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PubMed1996

A population association study of four candidate genes (hexokinase II, glucagon-like peptide-1 receptor, fatty acid binding protein-2, and apolipoprotein C-II) with type 2 diabetes and impaired glucose tolerance in Japanese subjects.

In order to define the major genetic factor(s) for the development of Type 2 (non-insulin-dependent) diabetes mellitus in Japanese subjects, a population association study of candidate genes involved in either glucose or lipid metabolism was carried out using microsatellite DNA polymorphisms. Each polymorphic locus near the four candidate genes, hexokinase II (HKII), glucagon-like peptide-1 receptor (GLP1R), fatty acid binding protein-2 (FABP-2), and apolipoprotein C-II (apoC-II) genes, were amplified by polymerase chain reaction (PCR) using 32P-labelled primers and each subject was genotyped for the association study. The HKII, GLP1R, FABP-2, and apoC-II polymorphisms exhibited 18, 10, 7, and 10 alleles, respectively. While polymorphism information contents (PICs) of these polymorphisms were relatively high, allele frequencies in these polymorphisms did not differ among subjects with Type 2 diabetes, impaired glucose tolerance (IGT) and non-diabetic controls. These results suggest that the HKII, GLP1R, FABP-2, and apoC-II genes are not the major inherited factors for the development of Type 2 diabetes or IGT in Japanese subjects, although minor contribution cannot be ruled out.

Apolipoprotein C-II↗
PubMed1996

Properties and localization of a tyrosine phosphorylated form of hexokinase in mouse sperm.

Mouse sperm possess a phosphotyrosine-containing hexokinase type 1 (HK1) that is associated with the plasma membrane fraction of these cells (Kalab et al., 1994; J. Biol Chem 269:3810-3817). This apparent plasma membrane association appears unique, since somatic HK1 is normally cytoplasmic or bound to the outer mitochondrial membrane via contact sites with a voltage-dependent anion channel (porin) through a porin-binding domain. In male germ cells, three cDNA clones have been described that encode unique HK1 isoforms (HK1-sa, HK1-sb, HK1-sc) that do not contain porin binding domains (Mori et al., 1993: Biol Reprod 49:191-203). This suggests that these proteins might not be localized to the outer mitochondrial membrane and could have alternative functions in germ cells and/or sperm. We demonstrate in the mouse that male germ cells and sperm could potentially express four HK1 isoforms (HK1-sa, HK1-sb, HK1-sc, and the somatic HK1). At the protein level, at least one of the HK1 isoforms becomes phosphorylated on tyrosine residues during spermatogenesis. Treatment of sperm membrane fractions to dissociate the phosphotyrosine-containing HK1 (pY-mHK1) yields results demonstrating that pY-mHK1 has properties of an integral membrane protein. Indirect immunofluorescence using a monoclonal antibody to HK1 demonstrates specific staining both in the head and tail regions of sperm. Surface biotinylation of intact sperm followed by precipitation with either polyclonal HK1 antiserum or with avidin-Sepharose suggests that pY-mHK1 possesses an extracellular domain. These results suggest that mouse sperm contain at least one HK1 isoform that is present on the sperm head, has an extracellular domain, and behaves as an integral membrane protein.

Animals↗
PubMed1996

Sperm from mice carrying two t haplotypes do not possess a tyrosine phosphorylated form of hexokinase.

Mouse sperm contain a tyrosine phosphorylated form of hexokinase type 1 (HK1; Kalab et al., 1994: J Biol Chem 269:3810-3817) that has properties consistent with an integral plasma membrane protein. Furthermore, this tyrosine phosphorylated form of HK1 has an extracellular domain and HK1 is localized to both the head and flagellum of nonpermeabilized cells (Visconti et al., 1995c). We have characterized HK1 in mature sperm from sterile tw32/tw5 mice (mutant sperm) that have defects in motility and sperm-egg interaction (Johnson et al., 1995: Dev Biol 168:138-149). Immunoprecipitation of mouse sperm extracts with an antiserum made against purified rat brain HK1 demonstrates the presence of HK1 in mutant sperm. Various biochemical and immunofluorescence assays indicate that at least a portion of the HK1 present in these cells is an integral membrane protein with an extracellular domain located on the sperm head and flagellum. However, immunoblot analysis with anti-phoshotyrosine antibodies demonstrates that HK1 in mutant sperm is not tyrosine phosphorylated. Northern blot and RT-PCR analysis does not indicate any obvious abnormalities in the transcription of somatic or germ cell-specific HK1 isoforms in mutant testes, and RFLP analysis of recombinant mice indicates that no genes specifying HK1 isoforms are located on chromosome 17. We have mapped the locus responsible for the lack of tyrosine phosphorylation of HK1 mutant sperm to the most proximal (to the centromere) of the four inversions within the t haplotype. A male sterility factor is located in this same inversion (Lyon, 1986: Cell 44:357-363). Since the mutant sperm are unable to complete fertilization, there could be a relationship between sterility and the lack of tyrosine phosphorylation of HK1 in these mutant sperm.

