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Query: EC:2.7.1.1 (hexokinase)
 5,274 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Enzymophoresis with coupled heterogeneous capillary enzyme reactor-capillary zone electrophoresis was developed and evaluated in the area of nucleic acids. Ribonuclease T1, hexokinase and adenosine deaminase were successfully immobilized on the inner walls of short fused-silica capillaries through glutaraldehyde attachment. These open-tubular capillary enzyme reactors were quite stable for a prolonged period of use under operation conditions normally used in capillary zone electrophoresis. The capillary enzyme reactors coupled in series with capillary zone electrophoresis served as peak locator on the electropherogram, improved the system selectivity, and facilitated the quantitative determination of the analytes with good accuracy. Also, they allowed the on-line digestion and mapping of minute amounts of transfer ribonucleic acids, and the simultaneous synthesis and separation of nanogram quantities of oligonucleotides.
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PMID:Enzymophoresis of nucleic acids by tandem capillary enzyme reactor-capillary zone electrophoresis. 140 Aug 40

A 1,820 bp full-length clone encoding for a new human protein was isolated from a lambda gt11 placental cDNA library using anti-human hexokinase antibodies. The cDNA complete sequence includes a 12 bp 5' non-coding region, a single open reading frame encoding a protein of 55 KDa (HP-10) and a 177 bp non-coding with two putative polyadenylation signals upstream of 3' poly(A)tail. The deduced amino acid sequence reveals a sequence of 492 amino acids that contains a stretch of 7 glutamic acid from position 169 and one potential glycosylation site at position 274. Although antibodies against hexokinase recognize the fusion protein and antibodies against the fusion protein recognize hexokinase, HP-10 is not human hexokinase, by a number of criteria including the alignment of determined amino acid sequences. In searching for a possible functional role of HP-10 its cDNA was inserted into a procaryotic vector which allows the expression of the non-fused protein. Bacteria expressing the HP-10 encoded protein were isolated and found to have a dramatic increase in endogenous phosphorylated proteins. Since HP-10 does not have a protein kinase activity per se it should be considered a new regulatory phosphorylation protein which is active in E. coli.
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PMID:Cloning and expression of a new human polypeptide which regulates protein phosphorylation in Escherichia coli. 179 27

Human brain hexokinase (hexokinase I) was produced in Escherichia coli from a synthetic gene under control of the bacteriophage T7 promoter. The expressed coding region derives from a human cDNA clone thought to specify hexokinase I based on amino acid sequence identity between the predicted translation product and hexokinase I from rat brain. The open reading frame from this cDNA was fused to the promoter and 5' flanking region of T7 gene 10, and expressed in E. coli by induction of T7 RNA polymerase. Induced cells contained a hexokinase activity and an abundant protein of apparent molecular weight 100,000, neither of which was present in cells lacking T7 RNA polymerase. Enzyme purified to near homogeneity consisted of a 100,000 Da protein, the size predicted from the nucleotide sequence of the expressed cDNA. The purified enzyme had Michaelis constants of 32 microM and 0.3 mM for glucose and ATP, respectively, and bound to rat liver mitochondria in the presence of MgCl2. Enzymatic activity was inhibited by glucose-6-P and this inhibition was relieved by inorganic phosphate. Deinhibition by phosphate is a property specific to brain hexokinase.
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PMID:Expression of human brain hexokinase in Escherichia coli: purification and characterization of the expressed enzyme. 204 17

Analogues of adenine nucleotides, containing an additional chloromethyl-pyrimidone ring fused to the purine base, were obtained by treatment of AMP, ADP and ATP with an alpha-acetylenic ester, methyl 4-chlorobut-2-ynoate. These compounds were tested for their ability to substitute for the natural substrates or cofactors of several enzymes. With the ADP analogue, pyruvate kinase showed a significant increase of the Km value and a moderate decrease of V, while the reverse was observed when hexokinase was tested with modified ATP. Adenylate kinase was active with the ATP analogue but not with the AMP derivative. Myosin accepted the ATP analogue as a substrate, but was irreversibly modified. Among the dehydrogenases tested, only glucose-6-phosphate dehydrogenase was inhibited by the nucleotide analogue. The structure--activity relationship of these nucleotide derivatives, which represent the largest dimensional deviation known from natural nucleotides, is discussed in comparison with some earlier described dimensional probes of enzyme-nucleotide binding sites.
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PMID:Reactions of nucleic acid bases with alpha-acetylenic esters. Synthesis and enzymatic evaluation of chemically modified nucleotides. 294 24

