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1.
Hexokinase family includes hexokinases I, II, III and IV, that catalyze the phosphorylation of glucose to produce glucose 6-phosphate. Hexokinase IV, also known as glucokinase, is only half size of the other types of hexokinases that contain two hexokinase domains. Despite the enormous progress in the study of hexokinases, the evolutionary relationship between glucokinase and other hexokinases is still uncertain, and the molecular processes leading to the emergence of hexokinases in vertebrates remain controversial. Here we clearly demonstrated the presence of a single hexokinase-like gene in the amphioxus Branchiostoma japonicum, Bjhk, which shows a tissue-specific expression pattern, with the most abundant expression in the hepatic caecum, testis and ovary. The phylogenetic and synteny analyses both reveal that BjHK is the archetype of vertebrate hexokinases IV, i.e. glucokinases. We also found for the first time that recombinant BjHK showed functional enzyme activity resembling vertebrate hexokinases I, II, III and IV. In addition, a native glucokinase activity was detected in the hepatic caecum. Finally, glucokinase activity in the hepatic caecum was markedly reduced by fasting, whereas it was considerably increased by feeding. Altogether, these suggest that Bjhk represents the archetype of glucokinases, from which vertebrate hexokinase gene family was evolved by gene duplication, and that the hepatic caecum plays a role in the control of glucose homeostasis in amphioxus, in favor of the notion that the hepatic caecum is a tissue homologous to liver. 相似文献
2.
Functional glucokinase isoforms are expressed in rat brain 总被引:5,自引:0,他引:5
3.
H Niemeyer M de la Luz Cárdenas E Rabajille T Ureta L Clark-Turri J Pe?aranda 《Enzyme》1975,20(6):321-333
Glucokinases obtained from the liver of several species of mammals and amphibians exhibit sigmoidal saturation functions for glucose. Hill coefficients (nH) are about 1.5, and half-saturation values (K0.5) lie between 1.5 and 8.5 mmol/l. The nH and K0.5 values are constant throughout the purification steps of rat glucokinase. A dimeric form of rat glucokinase appearing in aged preparations exhibits michaelian kinetics. Sigmoidal kinetics is considered as an adaptive feature of glucokinases to increase the efficiency of the liver uptake of glucose at the changeable concentrations in the blood resulting from variations in the amount of dietary glucose. 相似文献
4.
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. 相似文献
5.
The regulatory protein of rat liver glucokinase (hexokinase IV or D) behaved as a fully competitive inhibitor of this enzyme when glucose was the variable substrate, i.e. it increased the half-saturating concentration of glucose as a linear function of its concentration without affecting V (velocity at infinite concentration of substrate). The inhibition by the regulatory protein and that by palmitoyl-CoA were synergistic with that by N-acetyl-glucosamine, indicating that the two former inhibitors bind to a site distinct from the catalytic site. In contrast, the effects of the regulatory protein and palmitoyl-CoA were competitive with each other, indicating that these two inhibitors bind to the same site. The regulatory protein exerted a non-competitive inhibition with respect to Mg-ATP at concentrations of this nucleotide less than 0.5 mM. At higher concentrations, the latter antagonized the inhibition by the regulatory protein partly by decreasing the apparent affinity for fructose 6-phosphate. The following anions inhibited glucokinase non-competitively with respect to glucose: Pi, sulfate, I-, Br-, No3-, Cl-, F- and acetate. Pi and sulfate, at concentrations in the millimolar range, decreased the inhibition by the regulatory protein by competing with fructose 6-phosphate. Monovalent anions also antagonized the inhibition by the regulatory protein with the following order of potency: I- greater than Br- greater than NO3- greater than Cl- greater than F- greater than acetate and their effect was non-competitive with respect to fructose 6-phosphate. Glucokinase from Buffo marinus and pig liver were, like the rat liver enzyme, inhibited by the regulatory protein, as well as by palmitoyl-CoA at micromolar concentrations. In contrast, neither compound inhibited hexokinases from rat brain, beef heart or yeast, or the low-Km specific glucokinase from Bacillus stearothermophilus. 相似文献
6.
7.
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. 相似文献
8.