Animals↗
PubMed1998

Mouse spermatogenic cell-specific type 1 hexokinase (mHk1-s) transcripts are expressed by alternative splicing from the mHk1 gene and the HK1-S protein is localized mainly in the sperm tail.

Unique type 1 hexokinase (HK1) mRNAs are present in mouse spermatogenic cells (mHk1-s). They encode a spermatogenic cell-specific sequence region (SSR) but not the porin-binding domain (PBD) necessary for HK1 binding to porin on the outer mitochondrial membrane. This study determined the origin of the multiple Hk1-s transcripts in mouse spermatogenic cells and verified that they are translated in mouse spermatogenic cells. It also showed that a single mHk1 gene encodes the mHk1 transcripts of somatic cells and the mHk1-sa and mHk1-sb transcripts of spermatogenic cells, that alternative exons are used during mHk1 gene expression in mouse spermatogenic cells, and that mHK1-S is translated in mouse spermatogenic cells and is localized mainly with the fibrous sheath in the tail region, not with the mitochondria in the midpiece of mouse sperm.

Alternative Splicing↗
PubMed1990

ATP modifications in hexokinase deficient fibroblasts exposed to nutrient shifts.

Human fibroblasts from a patient homozygous for hexokinase (HK) deficiency and normal controls were submitted to different nutrient shifts to investigate the role of glucose metabolism on ATP levels. HK-deficient cells maintained in the presence of both glucose and L-glutamine had 20 per cent less ATP than controls and an ATP/ADP ratio of 18 instead of 37-40. Glucose alone was not able to maintain normal ATP levels neither in the controls nor in the HK-deficient cells. However while in control cells the ATP/ADP ratio was not modified, it was reduced to 10 in HK deficiency. The effect of several hexoses on ATP levels was also investigated. Mannose, which is metabolized by HK phosphorylation, was the least efficient for ATP maintenance in HK-deficient cells. In contrast galactose, which is metabolized by a specific galactokinase, provided ATP values close to those observed in the presence of glutamine. These results suggest that glucose metabolism is an important determinant in the maintenance of ATP levels in cultured cells and that glutamine, although being a good metabolic substrate, is not sufficient to maintain normal ATP concentrations.

Adenosine Diphosphate↗
PubMed1984

Isoenzyme pattern and subcellular localization of hexokinases in human breast cancer and nonpathological breast tissue.

Subcellular distribution of hexokinase (HK) isoenzymes in 22 human breast cancers (21 primary cancers and 1 axillary metastatic growth) and 7 non-pathological human mammary gland tissue samples was studied with starch gel electrophoresis on isolated cell fractions obtained by differential centrifugation. Fractions used were cytosol, mitochondria and microsomes. A comparison of two methods for detecting HK activity was made, yielding different results regarding HK II and HK III. A method based on the ability of NADPH to fluoresce in UV gave a constant pattern of HK isoenzymes. In non-pathological breast tissue, only HK I was seen, i.e. in the cytosol and the microsomal fractions. HK I was also seen in all fractions of the cancers, but another more anodal band of HK I, as well as HK II and HK III, consistently appeared in the cytosol fractions. The more commonly used staining technique with tetrazolium dye revealed HK II in 45% and HK III in 50% of the samples. The pattern of HK isoenzymes in the cancers was the same irrespective of estrogen and progesterone cytosolic receptor contents and the histology of the tumors. The fluorescence method is, therefore, much more sensitive than the tetrazolium technique for detecting HK activity after electrophoresis and could explain difference in results obtained by various laboratories.

Breast↗
PubMed1993

Detection of a progression-linked DNA restriction fragment in rat hepatoma cells probed with a hexokinase cDNA.

