase have been evaluated by site-directed mutagenesis, expression and purification of the wild-type and mutant enzymes, and steady-state kinetic analysis. Mutation of Asn-166 to arginine increased apparent affinity for both glucose and ATP by a factor of 3. Mutation of Ser-151 to cysteine, alanine, or glycine lowered the Km for glucose by factors of 2-, 26-, and 40-fold, respectively, decreased Vmax, abolished cooperativity for glucose, and also decreased Km for mannose and fructose. The Ser-151 mutants had hexose Km values similar to those of yeast hexokinase, hexokinase I, and the recombinantly expressed COOH-terminal half of hexokinase I. However, the Ki values for the competitive inhibitors, N-acetylglucosamine and glucose-6-P, were unchanged, suggesting that Ser-151 is not important for inhibitor binding. Mutation of Ser-151 also increased the Km for ATP about 5-fold and abolished the enzyme's low ATPase activity, which indicates it is essential for ATP hydrolysis. The substrate-induced change in intrinsic fluorescence of S151A occurred at a much lower glucose concentration than that for wild-type enzyme. The results implicate a dual role for Ser-151 as a determinant of hexose affinity and catalysis, exclusive of the glucose-induced conformational change, and suggest that the low hexose affinity of glucokinase is dependent on interaction of Ser-151 with other regions of the protein.
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PMID:Human beta-cell glucokinase. Dual role of Ser-151 in catalysis and hexose affinity. 773 Mar 77

Human beta-cell glucokinase recognition and phosphorylation of different sugars was investigated by steady-state kinetic analysis, measurements of substrate-induced intrinsic fluorescence changes, and molecular modeling and calculation of interaction energies. Measurements of kcat/Km showed that glucokinase phosphorylated the sugars in the order glucose = mannose > deoxyglucose > fructose = glucosamine. The mode of binding of these sugars to the open conformation of glucokinase was predicted from molecular modeling. Glucokinase is predicted to form similar interactions with the 6-OH, 4-OH, and 1-OH groups of all these sugars. The interactions of the 2-OH and 3-OH groups differ and depend on the type of sugar and reflect differences in cooperative behavior. For example, glucose and deoxyglucose exhibited cooperative behavior with Hill coefficients of 1.8 and 1.5, respectively, while mannose and fructose demonstrated Michaelis-Menten behavior. Galactose, allose, and 2,5-anhydroglucitol were not substrates under the assay conditions used, and the alpha- and beta-anomers of methylglucose were poor substrates with Km's greater than 1000 mM. Glucokinase exhibited an ATPase activity which was 1/2000th that of the rate of the kinase reaction, and unlike yeast hexokinase, it was not affected by the addition of lyxose. Glucosamine was a low affinity inhibitor as well as a substrate, while N-acetylglucosamine and mannoheptulose were high-affinity inhibitors. The change in intrinsic fluorescence that was induced by glucose, mannose, and mannoheptulose had the opposite sign for glucosamine, which implies a very different mode of binding from the other sugars. The calculated interaction energies of glucokinase with glucose, mannose, deoxyglucose, and fructose agree very well with the measured values of kcat/Km, which indicates that these sugars are recognized by binding to the open conformation of glucokinase.
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PMID:Sugar specificity of human beta-cell glucokinase: correlation of molecular models with kinetic measurements. 774 12

Streptococcus mutans, an important aetiological agent of dental caries, is known to transport glucose via the phosphoenolpyruvate (PEP) phosphotransferase system (PTS). An alternative non-PTS glucose transport system in S. mutans Ingbritt was suggested by the increased ATP-dependent phosphorylation of glucose and the presence of higher cellular concentrations of free glucose in cells grown in continuous culture under PTS-repressed conditions compared to those resulting in optimal PTS activity. A method was developed for the preparation of membrane vesicles in order to study this system in the absence of PTS activity. These vesicles had very low activity of the cytoplasmic enzymes, glucokinase, pyruvate kinase and lactate dehydrogenase. This, coupled with the lack of glycolytic activity and the inability to transport glucose, suggested that the vesicles would also be deficient in PTS activity because of the absence of the general soluble PTS proteins, Enzyme I and HPr, required for the transport of all PTS sugars. Freeze-fracture electron microscopy and membrane H(+)-ATPase analysis indicated that over 90% of the vesicles had a right-side-out orientation. Vesicles from cells grown in continuous culture under PTS-dominant and PTS-repressed conditions both exhibited glucose counterflow. This indicates the presence of a constitutive non-PTS carrier in the organism capable of transporting glucose and utilizing ATP for glucose phosphorylation. Analysis of growth yields of cells grown under PTS-repressed and PTS-optimal conditions suggests that ATP, or an equivalent high energy molecule, must be involved in the actual transport process. This analysis is consistent with an ATP-binding protein model such as the Msm transport system reported by R. R. B. Russell and coworkers (J Biol Chem 267, 4631-4637), but it does not exclude the possibility of a separate permease for glucose.
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PMID:Vesicles prepared from Streptococcus mutans demonstrate the presence of a second glucose transport system. 800 May 34