Glucokinase gene (HPGLK1) was cloned from a methylotrophic yeast Hansenula polymorpha by complementation of glucose-phosphorylation deficiency in a H. polymorpha double kinase-negative mutant A31-10 by a genomic library. An open reading frame of 1416 nt encoding a 471-amino-acid protein with calculated molecular weight 51.6 kDa was characterized in the genomic insert of the plasmid pH3. The protein sequence deduced from HPGLK1 exhibited 55 and 46% identity with glucokinases from Saccharomyces cerevisiae and Aspergillus niger, respectively. The enzyme phosphorylated glucose, mannose and 2-deoxyglucose, but not fructose. Transformation of HPGLK1 into A31-10 restored glucose repression of alcohol oxidase and catalase in the mutant. Transformation of HPGLK1 into S. cerevisiae triple kinase-negative mutant DFY632 showed that H. polymorpha glucokinase cannot transmit the glucose repression signal in S. CEREVSIAE: synthesis of invertase and maltase in respective transformants was insensitive to glucose repression similarly to S. cerevisiae DFY568 possessing only glucokinase. 相似文献
9.
1. An 870-fold purification of glucokinase from rat liver is described which involves ammonium sulphate fractionation and the use of DEAE-Sephadex, DEAE-cellulose and polyacrylamide columns. 2. The preparation is free of any interfering enzymes and has a specific activity of 8mumoles/min./mg. of protein. 3. Glucokinase catalyses the phosphorylation of glucose, mannose and 2-deoxyglucose. 4. The enzyme is inhibited by high concentrations of glucose 6-phosphate only; ADP is an inhibitor whose effect depends on the Mg(2+) concentration. 5. The properties of glucokinase are compared briefly with those of other phosphotransferases. 相似文献
10.
Cordeiro AT Cáceres AJ Vertommen D Concepción JL Michels PA Versées W 《Journal of molecular biology》2007,372(5):1215-1226
Glucose is an essential substrate for Trypanosoma cruzi, the protozoan organism responsible for Chagas' disease. The glucose is intracellularly phosphorylated to glucose 6-phosphate. Previously, a hexokinase responsible for this phosphorylation has been characterized. Recently, we identified an ATP-dependent glucokinase in T. cruzi exhibiting a tenfold lower substrate affinity compared to the hexokinase. Both enzymes, which belong to very different groups of the same family, are located inside glycosomes, the peroxisome-like organelles of Kinetoplastida that are known to contain the first seven glycolytic steps as well as enzymes of the oxidative branch of the pentose phosphate pathway. Here, we present the crystallographic structure of T. cruzi glucokinase, in complex with glucose and ADP. The structure suggests a loose tetrameric assembly formed by the association of two tight dimers. TcGlcK was previously reported to exist in a concentration-dependent equilibrium of monomeric and dimeric states. Here, we used mass spectrometry analysis to confirm the existence of TcGlcK monomeric and dimeric states. The analysis of subunit interactions and comparison with the bacterial glucokinases give insights into the forces promoting the stability of the different oligomeric states. Each T. cruzi glucokinase monomer contains one glucose and one ADP molecule. In contrast to hexokinases, which show a moderate preference for the alpha anomer of glucose, the electron density clearly shows the d-glucose bound in the beta configuration in the T.cruzi glucokinase. Kinetic assays with alpha and beta-d-glucose further confirm a moderate preference of the T. cruzi glucokinase for the beta anomer. Structural comparison of the glucokinase and hexokinases permits the identification of a possible mechanism for anomer selectivity in these hexose-phosphorylating enzymes. The preference for distinct anomers suggests that in T. cruzi hexokinase and glucokinase are not directly competing for the same substrate and are probably both present because they exert distinct physiological functions. 相似文献
11.
Precocious induction of hepatic glucokinase and malic enzyme in artificially reared rat pups fed a high-carbohydrate diet 总被引:2,自引:0,他引:2
P M Haney C R Estrin A Caliendo M S Patel 《Archives of biochemistry and biophysics》1986,244(2):787-794
Glucokinase and NADP:malate dehydrogenase (malic enzyme) first appear in liver when rat pups are weaned from milk which is high in fat to lab chow which is high in carbohydrate. To examine the influence of diet during the early neonatal period, before developmental changes in the circulating concentrations of thyroid and adrenocortical hormones occur, high-carbohydrate formula (56% of calories from carbohydrate), isocaloric and isonitrogenous with rat milk, was intermittently infused via gastrostomy starting on the second day of life. Pups had no further access to their dams. Body weights attained by these pups were at least 90% of those attained by mother-fed pups, which served as controls. In artificially reared rats fed the high-carbohydrate formula, on Day 4, glucokinase and malic enzyme were 30 and 18% of adult activity, respectively; on Day 10, glucokinase and malic enzyme were 71 and 96% of adult activity, respectively. On Days 4 and 10 glucose-6-phosphate dehydrogenase was elevated four- to fivefold in pups fed the high-carbohydrate formula compared to mother-fed pups. A second isocaloric formula, with 22% of calories from carbohydrate but low in protein, resulted in intermediate levels of all three enzymes on Day 10. Pups fed the high-carbohydrate formula has plasma insulin concentrations four- to fivefold greater than mother-fed pups on both Days 4 and 10. Triiodothyronine administration (1 microgram/g body wt) on Day 1 enhanced the induction of malic enzyme but not glucokinase on Day 4 in pups fed the high-carbohydrate formula. The results demonstrate that neonatal rat liver is competent to respond to high carbohydrate intake by induction of glucokinase and malic enzyme. 相似文献
12.