We have compared genomic DNA isolated from a series of rat hepatomas and from normal rat liver tissue by restriction fragment analysis. Our objective was to identify a tumor-specific restriction fragment that would be present in rapidly growing tumors and missing in normal tissues. Using a cDNA probe specific for the C-terminal half of rat brain hexokinase, we have identified several changes in DNA restriction fragment lengths in rat hepatomas vs. normal rat liver. Most of these involved restriction fragments that were present in normal rat DNA and missing in tumor DNA. However, one HinfI fragment of M(r) = 13.4 kilobase pairs (kb) was found to be present in rapidly growing rat hepatomas, but missing in normal rat tissue and in hepatomas of slow or intermediate growth rate.

Animals↗
PubMed2003

Sorting specificity of spermatogenic cell specific region of mouse hexokinase-s (mHk1-s).

Hexokinase is the first enzyme involved in the glycolysis process that produces glucose phosphorylate. Our previous study reported on our cloning of mouse Hk1-s (mHk1-s) cDNA, which were expressed only in testis cells, and noted that this cDNA has a spermatogenic cell-specific region (SSR) that replaces the porin binding domain (PBD) in the Hk1of somatic cells. Although we know that PBD binds to the outer membrane of a mitochondrion, the role of the SSR is not yet understood. To investigate the intracellular localization of SSR, we constructed expression vectors with the epitope tag (GFP-, HA-), subcloned SSR, or PBD cDNA. We transfected these vectors in mouse fibroblast, NIH3T3 cells, after which we observed the localization of the SSR and PBD in the NIH3T3 cells. Our current study using the immunocytochemical method revealed that PBD is concentrated around the mitochondrion. However, the SSR could not be ascribed to the mitochondrion, ER, or nuclear colocalization. Moreover, subcellular fractionation analysis showed that PBD was detected in the mitochondrial fraction, and that SSR was detected in the cytosolic fraction. Our findings suggest that PBD of Hk1 targets mitochondrion, but the SSR of mHk1-s targets some specific organellae.

3T3 Cells↗
PubMed2001

Hexokinase PII: structural analysis and glucose signalling in the yeast Saccharomyces cerevisiae.

Hexokinase PII (Hxk2) is a yeast glucose phosphorylating enzyme that, besides its role in glycolysis, seems to have an additional role in glucose signalling. To study the domains in Hxk2 that may participate in this latter process, we have constructed 11 mutant alleles using site-directed mutagenesis. Six of them were clustered charged-to-alanine mutants in which clusters of charged residues were changed to alanine residues. Two of them contained substitutions in Ser15 to either alanine or glutamic acid and three of them had deletions at either the N-terminus or the C-terminus of the protein. In most of them, the catalytic activity correlated directly with their functionality in glucose signalling. However, we found two mutants (Delta1-15 and Delta476-486) that, having low catalytic activity, were still fully functional in glucose signalling. This may indicate that other factors and not just the catalytic activity of the enzyme may be important for the functionality of the protein in glucose signalling.

Amino Acid Sequence↗
PubMed1997

Two Sp sites are important cis elements regulating the upstream promoter region of the gene for rat type I hexokinase.

Multiple transcriptional start sites have been identified for the gene encoding the rat Type I isozyme of hexokinase (White, J.A., Liu, W., and Wilson, J. E., Arch. Biochem. Biophys. 335, 161-172, 1996); these are clustered at positions approximately -460, -300, and -100 relative to the translational start codon (ATG, with A being +1). PC12 cells and H9c2 cells were transfected with luciferase reporter constructs containing genomic sequence between positions -3366 and -171. Marked (85%) decrease in promoter activity was associated with deletion of sequence between -742 and -516. In DNase I footprinting experiments, two regions, called P1 (-552 to -529) and P2 (-480 to -458) boxes, were protected by proteins present in nuclear extracts from PC12 cells. Mutation or deletion of the P2 box had no effect on promoter activity; protection in this region, which includes the most upstream cluster of transcriptional start sites, is attributed to binding of RNA polymerase II or associated factors. In contrast, mutations or deletions in the P1 box had markedly detrimental effects on promoter activity and on binding of proteins in PC12 cell nuclear extracts. Maintenance of a consensus Sp1 binding site centrally located in the P1 box was critical for both promoter activity and binding. A second Sp1 site (-570), just upstream from the P1 box, was also shown to be functionally important but no protection of this region was detected in footprinting experiments, presumably reflecting lower affinity at this site under the conditions used. Supershift experiments demonstrated the involvement of Sp1, Sp3, and Sp4 in formation of complexes with the P1 box region and implicate these transcription factors in regulating promoter activity associated with this region. Another series of reporter constructs, including sequence between -171 and -1, permitted detection of an additional promoter activity downstream from -364. While not yet extensively characterized, it is already evident that the cis elements influencing the downstream promoter activity are distinct from the Sp factors determined to be important in expression from the upstream promoter region.