Equilibrium calculations on biochemical reaction systems can be made at three levels. Level 1 is the usual chemical calculation with species at specified temperature and pressure using standard Gibbs energies of formation of species or equilibrium constants K. Level 2 utilizes reactants such as ATP (a sum of species) at specified T, P, pH, and pMg with standard transformed Gibbs energies of formation of reactants or apparent equilibrium constants K'. Calculations at this level can also be made on the enzymatic mechanism for a biochemical reaction. Level 3 utilizes reactants at specified T, P, pH, and pMg, but the equilibrium concentrations of certain reactants are also specified. The fundamental equation of thermodynamics is derived here for Level 3. Equilibrium calculations at this level use standard transformed Gibbs energies of formation of reactants at specified concentrations of certain reactants or apparent equilibrium constants K". Level 3 is useful in calculating equilibrium concentrations of reactants that can be reached in a living cell when some of the reactants are available at steady-state concentrations. Calculations at all three levels are facilitated by the use of conservation matrices and stoichiometric number matrices for systems. Three cases involving glucokinase, glucose-6-phosphatase, and ATPase are discussed.
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PMID:Levels of thermodynamic treatment of biochemical reaction systems. 824 5

Physiologically, a postprandial glucose rise induces metabolic signal sequences that use several steps in common in both the pancreas and peripheral tissues but result in different events due to specialized tissue functions. Glucose transport performed by tissue-specific glucose transporters is, in general, not rate limiting. The next step is phosphorylation of glucose by cell-specific hexokinases. In the beta-cell, glucokinase (or hexokinase IV) is activated upon binding to a pore protein in the outer mitochondrial membrane at contact sites between outer and inner membranes. The same mechanism applies for hexokinase II in skeletal muscle and adipose tissue. The activation of hexokinases depends on a contact site-specific structure of the pore, which is voltage-dependent and influenced by the electric potential of the inner mitochondrial membrane. Mitochondria lacking a membrane potential because of defects in the respiratory chain would thus not be able to increase the glucose-phosphorylating enzyme activity over basal state. Binding and activation of hexokinases to mitochondrial contact sites lead to an acceleration of the formation of both ADP and glucose-6-phosphate (G-6-P). ADP directly enters the mitochondrion and stimulates mitochondrial oxidative phosphorylation. G-6-P is an important intermediate of energy metabolism at the switch position between glycolysis, glycogen synthesis, and the pentose-phosphate shunt. Initiated by blood glucose elevation, mitochondrial oxidative phosphorylation is accelerated in a concerted action coupling glycolysis to mitochondrial metabolism at three different points: first, through NADH transfer to the respiratory chain complex I via the malate/aspartate shuttle; second, by providing FADH2 to complex II through the glycerol-phosphate/dihydroxy-acetone-phosphate cycle; and third, by the action of hexo(gluco)kinases providing ADP for complex V, the ATP synthetase. As cytosolic and mitochondrial isozymes of creatine kinase (CK) are observed in insulinoma cells, the phosphocreatine (CrP) shuttle, working in brain and muscle, may also be involved in signaling glucose-induced insulin secretion in beta-cells. An interplay between the plasma membrane-bound CK and the mitochondrial CK could provide a mechanism to increase ATP locally at the KATP channels, coordinated to the activity of mitochondrial CrP production. Closure of the KATP channels by ATP would lead to an increase of cytosolic and, even more, mitochondrial calcium and finally to insulin secretion. Thus in beta-cells, glucose, via bound glucokinase, stimulates mitochondrial CrP synthesis. The same signaling sequence is used in the opposite direction in muscle during exercise when high ATP turnover increases the creatine level that stimulates mitochondrial ATP synthesis and glucose phosphorylation via hexokinase. Furthermore, this cytosolic/mitochondrial cross-talk is also involved in activation of muscle glycogen synthesis by glucose. The activity of mitochondrially bound hexokinase provides G-6-P and stimulates UTP production through mitochondrial nucleoside diphosphate kinase. Pathophysiologically, there are at least two genetically different forms of diabetes linked to energy metabolism: the first example is one form of maturity-onset diabetes of the young (MODY2), an autosomal dominant disorder caused by point mutations of the glucokinase gene; the second example is several forms of mitochondrial diabetes caused by point and length mutations of the mitochondrial DNA (mtDNA) that encodes several subunits of the respiratory chain complexes. Because the mtDNA is vulnerable and accumulates point and length mutations during aging, it is likely to contribute to the manifestation of some forms of NIDDM.(ABSTRACT TRUNCATED)
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PMID:Mitochondria and diabetes. Genetic, biochemical, and clinical implications of the cellular energy circuit. 854 53