Starting with a mutant of Saccharomyces cerevisiae lacking glucokinase and both the hexokinase isozymes P1 and P2, strains were constructed, by genetic crosses, that carry single glucose-phosphorylating enzymes. The P1 and P2 isozymes and a structurally altered form of P1 hexokinase were partially purified from these strains. Hexokinases P1, P2, and the altered P1 enzyme, respectively, phosphorylate fructose nearly four, two, and ten times as fast as they phosphorylate glucose. Strains bearing P1 show a pronounced Pasteur reaction and phosphorylate glucose, fructose, and mannose faster than those bearing the P2 isozyme. However, there is no appreciable difference between these two hexokinases in regard to the rate and the extent of growth that they sustain. The ability of yeast to grow on a particular sugar is contingent only upon the presence of an enzyme that phosphorylates it. Glucokinase seems to be responsible for catalyzing nearly half of the glucose flux in the wild type yeast. Strains bearing glucokinase alone do show a Pasteur effect. 相似文献
13.
I E O'Neill D R Langslow 《Comparative biochemistry and physiology. B, Comparative biochemistry》1978,59(4):317-325
1. Glucokinase was absent from chicken liver and only the low Km hexokinases, inhibited by AMP, ADP but not ATP, were present. 2. The Km of chicken liver glucose-6-phosphatase for glucose-6-phosphate was reduced from 5.65 to 3.75 mM following starvation, and the enzyme was inhibited by glucose. 3. Starvation of chickens for 24 hr slightly lowered the hexokinase activity and doubled glucose-6-phosphatase activity; it did not change subcellular distribution of the enzymes. Oral glucose rapidly restored the activities to fed values. 4. It was concluded that glucose uptake into, and efflux from, chicken hepatocytes, was regulated by the activity and kinetic characteristics of glucose-6-phosphatase and by the glucose-6-phosphate concentration, and that the hexokinases had little regulatory function. 相似文献
14.
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. 相似文献
15.
About ScienceDirect 《FEBS letters》1982,140(2):325-328
The effects of varied durations of food deprivation on the rates and kinetics of glucose phosphorylation by isolated rat hepatocytes have been examined. Glucokinase activity was measured concurrently in extracts from these cells prepared from livers of rats which had fasted for 0, 24, 48 and 72 h. Significant levels of hepatocyte glucose phosphorylation were noted even when glucokinase levels were extrapolated to zero. The K0.5-glucose value of 33 mM in cells from fed rats increased to 48 mM after a 72-h fast. It is concluded that a high K0.5 glucose-phosphorylating enzyme or enzymes compensatory to insulin-dependent glucokinase is/are involved in rat liver glucose phosphorylation. 相似文献
16.
Ichitomo Miwa Sachie Mitsuyama Yukiyasu Toyoda Tomiyasu Murata Jun Okuda 《Preparative biochemistry & biotechnology》2013,43(2):163-178
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. 相似文献
17.
1. It was shown that the development of liver glucokinase in the rat coincided with a peak in the levels of circulating thyroid hormone at about the 16th postnatal day. 2. Administration of thyroid inhibitors blocked the development of the enzyme and administration of thyroid hormone restored activity to normal levels. 3. Glucokinase could be induced prematurely as early as the 2nd postnatal day by the administration of thyroid hormone followed by daily injection of glucose (10 mg/g body weight). 4. Glucocorticoids and corticotropin failed to induce glucokinase activity prematurely. 5. The postnatal increase in circulating thyroid hormone levels together with increased intake of carbohydrate at weaning may be the normal physiological stimulus for induction of this enzyme. 相似文献
18.
T L Andreone R L Printz S J Pilkis M A Magnuson D K Granner 《The Journal of biological chemistry》1989,264(1):363-369
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. 相似文献
19.
Hexokinase isoenzymes of RIN-m5F insulinoma cells. Expression of glucokinase gene in insulin-producing cells. 总被引:4,自引:1,他引:3
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U Vischer B Blondel C B Wollheim W H?ppner H J Seitz P B Iynedjian 《The Biochemical journal》1987,241(1):249-255
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. 相似文献