Animals↗
PubMed1999

Allosteric regulation of type I hexokinase: A site-directed mutational study indicating location of the functional glucose 6-phosphate binding site in the N-terminal half of the enzyme.

The Type I isozyme of mammalian hexokinase has evolved by a gene duplication-fusion mechanism, with resulting internal duplication of sequence and ligand binding sites. However, 1:1 binding stoichiometry indicates that only one of these is available for binding the product inhibitor, Glc-6-P; the location of that site, in the N- or C-terminal half, remains under debate. Recent structural studies (Aleshin et al., Structure 6, 39-50, 1998; Mulichak et al., Nature Struct. Biol. 5, 555-560, 1998) implicated Asp 84 or its analog in the C-terminal half, Asp 532, in binding of Glc-6-P. Zeng et al. (Biochemistry 35, 13157-13164, 1996) demonstrated that mutation of Asp 532 to Lys or Glu did not affect inhibition by the Glc-6-P analog, 1,5-anhydroglucitol-6-P. These same mutations, as well as mutation to Ala, at the Asp 84 position are now shown to result in increased Ki for 1,5-anhydroglucitol-6-P. The ability of Pi to antagonize inhibition by the Glc-6-P analog is severely diminished or abolished by these mutations, suggesting that antagonism is dependent on precise positioning of the inhibitory hexose 6-phosphate. The structure of the enzyme complexed with Glc and Pi has been determined, and shows that Pi occupies the same site as the 6-phosphate group in the complex with Glc-6-P. Thus, antagonism between these ligands results from competition for a common anion binding site in the N-terminal half.

Adenosine Triphosphate↗
PubMed2002

Kinetic and regulatory properties of HK I(+), a modified form of the type I isozyme of mammalian hexokinase in which interactions between the N- and C-terminal halves have been disrupted.

A modified form (HK I(+)) of rat Type I hexokinase (HK I) has been expressed. HK I(+) contains a centrally located polyalanine insert which, along with the known helical propensity of adjacent sequence, was expected to lead to alpha-helix formation, with resulting distension of the molecule and disruption of interactions between the N- and C-terminal halves. The properties of HK I(+) are consistent with this expectation and with previous proposals that (1) inhibition of HK I by Glc-6-P or its analogs and antagonism of this inhibition by P(i) result from competition of these ligands for a binding site in the N-terminal half of HK I, with resulting conformational changes propagated through interactions with the catalytic C-terminal half, and (2) binding of Glc-6-P to a site in the C-terminal half of HK I is obstructed by interactions between the halves, present in HK I but not HK I(+).

Amino Acid Sequence↗
PubMed2000

Anabolic function of the type II isozyme of hexokinase in hepatic lipid synthesis.

The mRNA encoding the Type II isozyme of hexokinase was markedly elevated in livers of transgenic mice overexpressing the transcriptionally active nuclear form of sterol regulatory element binding protein-1a (nSREBP-1a), but not in transgenic mice overexpressing the nSREBP-1c or nSREBP-2 isoforms. Cellulose acetate electrophoresis and immunoblotting results confirmed selective increase of the Type II isozyme in livers of transgenic mice expressing nSREBP-1a. SREBP-1a has previously been shown to activate transcription of genes encoding enzymes involved in biosynthesis of fatty acids and glycerolipids and to a lesser extent the enzymes of cholesterol biosynthesis. Thus, these results are consistent with the view that the Type II isozyme serves an anabolic function, providing precursors and reducing equivalents required for increased rates of hepatic lipid synthesis.

Animals↗
PubMed1993

DL-propranolol inhibits lens hexokinase activity and affects lens optics.

A clinico-biochemical study indicated that the beta-blocker DL-propranolol may affect human lens epithelial hexokinase (HK) activity. In that study five key enzymes were analysed in 192 freshly excised human lens epithelia obtained during cataract surgery. In a large number of patients the epithelial HK was found to be inactive. Medical records of these patients showed widespread use of the drug DL-propranolol. In vitro experiments demonstrated a direct inhibitory effect of the drug on human lens HK activity. Lens refractive function was monitored during long term bovine lens culture experiments in which the potential cataractogenic agent was added to the culture media. DL-propranolol in a concentration of 0.1 mM reduced HK activity in bovine lens epithelium after 72 hr in organ culture and disrupted lens light focusing ability after 250 hr of incubation. Kinetic studies of HK inhibition suggested a competitive inhibitory effect of the drug on the enzyme.