Special features of glucose metabolism in pancreatic beta-cells are central to an understanding of the physiological role of these cells in glucose homeostasis. Several of these characteristics are emphasized: a high-capacity system for glucose transport; glucose phosphorylation by the high-Km glucokinase (GK), which is rate-limiting for glucose metabolism and determines physiologically the glucose dependency curves of many processes in beta-cell intermediary and energy metabolism and of insulin release and is therefore viewed as glucose sensor; remarkably low activity of lactate dehydrogenase and the presence of effective hydrogen shuttles to allow virtually quantitative oxidation of glycolytic NADH; the near absence of glycogen and fatty acid synthesis and of gluconeogenesis, such that intermediary metabolism is primarily catabolic; a crucial role of mitochondrial processes, including the citric acid cycle, electron transport, and oxidative phosphorylation with FoF1 ATPase governing the glucose-dependent increase of the ATP mass-action ratio; a Ca(2+)-independent glucose-induced respiratory burst and increased ATP production in beta-cells as striking manifestations of crucial mitochondrial reactions; control of the membrane potential by the mass-action ratio of ATP and voltage-dependent Ca2+ influx as signal for insulin release; accumulation of malonyl-CoA, acyl-CoA, and diacylglycerol as essential or auxiliary metabolic coupling factors; and amplification of the adenine nucleotide, lipid-related, and Ca2+ signals to recruit many auxiliary processes to maximize insulin biosynthesis and release. The biochemical design also suggests certain candidate diabetes genes related to fuel metabolism: low-activity and low-stability GK mutants that explain in part the maturity-onset diabetes of the young (MODY) phenotype in humans and mitochondrial DNA mutations of FoF1 ATPase components thought to cause late-onset diabetes in BHEcdb rats. These two examples are chosen to illustrate that metabolic reactions with high control strength participating in beta-cell energy metabolism and generating coupling factors and intracellular signals are steps with great susceptibility to genetic, environmental, and pharmacological influences. Glucose metabolism of beta-cells also controls, in addition to insulin secretion and insulin biosynthesis, an adaptive response to excessive fuel loads and may increase the beta-cell mass by hypertrophy, hyperplasia, and neogenesis. It is probable that this adaptive response is compromised in diabetes because of the GK or ATPase mutants that are highlighted here. A comprehensive knowledge of beta-cell intermediary and energy metabolism is therefore the foundation for understanding the role of these cells in fuel homeostasis and in the pathogenesis of the most prevalent metabolic disease, diabetes.
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PMID:Banting Lecture 1995. A lesson in metabolic regulation inspired by the glucokinase glucose sensor paradigm. 854 69

