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PubMed1996

Sequence of human hexokinase III cDNA and assignment of the human hexokinase III gene (HK3) to chromosome band 5q35.2 by fluorescence in situ hybridization.

Complementary DNA clones encoding human hexokinase III were isolated from a liver cDNA library. There was 84.7% identity between the amino acid sequences of human and rat hexokinase III. RNA blotting showed the presence of hexokinase III mRNA in liver and lung. Fluorescence in situ hybridization localized the human hexokinase III gene (HK3) to chromosome 5, band q35.2.

Amino Acid Sequence↗
PubMed1984

Cloning of hexokinase isoenzyme PI from Saccharomyces cerevisiae: PI transformants confirm the unique role of hexokinase isoenzyme PII for glucose repression in yeasts.

Hexokinase isoenzyme PI was cloned using a gene pool obtained from a yeast strain having only one functional hexokinase, isoenzyme PI. The gene was characterized using 20 restriction enzymes and located within a region of 2.0 kbp. The PI plasmid strongly hybridized with the PII plasmids isolated previously (Fröhlich et al. 1984). Hence there was a close relationship between the two genes, one of which must have been derived from the other by gene duplication. In contrast, glucose repression was restored only in hexokinase PII transformants; PI transformants remained non-repressible. This observation provided additional evidence for the hypothesis of Entian (1980) that only hexokinase PII is necessary for glucose repression. Furthermore, glucose phosphorylating activity in PI transformants exceeded that of wild-type cells, giving clear evidence that the phosphorylating capacity is not important for glucose repression.

Cloning, Molecular↗
PubMed1991

Complete amino acid sequence of the type II isozyme of rat hexokinase, deduced from the cloned cDNA: comparison with a hexokinase from novikoff ascites tumor.

The 917-residue amino acid sequence of the Type II isozyme of rat hexokinase has been deduced from the nucleotide sequence of cloned cDNA. The sequences of 197 nucleotides in the 5' untranslated region and 687 bases of the 3' untranslated region have also been determined. A region of overlap between two discrete cDNA clones was confirmed by isolation and sequencing of a genomic DNA clone that spanned the region. Within this region, the 634-nucleotide coding sequence was divided into three exons, each of 150-250 nucleotides; these results suggest that the gene encoding Type II hexokinase is likely to be quite complex. There is extensive similarity between the sequences of the N- and C-terminal halves of the Type II isozyme, as previously seen with the Type I and III isozymes; this is consistent with the view that these enzymes evolved by a process of gene duplication and fusion. A cDNA encoding the entire C-terminal half of a hexokinase from Novikoff ascites tumor cells was also isolated and found to be identical to a cDNA encoding the corresponding region of the Type II isozyme of skeletal muscle. Northern analysis indicated that a single mRNA, approx 5200 nucleotides in length, encoded both the skeletal muscle and the tumor enzymes. These results do not support previous speculation that the hexokinase isozymes of normal tissue are distinct from those of tumors, and suggest the possibility that post-translational modifications of a single protein species might account for apparent differences between the isozymes of normal and tumor tissues.

Amino Acid Sequence↗
PubMed1996

Schizosaccharomyces pombe possesses an unusual and a conventional hexokinase: biochemical and molecular characterization of both hexokinases.

Two hexokinases were characterized in Schizosaccharomyces pombe: hexokinase 1, with a low phosphorylation coefficient on glucose (Km 8.5 mM) and hexokinase 2, a kinetically conventional hexokinase. Genes hxk1+ and hxk2+ encoding these enzymes were cloned and sequenced. Disruption of hxk1+ had no effect on growth but disruption of hxk2+ doubled the generation time in glucose. Spores carrying the double disruption hxk1+ hxk2+ did not grow on glucose or fructose after one week. Expression of hxk1+ increased strongly during growth in fructose or glycerol. Expression of hxk2+ was highest during growth in glycerol. A NADP-dependent glucose dehydrogenase was detected, but not a glucokinase.

Amino Acid Sequence↗
PubMed1995

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.

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.

Adenosine Triphosphate↗
PubMed1999

Expression of hexokinase 1 and hexokinase 2 in mammary tissue of nonlactating and lactating rats: evaluation by RT-PCR.

Because the initial step in the metabolism of glucose involves phosphorylation by hexokinase (HK), we tested the hypothesis that the expression of the isozymes, hexokinase type 1 (HK1) and hexokinase type 2 (HK2), would be different in rat mammary tissue during pregnancy and lactation. RNA was extracted from mammary tissue dissected from timed pregnant rats (from gestional days 10 to 21) and nursing rat mothers (up to postnatal day 5) for mRNA examination by reverse transcriptase and polymerase chain reaction (RT-PCR) using isozyme specific oligonucleotide primers to the HK1 and HK2 cDNAs. The HK1 mRNA was expressed in both the nonlactating and lactating mammary gland tissue, but HK2 mRNA was found only during lactation. We speculate that the pattern of HK expression might affect human milk production and quality.

