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PubMed1987

Interaction of creatine kinase and hexokinase with the mitochondrial membranes, and self-association of creatine kinase: crosslinking studies.

Covalent coupling of protein by crosslinking reagents have been used to study the interaction of mitochondrial creatine kinase (CKm) and hexokinase (HK) with the mitochondrial membranes. The effects of crosslinkers were studied either by following the inhibition of solubilization of enzymatic activities or by modification of the electrophoretic patterns of proteins solubilized from mitochondria after treatment with different crosslinkers. Dimethylsuberimidate (DMS) efficiently reduced the amount of HK activity solubilized by various agents but it did not modify solubilization of CKm from mitochondria. The effect of DMS on HK solubilization did not result from non specific crosslinking since it did not impede the solubilization of adenylate kinase. Bissuccinimidyl another class of crosslinker has been tested. Ethyleneglycol bis (succinimidyl succinate)(EGS) efficiently reduced HK solubilization, but in addition it induced osmotic stabilization of mitochondria and thus impeded release of soluble or solubilized proteins from the intermembrane space. Furthermore this agent drastically inhibited CKm activity and thus, in a second set of experiments the effect of crosslinkers have been studied by the disappearance of protein bands in the electrophoretic pattern of soluble fractions obtained from mitochondria, the outer membranes of which have been ruptured to allow free release of soluble proteins. Results of these experiments showed that succinimidyl reagents and Cu++-Phenanthroline substantially reduced the amount of CKm released from mitochondria and confirmed that bisimidates were ineffective in inhibiting CKm solubilization. In addition crosslinking reagents have been used to study subunits interactions in purified CKm. Our results showed, in contrast with control experiments with a non oligomeric protein (ovalbumin) which did not give rise to polymers, that in the same conditions electrophoresis of crosslinked CKm resolved a set of species with molecular weights roughly equal to integral multiples of the protomer. These results proved that the polymeric form of CKm was an octamer.

Adenylate Kinase↗
PubMed1990

Microphotometric determination of enzymes in brain sections. I. Hexokinase.

A histochemical procedure was established for the microphotometric determination of hexokinase (HK) in sections of the rat hippocampus, which served as an exemplary brain region. For this quantitative procedure, slides were coated with glucose 6-phosphate dehydrogenase (G6PDH) as an auxiliary enzyme and sections were mounted onto this enzyme film. The sections were then incubated with the following adapted incubation medium: 5 mM D-glucose, 1.5 mM NADP, 7.5 mM ATP, 4 mM nitroblue tetrazolium chloride, 10 mM NaN3, 10 mM MgCl2, 0.25 mM phenazine methosulfate, 1 U/ml G6PDH, 22% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 7.5. A linear response of the reaction was observed in the initial 10 min of reaction (kinetic and end-point measurements). The relationship between HK activity and section thickness was linear up to 5 microns. The need for such thin sections is discussed in relation to the limited penetration of the auxiliary enzyme into the section. It is concluded that the quantitative demonstration of HK in brain sections could be a valuable tool for studying the local metabolic entrance of glucose in the glycolytic pathway.

Animals↗
PubMed1984

A de novo case of trisomy 10p: gene dosage studies of hexokinase, inorganic pyrophosphatase and adenosine kinase.

A female infant with multiple congenital anomalies is presented. Cytogenetic study revealed the presence of a de novo, supernumerary, small telocentric chromosome exhibiting the banding pattern of the short arm of chromosome no. 10 [47,XX,+10p(pter----cen)]. Her clinical features were compatible with the 10p trisomy syndrome. Hexokinase (HK-1) activity was elevated in the patient's erythrocytes, which is consistent with an assignment of HK-1 to 10pter---cen10. The absence of a gene dosage effect for inorganic pyrophosphatase (PP) in this study indicates exclusion of PP from 10pter ----cen10, and therefore implies a regional assignment of cen10----10q24 for PP. Adenosine kinase (ADK) activity was within control limits, which is consistent with exclusion of ADK from 10pter----cen10.

Abnormalities, Multiple↗
PubMed1994

Effect of exercise on hexokinase distribution and mitochondrial respiration in skeletal muscle.