In an attempt to seek out new factors that are related to colorectal carcinogenesis at the molecular level, subtractive hybridization between cDNA of normal mucosal tissues and mRNA of colorectal carcinoma tissues was performed. Subsequent screenings of the cDNA libraries, constructed from normal mucosal tissues, using the "subtractive probes" generated a total of 46 clones that were expressed in normal mucosa but were either expressed at a significantly reduced level or not expressed at all in cancer tissues. Partial nucleotide sequences of all of these cDNA clones were determined, and sequence homology analyses were performed with the Genbank database. Of the 46 cDNA samples, 44 contained substantial sequence homologies with 32 immunoglobulin gene fragments, a helix-loop-helix basic phosphoprotein gene, an acidic ribosomal phosphoprotein P2 gene, a BLR1 gene for Burkitt's lymphoma receptor 1 gene, D5S419 DNA segment containing (C-A) repeats, a glucokinase (GCK) gene, a Na+, K+-ATPase alpha-subunit gene, a histocompatibility system HLA-DR heavy-chain gene, a dystrophic gene, a mucin (MUC2) gene, a mu-glutathione S-transferase gene, a Menkes disease protein gene, and a 40-kDa keratin intermediate filament precursor gene. The remaining two cDNA clones (now registered under GenBank accession numbers U17714 and U20428) showed few (less than 60%) sequence homologies with any known sequences in the GenBank database and, therefore, may represent novel genes whose expression was down-regulated in human colorectal carcinomas. The possible clinical significance of these findings and the involvement of these two genes in the carcinogenesis of colorectal as well as other cancers are being investigated.
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PMID:Characterization of colorectal-cancer-related cDNA clones obtained by subtractive hybridization screening. 929 8

The activities of Na+,K(+)-ATPase in plasma membrane, of cytosolic enzymes and of glutamate dehydrogenase (GlGD) in mitochondria were measured in leukocytes (WBC) from dogs and cats to clarify the differences in energy metabolism in these cells. Feline WBC had significantly higher activities of hexokinase (HK), pyruvate kinase (PK) and LDH with pyruvate as substrate than did canine WBC. Canine WBC had significantly higher activities of glucokinase (GK) and GlDH than did feline WBC. Feline WBC had unique characteristics of energy metabolism in that the activities of the cytosolic enzymes under anaerobic conditions were significantly higher than those in canine WBC. It therefore appears that there are distinct differences in glucose-metabolism in WBC between dogs and cats. WBC enzyme activities are considered to reflect the metabolic state in the whole body of the animal. It is therefore suggested that changes in the activities of certain glycolytic enzymes in WBC may be useful as a diagnostic indicator in some types of metabolic disease in dogs and cats.
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PMID:A comparison of the activities of certain enzymes related to energy metabolism in leukocytes in dogs and cats. 961 90

We have proposed that hyperglycemia-induced dedifferentiation of beta-cells is a critical factor for the loss of insulin secretory function in diabetes. Here we examined the effects of the duration of hyperglycemia on gene expression in islets of partially pancreatectomized (Px) rats. Islets were isolated, and mRNA was extracted from rats 4 and 14 weeks after Px or sham Px surgery. Px rats developed different degrees of hyperglycemia; low hyperglycemia was assigned to Px rats with fed blood glucose levels less than 150 mg/dl, and high hyperglycemia was assigned above 150 mg/dl. beta-Cell hypertrophy was present at both 4 and 14 weeks. At the same time points, high hyperglycemia rats showed a global alteration in gene expression with decreased mRNA for insulin, IAPP, islet-associated transcription factors (pancreatic and duodenal homeobox-1, BETA2/NeuroD, Nkx6.1, and hepatocyte nuclear factor 1 alpha), beta-cell metabolic enzymes (glucose transporter 2, glucokinase, mitochondrial glycerol phosphate dehydrogenase, and pyruvate carboxylase), and ion channels/pumps (Kir6.2, VDCC beta, and sarcoplasmic reticulum Ca(2+)-ATPase 3). Conversely, genes normally suppressed in beta-cells, such as lactate dehydrogenase-A, hexokinase I, glucose-6-phosphatase, stress genes (heme oxygenase-1, A20, and Fas), and the transcription factor c-Myc, were markedly increased. In contrast, gene expression in low hyperglycemia rats was only minimally changed at 4 weeks but significantly changed at 14 weeks, indicating that even low levels of hyperglycemia induce beta-cell dedifferentiation over time. In addition, whereas 2 weeks of correction of hyperglycemia completely reverses the changes in gene expression of Px rats at 4 weeks, the changes at 14 weeks were only partially reversed, indicating that the phenotype becomes resistant to reversal in the long term. In conclusion, chronic hyperglycemia induces a progressive loss of beta-cell phenotype with decreased expression of beta-cell-associated genes and increased expression of normally suppressed genes, these changes being present with even minimal levels of hyperglycemia. Thus, both the severity and duration of hyperglycemia appear to contribute to the deterioration of the beta-cell phenotype found in diabetes.
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PMID:Critical reduction in beta-cell mass results in two distinct outcomes over time. Adaptation with impaired glucose tolerance or decompensated diabetes. 1243 14


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