Animals↗
PubMed1976

Yeast hexokinase: substrate-induced association--dissociation reactions in the binding of glucose to hexokinase P-II.

A method is described for the purification of native hexokinases P-I and P-II from yeast using preparative isoelectric focussing to separate the isozymes. The binding of glucose to hexokinase P-II, and the effect of this on the monomer--dimer association--dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation. Association constants for the monomer-dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupired in the dimer. At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme-glucose complex was 3 X 10(-4) mol 1(-1) and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I=0.02 mol 1(-1), pH less than 7.5) was also independent of enzyme concentration (in the range 10-1000 mug ml-1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non-cooperative; values of the intrinsic dissociation constant varied from 2.5 X 10(-3) mol 1(-1) at pH 7.0 to 6 X 10(-3) at pH 6.5. Under intermediate conditions (pH 7.0, ionic strength=0.15 mol 1(-1)), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperatively (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations. The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.

Binding Sites↗
PubMed1988

Effect of oleate on the apparent Km of monoamine oxidase and the amount of membrane-bound hexokinase in isolated rat hepatocytes: further evidence for the controlling role of the surface charge in hexokinase binding.

Brief incubation of isolated rat hepatocytes in the presence of the oleate-bovine serum albumin complex resulted in a release to the cytosol of a portion of hexokinase (EC 2.7.1.1) normally bound to intracellular membranes. This was correlated with an increase of the negative surface potential of the outer mitochondrial membrane, as measured in situ by determining changes of Km of monoamine oxidase (EC 1.4.3.4). It is suggested that non-esterified fatty acids produce a partial release of bound hexokinase in the liver cell by changing the surface charge of intracellular membranes.

Animals↗
PubMed2001

Ontogenesis of hexokinase I and hexokinase IV (glucokinase) gene expressions in common carp (Cyprinus carpio) related to diet.

The expressions of hexokinase IV (glucokinase, GK) and hexokinase (HK)-I genes were analysed during early ontogenesis of common carp (Cyprinus carpio). Unlike HK-I gene, which was expressed during all the stages of the development, GK was only induced by the first feeding with high levels of dextrin as a source of carbohydrate. This study confirms the high capacity of common carp to use glucose even at the very early stages of development.

Animal Nutritional Physiological Phenomena↗
PubMed1995

Determination of hexokinase isoenzyme I and II composition by RT-PCR: increased hexokinase isoenzyme II in human renal cell carcinoma.

Hexokinase isoenzyme composition has been noted to vary in different tissues and with the developmental and metabolic status of the cell. Until now these investigations were performed either by isoenzyme electrophoresis or by column chromatography. In this report we described an RNA-PCR method to evaluate the percentage of HK-1 and HK-2 in different rat tissues. Furthermore we applied the method to determine if a shift in isoform composition is detectable in human renal carcinomas compared to normal kidney tissue. In all of our specimens we were able to detect a shift toward HK-2 in the carcinoma specimens. We discuss a possible role for the detection of the shift in isoenzyme composition as a possible marker to discriminate between normal and malignant specimens.

Adult↗
PubMed1995

Human hexokinase II gene: exon-intron organization, mutation screening in NIDDM, and its relationship to muscle hexokinase activity.

In muscle, hexokinase II (HK2) regulates phosphorylation of glucose to glucose 6-phosphate, which has been reported to be impaired in patients with non-insulin-dependent diabetes mellitus (NIDDM). Here we report decreased HK2 enzyme activity in skeletal muscle biopsies from patients with impaired glucose tolerance compared with healthy control subjects (2.7 +/- 0.9 vs 4.9 +/- 1.1 nmol.min-1.mg protein-1). Therefore, mutations in the HK2 gene could contribute to skeletal muscle insulin resistance in NIDDM. To address this question, we first determined the exon-intron structure of the human HK2 gene and using this information, we screened all 18 exons with single-strand conformation polymorphism technique in 80 Finnish NIDDM patients. Nine nucleotide substitutions were found, one of which was a missense mutation (Gln142-His142) in exon 4. In human muscle, a single HK2 mRNA transcript with a size of approximately 5500 nucleotides was detected with Northern blot analysis. We also describe an HK2 pseudogene (HK2P1), which was mapped to chromosome 4, band q26, by fluorescence in situ hybridization to metaphase chromosomes. The clinical characteristics and HK2 enzyme activities of the subjects with either Gln or His at residue 142 did not differ from each other. Instead, HK2 activity correlated inversely with fasting blood glucose levels, suggesting that changes in HK2 activity could be secondary to other metabolic abnormalities (r = 0.55; p < 0.0003; n = 39). In conclusion; the data suggest that impaired HK2 activity in prediabetic individuals is a consequence of impaired glucose tolerance rather than of a genetic abnormality. The data thus seem to rule out mutations in the HK2 gene as a major cause of inherited insulin resistance in NIDDM.