Horses were subjected to treadmill running at 65% (submaximal) or 100% (maximal) VO2,max to examine the effects of exercise on subcellular distribution of hexokinase (HK) and on mitochondrial respiration. It is hypothesized that the fraction of HK bound to mitochondria will be reduced due to an elevation of glucose-6-phosphate (G-6-P) concentration in the exercising muscle and that such release of HK from mitochondria will depress oxidative phosphorylation. Changes in muscle G-6-P concentration, pH, subcellular HK distribution, mitochondrial respiration and other metabolites were determined in biopsy samples pre-exercise, immediately post-exercise and during the recovery phase. The fraction of HK associated with mitochondria decreased from 38% to 7% at the end of maximal exercise; exercise at VO2,max also reduced respiratory capacity of muscle homogenates by 20% and was associated with a fivefold increase in muscle [G-6-P], a potent agent known to dissociate HK from mitochondria. The HK distribution returned to normal within 60 min after exercise and the reassociation of the HK with mitochondria parallelled the removal of muscle G-6-P. No changes in muscle HK distribution and respiration were found following the submaximal exercise despite the fact that G-6-P was slightly elevated. Muscle concentrations of adenosine triphosphate, creatine phosphate and glycogen and pH dropped after exercise while lactate concentration increased. The amount of mitochondria-bound HK was also altered in vitro in a preparation of mitochondria isolated from rat skeletal muscle to examine the effect of the bound HK on mitochondrial respiration.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1987

Decrease in glucokinase and glucose-6-phosphatase and increase in hexokinase in putative preneoplastic lesions of rat liver.

Preneoplastic liver lesions were produced in female Wistar rats by oral administration of 2-acetylaminofluorene for 165 days succeeded by a carcinogen-free standard diet up to 420 days. During the treatment numerous altered hepatic foci (AHF) and hyperplastic nodules (HN) were detected histochemically by a focal decrease or lack of adenosine-5-triphosphatase and glucose-6-phosphatase (G-6-Pase) activities. In addition, the immunohistochemically demonstrable amount of L-type pyruvate kinase was clearly reduced. The histochemically demonstrated decrease of G-6-Pase was substantiated by microbiochemical determination of the enzyme activity in microdissected material. Moreover, during the experimental period a continuous decrease in glucokinase and an increase in hexokinase was detected microbiochemically within AHF and HN. These alterations indicate a shift in the carbohydrate metabolism from gluconeogenesis to glucose utilization and pentose-phosphate-pathway for biosynthesis of nucleic acids. Beside other oncofetal markers, HK may be used as indicator of the early stages of liver carcinogenesis.

2-Acetylaminofluorene↗
PubMed1996

Variant sequences of the Hexokinase II gene in familial NIDDM.

UNLABELLED: Hexokinase II (HKII) plays a central role in the intracellular metabolism of glucose in skeletal muscle, catalysing the phosphorylation of glucose to glucose 6-phosphate. It is therefore considered to be a potentially important candidate gene in the development of insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM). The aim of this study was to screen the HKII gene for mutations in NIDDM subjects from insulin-resistant families. Insulin sensitivity was assessed in unaffected first degree relatives from families with two or more living NIDDM subjects, and 15 families were identified as being insulin resistant. In 15 NIDDM subjects (one from each family) and 4 normoglycaemic control subjects, all 18 exons of the HKII gene were amplified by the polymerase chain reaction, and the products screened for mutations using a combination of single-stranded conformational polymorphism analysis and direct sequencing. Six sequence variations were detected in the NIDDM subjects; four silent polymorphisms [GAT vs GAC at codon 251 in exon 7, AAT vs AAC at codon 692 in exon 15, CCG vs CCC at codon 736 in exon 15, and CTG vs CTA at codon 766 in exon 16]; a single base change [T-->C], 22 base pairs distal to the exon-intron junction of exon 17 in the 5'-splice donor; and a single amino acid substitution [Gln142-->His] in exon 4, which was identified in 6 of the 15 NIDDM subjects. The frequency of the mutated codon 142 allele however, was comparable between NIDDM subjects with familial NIDDM (n = 56) and normoglycaemic control subjects (n = 48) (18.8% and 14.6% for NIDDM subjects and control subjects respectively; chi 2 = 0.6, p > 0.25). In addition, measures of insulin sensitivity were comparable in normal glucose tolerant subjects with (n = 20) and without (n = 40) the codon 142 polymorphism. IN CONCLUSION: (1) mutations in the coding regions of the HKII gene are unlikely to be major determinants in the development of insulin resistance and familial NIDDM; although (2) the influence of the codon 142 mutation in combination with other abnormalities of the insulin-signalling pathway on insulin action remain to be addressed.