1. Multiple hexokinase isozymes have been found in most vertebrates. Since each isozyme displays distinctive structural, kinetic and regulatory characteristics, the system qualifies as a useful probe for studies on molecular evolution. 2. At least seven types of chromatographic patterns of liver hexokinases have been observed in mammals. In contrast, each Class of lower vertebrates present only two or three distinct profiles. 3. Aves and higher Reptiles do not have the same hexokinase isozymes as other vertebrates. The nature of the differences is poorly understood. 4. Ontogenetic changes of liver hexokinase profiles are quite different in rat, chick and frog. 5. Structural comparisons of three vertebrate hexokinases having a molecular weight of approximately 100,000 suggest that those isozymes originated from a pre-vertebrate ancestor through gene duplication followed by fusion and further duplication events. Another hexokinase (the so-called glucokinase), with half the molecular weight, may have arisen either as the result of subsequent even splitting of the fused gene or, less probably, by divergence from a duplicated gene before the fusion event.
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PMID:The comparative isozymology of vertebrate hexokinases. 704 67

Chimeric hexokinases consisting of either the N-terminal half of Type I hexokinase fused with the C-terminal half of the Type II isozyme (NICII) or the inverse pair (NIICI), along with the parental isozymes, were expressed in COS-1 cells. The thermal stability of the chimeras was intermediate between that of the highly labile Type II isozyme and the relatively stable Type I hexokinase. In their Kms for substrates, Glc and ATP, the chimeric enzymes were similar to the parental isozyme from which the C-terminal half was derived. Although the Type I and Type II isozymes were similar in their sensitivity to inhibition (competitive vs ATP) by the Glc-6-P analogs, 1,5-anhydroglucitol 6-phosphate (AnGlc-6-P), and Glc-1,6-bisphosphate, the chimeric enzymes differed markedly, with the NIICI chimera being much more sensitive and the NICII chimera much less sensitive than either parental form to these inhibitors. In contrast, the response of the chimeras to Pi, either as an antagonist of inhibition by AnGlc-6-P or, at higher concentrations, as an inhibitor, was correlated with the origin of the N-terminal domain. The results are consistent with the view that catalytic function is associated with the C-terminal domain of the Type I isozyme, with regulatory function--inhibition by Glc-6-P and its analogs and antagonism of this inhibition by Pi--being mediated by the N-terminal domain.
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PMID:Functional organization of mammalian hexokinases: characterization of chimeric hexokinases constructed from the N- and C-terminal domains of the rat type I and type II isozymes. 784 Jun 18

Sucrose is the main transported form of assimilates, but, significantly, it also regulates a variety of processes such as photosynthesis and carbon or nitrogen storage. The effects of high sucrose levels are mediated directly by modulation of gene expression. The regulation of storage protein accumulation, here patatin from potato tubers, was used as a model system to study sucrose mediated signal transduction. The transcriptional regulation of patatin genes in conserved in transgenic Arabidopsis, as shown by the analysis of expression of two classes of patatin promoters fused to uidA. Two distinctly different patterns of gene expression were observed. In roots, class I promoter expression is strongly dependent on the exogenous supply of sugars. 3-O-methylglucose induction indicates that the sensor is located upstream of hexokinase. In contrast, the class II promoter is constitutively active in root tips and hydatodes. The progeny of a homozygous class I line was mutagenized with ethyl methane sulphonate and screened for signal transduction mutants using a non-destructive screening system for GUS activity. Four mutants showing reduced sucrose responses (rsr) and two mutants with modified expression patterns (mep) regarding the root tip were identified. In backcross analyses, it was shown that rsr1-1 carries a recessive trans mutation whereas rsr4-1 seems to be a semi-dominant trans mutation in sugar-mediated gene regulation.
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PMID:Identification of mutants in metabolically regulated gene expression. 902 2