Animals↗
PubMed1999

Human HKR isozyme: organization of the hexokinase I gene, the erythroid-specific promoter, and transcription initiation site.

We previously described a cDNA for the human HKR isozyme, whose sequence is identical to that of the ubiquitous HKI isozyme, except for a unique 5' end sequence. Screening a human genomic library with a DNA fragment containing an erythroid-specific sequence we found one clone including 5' ends for both HKR and HKI genes. The first HKR exon was located 3 kb 5' of the first HKI exon. These results confirmed that HKR is produced from the HKI gene by alternate promoter and splicing. The HKI gene consisted of 19 exons. All exon-intron boundaries are conserved among the genes for hexokinase and glucokinase. The HKI gene length was estimated at over 67 kb. The initiation site for the HKR was identified by primer extension. Its promoter did not have a canonical TATA box, but an inverted GATA at nt -177 (i.e., 36 nt 5' to the transcription initiation site). In the HKR promoter a DNA fragment spanning nt -275 to nt -107 exhibited erythroid-specific activity. However, this was absent in shorter promoter fragments (nt -206 to -107 or nt -229 to -107). The sequence nt -275 to -229, which appeared critical for the erythroid-specific expression of the HKR gene, contained a consensus motif for Sp-1 and GATA, CCAAT, and GGAA motifs. The electrophoretic mobility shift assay (EMSA) suggested erythroid-specific cooperative protein-protein interaction in this region. Deletion of the GATA sequence as well as reaction with a specific antibody identified GATA-1 as one of the interacting proteins.

5' Untranslated Regions↗
PubMed2002

Purification of the Type II and Type III isozymes of rat hexokinase, expressed in yeast.

C-terminally His-tagged versions of the Type II and Type III isozymes of rat hexokinase were expressed in Pichia pastoris and Schizosaccharomyces pombe, respectively. Milligram amounts of the homogeneous isozymes were readily obtained in good yield by chromatography on Ni-NTA columns. The specific activities were 133 +/- 4 and 76 +/- 3 u/mg for the purified Type II and Type III isozymes, respectively. The K(m)'s for glucose and ATP were in good agreement with values in the literature for the isozymes isolated from mammalian tissues. The Type III isozyme exhibited substrate inhibition at elevated levels of glucose, as previously observed for this isozyme isolated from mammalian tissue sources.

Animals↗
PubMed1996

Hexokinase production from S. cerevisiae. Culture conditions.

The effects of pH (4.0, 4.5, or 5.0), temperature (T) (30, 35, or 40 degrees C) and dissolved oxygen (DO) (0.2, 2.0, 4.0,or 6.0 mg O2/L) on hexokinase and invertase formation by yeast were studied. The highest enzyme activities were attained at pH 4.0, DO = 4.0 mg O2/L, and T = 35 or 40 degrees C.

Glycoside Hydrolases↗
PubMed1994

Cytochrome oxidase and hexokinase activities in an infusion edema model with preserved blood flow.

Despite numerous investigations, the mechanisms underlying the neurological deficits observed in association with interstitial edema remain unclear. A recent study has demonstrated that the cerebral blood flow (CBF) in edematous white matter is unchanged if the blood flow values are corrected for dilution. In contrast, the cerebral glucose metabolism (CMRgl) has been found to be increased. In order to examine the effects of interstitial edema on the oxidative metabolism, we measured the cytochrome oxidase (CYO) activity, a marker of mitochondrial respiration, and the hexokinase (HK) activity, a marker of glycolysis, together with CBF and CMRgl employing the iodoantipyrine and deoxyglucose autoradiography in an infusion edema model in rats. In agreement with the previous study, CBF was not significantly changed in the edematous hemisphere. No significant alterations in CMRgl and HK activity were noted. In contrast, there was a significant decrease in CYO activity in the edematous hemisphere (-17%; p < 0.01), which was correlated to the edema. These findings suggest that interstitial edema causes a decreased mitochondrial respiratory function despite a maintained circulation. This may be explained by postulating a decreased oxygen delivery and/or accumulation of lactate, both of which have been shown to interfere with mitochondrial respiratory function.

Animals↗