Amino Acid Sequence↗
PubMed1978

Identification and linkage relationships of three hexokinase genes in Aedes aegypti.

Four regions of hexokinase activity are detected by starch gel electrophoresis of adult Aedes aegypti. Three of the regions, Hk-2, Hk-3, and Hk-4, are produced by three tightly linked loci, located on the third chromosome 7.25 map units from the locus fuzzy. The three loci show developmental differences as well as differences in substrate specificity.

Aedes↗
PubMed1995

Isoenzymes related to flight activity in Tetragonisca angustula (Hymenoptera:Apidae:Meliponinae): evidence of postranslational modification of the hexokinase and detection of new glycerol-3-phosphate dehydrogenase variants.

To detect genetic markers, electrophoretic analyses were carried out on Tetragonisca angustula, a stingless bee well adapted to its wide area of occurrence, which is becoming economically important because of the quality of its honey. Polymorphism of the enzyme hexokinase was detected and it was demonstrated that, although it presents two bands in meliponines, this enzyme is under the control of a single locus whose product probably undergoes postranslational modification. The more common allele occurs in the two Tetragonisca subspecies studied here, but the other seems to be characteristic of only one subspecies. Four variants involving the two glycerol-3-phosphate dehydrogenase loci are also described. These two enzymes share the property of presenting two electrophoretic bands, one of which is characteristic of actively flying adult thoraces.

Animals↗
PubMed1988

The effect of pretreatment with pentobarbital on the extent of [14C] incorporation from [U-14C]glucose into various rat brain glycolytic intermediates: relevance to regulation at hexokinase and phosphofructokinase.

In the present investigation we monitored the incorporation of [14C] from [U-14C]glucose into various rat brain glycolytic intermediates of conscious and pentobarbital-anesthetized animals. Labeled glucose was delivered to brain by single bolus intracarotid injection and brain tissue was subsequently prepared at 15, 30, and 45 sec by freeze-blowing. Glycolytic intermediates were then separated by column chromatography. Our results showed a gradual decrease with time of 14C-labeled glucose which gave a calculated rate for glucose metabolism of 0.86 mumol/min/g and 0.56 mumol/min/g in conscious and anesthetized animals, respectively. Compared to the results obtained using conscious animals the administration of pentobarbital not only resulted in a significant attenuation of the rate of glucose metabolism but also caused a similar reduction in the amount of 14C incorporated into several glycolytic intermediates. These intermediates included: glucose 6-phosphate, fructose 6-phosphate, fructose 1,6 diphosphate, dihydroxyacetone phosphate and post glycolytic compounds. In addition, pretreatment with pentobarbital resulted in a 75% increase in the endogenous concentration of glucose, 10% increase in glucose 6-phosphate, no change in fructose 6-phosphate and 42% decrease in lactate compared to levels in brains obtained from conscious animals. These results are discussed in relation to control of glycolysis through coupled regulation at hexokinase-phosphofructokinase.

Animals↗
PubMed1983

The effect of endurance-training on the maximum activities of hexokinase, 6-phosphofructokinase, citrate synthase, and oxoglutarate dehydrogenase in red and white muscles of the rat.