The presumptive coronal sutures of rat fetuses at gestation days 19 and 20 have been shown to fuse prematurely when grown in the absence of dura mater in culture. In the present study, the representative enzymes of glucose metabolism and the antioxidative pathway were assayed during the process of suture fusion. The coronal sutures of fetal day 19.5 (F19) and neonatal day 1 rats were grown in the presence or absence of dura mater in serum-free culture. The enzymes assayed were hexokinase (HK) and pyruvate kinase (PK) of glycolysis, and glucose 6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) of the antioxidative pathway. F19 sutures cultured without dura mater, which fused, showed significant increases in enzyme activities over the preculture levels. HK increased by 200% to 300% of the preculture levels, G6PD by 400% to 500%, GR by 200%, and PK by 400% to 500%. The fetal sutures cultured with dura mater, which did not fuse, showed little alterations of HK, G6PD, and GR activities, but showed a significant 200% to 400% increase in PK activity. Neonatal sutures showed significant increases in enzyme activities during culture, but the presence of dura mater did not significantly affect enzyme activities. High activity levels of enzymes of the antioxidative pathway in F19 sutures coincided with the period of premature suture fusion. Treatment of fetal calvaria with prooxidant (induced by ferrous iron and ascorbic acid) produced suture fusion even in the presence of dura mater. Treatment with deferoxamine (an iron chelator and antioxidant) during the culture prevented suture fusion. The results suggest that fusing sutures experience increased biosynthetic demands and are placed under oxidative stress. When oxidative stress overwhelms the dural influence, the sutures undergo premature fusion.
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PMID:Iron-induced rat coronal suture fusion in vitro: the role of redox regulation. 948 49

Whole cell lysates of pathogenic and nonpathogenic strains of Cryptobia salmositica were subjected to subcellular fractionation using differential and isopycnic centrifugation in sucrose. The glycolytic enzymes hexokinase, fructose-1,6-biphosphate aldolase, triosephosphate isomerase, glucosephosphate isomerase and glyceraldehyde-3-phosphate-dehydrogenase and the peroxisomal enzyme catalase were associated with a microbody that had a buoyant density in sucrose of 1.21 g cm-3. Lactate dehydrogenase was detected in whole cell lysates, but not in purified organelles. A microbody with a positive reaction for catalase was detected in electron microscope sections of the pathogenic and nonpathogenic strains. These catalase-containing microbodies fused with lipid bodies and vacuoles, arose by division from pre-existing microbodies and expelled their contents into the cytoplasm of the cell. Both strains also modified the catalase content in their microbodies. Under aerobic conditions, they metabolized glucose to pyruvate and lactate. We conclude that part of the glycolytic pathway in C. salmositica is compartmentalized in a microbody called the glycosome.
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PMID:Identification of glycosomes and metabolic end products in pathogenic and nonpathogenic strains of Cryptobia salmositica (Kinetoplastida: Bodonidae). 1098 44

Inhibition by its product, glucose, is a kinetic property of hexokinase type III. In this paper, we report the overexpression in Escherichia coli of human hexokinase type III. The recombinant enzyme was genetically fused with a hexahistidine peptide at the C-terminal end. This modification confers to the product the ability to bind the Ni2+ ion immobilised into agarose by nitrilotriacetic acid (NTA) groups. The purification was performed by one-step column chromatography using ammonium sulphate as stabilising agent. Recombinant hexokinase type III appears as a single band of approximately 100 kDa on a SDS-PAGE gel and shows specific activity of 16 U/mg. Its kinetic parameters are comparable to those of the native enzyme, including the fact that it can be inhibited by glucose. The comparison of these results with the properties of the overexpressed carboxyl-domain led us to suppose that the inhibition site for glucose required the presence of the N-terminal domain.
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PMID:The overexpressed hexahistidine-tagged human hexokinase type III is inhibited by D-glucose. 1245 31


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