Aged↗
PubMed2002

Regulation of mesangial cell hexokinase activity and expression by heparin-binding epidermal growth factor-like growth factor: epidermal growth factors and phorbol esters increase glucose metabolism via a common mechanism involving classic mitogen-activated protein kinase pathway activation and induction of hexokinase II expression.

Heparin-binding epidermal growth factor -like growth factor (HB-EGF) expression and hexokinase (HK) activity are increased in various pathologic renal conditions. Although the mitogenic properties of HB-EGF have been well characterized, its effects on glucose (Glc) metabolism have not. We therefore examined the possibility that HB-EGF might regulate HK activity and expression in glomerular mesangial cells, which constitute the principal renal cell type affected by a variety of pathologic conditions. Protein kinase C (PKC)-dependent classic mitogen-activated protein kinase (MAPK) pathway activation has been associated with increased HK activity in this cell type, so we also examined dependence upon these signaling intermediates. HB-EGF (> or =10 nm) increased total HK activity over 50% within 12-24 h, an effect mimicked by other EGF receptor agonists, but not by IGF-1 or elevated Glc. EGF receptor and classic MAPK pathway antagonists prevented this increase, as did general inhibitors of gene transcription and protein synthesis. Both HB-EGF and phorbol esters activated the classic MAPK pathway, albeit via PKC-independent and PKC-dependent mechanisms, respectively. Both stimuli were associated with increased HK activity, selectively increased HKII isoform expression, and increased Glc metabolism via both the glycolytic-tricarboxylic acid cycle route and the pentose phosphate pathway. HB-EGF thus constitutes a novel regulator of mesangial cell HK activity and Glc metabolism. HKII is the principal regulated isoform in these cells, as it is in insulin-sensitive peripheral tissues, such as muscle. However, the uniform requirement for classic MAPK pathway activation distinguishes HKII regulation in mesangial cells from that observed in muscle. These findings suggest a novel mechanism whereby growth factors may couple metabolism to glomerular injury.

Animals↗
PubMed1976

Yeast hexokinase. A fluorescence temperature-jump study of the kinetics of the binding of glucose to the monomer forms of hexokinases P-I and P-II.

The binding of glucose to the monomeric forms of hexokinases P-I and P-II in Tris and phosphate buffers at pH 8.0 in the presence of 1 mol l-1 KCl has been studied using the fluorescence temperature-jump technique. For both isozymes only one relaxation time was observed; values of tau-1 increased linearly with increasing concentration of free reacting partners. The apparent second-order rate constant for association was about 2 X 10(6) 1 mol-1 s-1 for both isozymes; the differences in the stabilities of the complexes with P-I and P-II are entirely attributable to the fact that glucose dissociates more slowly from its complex with P-I than P-II (approximately 300 s-1 and 1100 s-1 respectively). Although the kinetic data are compatible with a single-step mechanism for glucose binding the association rate constant was much lower than that expected for a diffusion-limited rate of encounter. Other mechanisms for describing an induced-fit are discussed. It is shown that the data are incompatible with a slow 'prior-isomerization' pathway of substrate binding, but are consistent with a 'substrate-guided' pathway involving isomerization of the enzyme-substrate complex.

Binding Sites↗
PubMed2004

Proinflammatory interleukin-1 cytokines increase mesangial cell hexokinase activity and hexokinase II isoform abundance.

Mesangial cell hexokinase (HK) activity is increased by a diverse array of factors that share both an association with pathological conditions and a common requirement for classic MAPK pathway activation. To better understand the relationship between glucose (Glc) metabolism and injury and to indirectly test the hypothesis that these changes constitute a general adaptive response to insult, we have sought to identify and characterize injury-associated factors that couple to mesangial cell HK regulation. Proinflammatory interleukin-1 (IL-1) cytokines activate the MAPK pathway and have known salutary effects in this cell type. We therefore examined their ability to influence mesangial cell HK activity, Glc utilization, MAPK pathway activation, and individual HK isoform abundance. IL-1beta increased HK activity in both a time- and concentration-dependent manner: activity increased maximally by approximately 50% between 12 and 24 h with an apparent EC(50) of 3 pM. IL-1alpha mimicked, but did not augment, the effects of IL-1beta. Specific IL-1 receptor antagonism and selective MAPK/ERK kinase or upstream Ras inhibition prevented these increases, whereas PKC inhibition did not. Changes in HK activity were associated with both increased Glc metabolism and selective increases in HKII isoform abundance. We conclude that IL-1 cytokines can regulate cellular Glc phosphorylating capacity via an IL-1 receptor-, Ras-, and classic MAPK pathway-mediated increase in HKII abundance. These findings suggest a novel, previously undescribed mechanism whereby metabolism may be coupled to inflammation and injury.

Animals↗