Adult female rats were subjected to an eleven-week endurance-training programme, and, for the first time, the maximum activities of enzymes that can indicate the quantitative capacities of both anaerobic glycolysis and the Krebs cycle in muscle (viz. 6-phosphofructokinase and oxoglutarate dehydrogenase respectively) were measured in heart plus white and fast-oxidative skeletal muscle. No changes were observed in heart muscle. In fast-oxidative skeletal muscle, activities of hexokinase, citrate synthase, and oxoglutarate dehydrogenase were increased by 51, 26, and 33% respectively but there was no effect on 6-phosphofructokinase. These results demonstrate that in red muscle there is no effect of this training programme on the anaerobic capacity but that of the aerobic system is increased by one third. In white skeletal muscle, only the activity of citrate synthase was increased, which indicates that this activity may not provide even qualitative information about changes in capacity of the Krebs cycle.

Aerobiosis↗
PubMedDate not supplied

Thermostability of yeast hexokinase and yeast glucose-6-phosphate dehydrogenase.

Kinetic study of the mechanism of the temperature-induced loss of the catalytic activity by yeast hexokinase (HK) and yeast glucose-6-phosphate dehydrogenase (G-6-PDG) has shown the dissociative nature of the processes. In the temperature range 40-47 degrees C, they are satisfactorily described in terms of consecutive reactions in which steps of irreversible denaturation of the monomeric units follow the reversible dissociation of inactive oligomeric forms into the active units, resulting in an increase in catalytic activity. The experimental data have been analyzed in the framework of the dissociative mechanism, and a semiquantitative method has been developed for calculating the individual rate constants.

Enzyme Stability↗
PubMed2005

Hexokinase-II expression in untreated oral squamous cell carcinoma: comparison with FDG PET imaging.

Hexokinase is thought to be one of the key factors of glucose catabolism in the cell. The aim of this study was to investigate the relationship between HK-II expression and 18F-fluoro-2-deoxy-D-glucose (FDG) uptake in human untreated oral squamous cell carcinoma (OSCC). Pre-operatively FDG positron emission tomography (PET) was performed 60 min after FDG injection in all the patients. Maximum standardized uptake value (SUV) was used for evaluation of tumor FDG uptake. Tumor sections were stained immunohistochemically for HK-II. All the tumor sections stained positive for HK-II. Eighteen (95%) tumors in HK-II showed immunostained positive area >50%. HK-II findings revealed eleven (58%) tumors with strong intensity, six (32%) with moderate intensity and two with weak intensity (10%). There was no statistically significant correlation between SUV and the expression of HK-II (p = 0.46). In conclusion, OSCC showed increased FDG accumulation and overexpression of HK-II. However, we did not find any significant relationship between high FDG uptake and overexpression of HK-II in this patient population, and thus other properties need to be evaluated in order to elucidate key factors responsible for FDG activity in OSCC.

Adult↗
PubMed2005

Influence of streptozotocin-induced diabetes on hexokinase activity of rat salivary glands.

The influence of diabetes on the enzyme hexokinase (HK) was examined in the salivary glands of rats. Diabetes was induced by an intraperitoneal injection of streptozotocin (60 mg/Kg body weight) in overnight fasted rats (180-200 g). The animals were killed 48 hours and 30 days after the induction of diabetes and the submandibular and parotid salivary glands extracted for use. Hyperglycemia was evaluated by determining the blood sugar. The area occupied by each intralobular component, acini, ducts, total parenchyma and stroma was measured, and no differences were observed compared with control. In the soluble fraction of the submandibular gland, no difference in the specific activity of HK was observed, between the diabetic and control animals, however, the activity per gland and per g of tissue showed lower values than control. The specific activity of the bound form was reduced in the diabetic gland. The results obtained for the parotid gland were different from the submandibular. The specific activity of both the soluble and bound forms were increased in the diabetic animals. The DEAE-cellulose column chromatography of the soluble and bound forms of the enzyme from both glands showed a first peak appearing during the washing of the column and two other peaks were eluted by the gradient. Thus, three isoenzymes in the submandibular and parotid salivary glands for the control and diabetic rats have been found.

Animals↗
PubMed1997

The human hexokinase II gene promoter: functional characterization and detection of variants among patients with NIDDM.

Hexokinase II (HKII) plays an important role in facilitating glucose uptake by skeletal muscle, heart, and adipose tissue in response to insulin. We have cloned and sequenced the proximal promoter region of the human HKII gene, determined the transcription start sites and screened the 2.0 kb of the proximal 5' flanking region for variants in non-insulin-dependent diabetic patients and control subjects. We found three variants in this region, one in the 5' untranslated region (G-->C at +217) and two in the promoter region (T-->G at -1043 and G-->A at -1159). The allele frequencies of these variants did not differ between the diabetic and control subjects and these variants are not associated with insulin resistance. Various segments of the human HKII promoter were tested for driving expression of the luciferase reporter gene. The proximal 500 bp and 400 bp of the promoter were sufficient to drive maximal activity in adipocyte (3T3F442A) and myocyte (C2C12F3) cell lines, respectively. This region of the promoter is GC-rich and contains eight consensus binding sites for the transcription factor Sp-1, five for AP-2, two putative response elements for each of insulin and cyclic AMP. The proximal 175 bases of the promoter retained only 7-15% of maximal activity. Sequence elements located between positions -304 and -215 accounted for approximately 80% of the basal HKII promoter. In addition, the region between -215 and -184 contains a negative regulatory element for expression in 3T3F442A but not in C2C12F3 cells.

Adipocytes↗
PubMed2000

Mouse hexokinase II gene: structure, cDNA, promoter analysis, and expression pattern.

In mammalian tissues, the phosphorylation of intracellular glucose to glucose-6-phosphate (Glu-6-P) is facilitated by four distinct hexokinase (HK) isoenzymes, designated as HKI-IV. Because of the role of HKII as a leading glycolytic enzyme in insulin-sensitive tissues such as skeletal muscle, heart, and adipose tissue, defects in HKII function could contribute to the development of insulin resistance and perhaps Type 2 diabetes. As a first step towards elucidation of the physiological role of HKII in insulin resistance and type 2 diabetes using mouse knock-out models, we determined the genomic structure, sequence of the cDNA and of 4.8 kb of the 5' regulatory region, and tissue-specific expression of the mouse HKII gene. The gene comprises 18 exons that span approximately 50 kb of DNA. Nucleotide sequence of the proximal promoter revealed a number of conserved putative transcription factor binding motifs. We also found numerous repeat elements throughout the mouse HKII gene. The mouse HKII cDNA is approximately 5.5 kb in length and contains an open reading frome of 2751 bp encoding a protein of 917 amino acids. The mouse HKII gene is predominantly expressed in skeletal muscle, heart, and adipose tissue. The transcription initiation and polyadenylation sites for the mouse HKII mRNA were similar to those of the rat and human genes.

Animals↗
PubMed1998

Developmental expression of hexokinase 1 and 3 in rats.

Mammalian hexokinase types one and three (HK1 and HK3) are 100 kDa isozymes that phosphorylate glucose to glucose-6-phosphate. HK1 is present in most tissues but is especially prominent in brain and kidney. HK3 is less well studied, but may be most prominent in the spleen and lymphocytes. In this study, we determined the ontogeny of the expression of these isoforms in the rat. Using immunohistochemistry, we identified HK1 and HK3 immunoreactivity in the brain, heart, kidney, liver, skeletal muscle and spleen from gestational day 14 (E14) to 45 days after birth (P45). With the exception of the liver and spleen, we observed a similar age- and cell-dependent staining pattern for both isoforms in all organs studied. The brain and spleen were analyzed in more detail to identify specific regions of immunoreactivity during maturation. A transient expression of HK1 and HK3 was noted in the cell bodies of mature neurons, including layers V and VI of the cerebral cortex and the cerebellar Purkinje cells followed by localization to the white matter of the cerebrum and cerebellum. In the spleen, HK3 immunoreactivity was detected postnatally and appeared to track with the infiltration of B cells. Our demonstration of changing patterns of immunoreactivity for HK1 and HK3 in fetal and postnatal organs suggests that these HK isoforms are involved the process of development. We speculate that HK1 and HK3 share a complex interaction during development of these organs and regulate glucose metabolism at multiple levels during development.

Animals↗