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At least 217 records · Page 12Linked to original sources
PubMed1991

Hexokinase levels in discrete regions of rat brain: evaluation by quantitative immunofluorescence using interactive laser cytometry and comparison with basal rates of glucose utilization.

Relative levels of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) have been determined in 16 discrete regions of adult rat brain by a quantitative immunofluorescence method. The distribution of immunofluorescence in brain sections was determined by interactive laser cytometry and related to hexokinase content by comparison with standard sections containing known amounts of the enzyme. In many of these regions, referred to here as group I regions, hexokinase content was correlated with previously reported basal rates of glucose utilization. However, several regions (group II regions) in which hexokinase content exceeded that expected from basal rates of glucose utilization were also detected. Compared with the corresponding regions from albino rat brain, higher hexokinase levels were found in the dorsal and ventral lateral geniculate (group I regions) of pigmented Norway rats, a result reflecting previously reported increased glucose utilization by these regions in pigmented rats. There was no difference in hexokinase levels in the superior colliculus, a group II structure, from albino and pigmented rats, a finding implying that a reported increase in rate of glucose utilization in the superior colliculus of pigmented rats is effected without an increase in hexokinase content. It is suggested that group II regions may be adapted to sustain increases in rates of glucose utilization that are, relative to basal rates, considerably greater than those experienced by group I regions.

Animals↗
PubMed1995

Glucose transporter expression and functional role of hexokinase in insulin biosynthesis in mouse beta TC3 cells.

It was previously reported that insulin biosynthesis in mouse beta TC3 cells was regulated by glucose (Nagamatsu, S., and D. F. Steiner. Endocrinology 130: 748-754, 1992). In the present study, we examined the effect of glucose on the glucose transporter expression and hexokinase activities and determined the relationship between them and glucose-stimulated insulin biosynthesis in beta TC3 cells. Reverse transcriptase-polymerase chain reaction and Northern blot analysis revealed that beta TC3 cells expressed GLUT-1 and GLUT-3 glucose transporter mRNAs, but not GLUT-2. The levels of GLUT-1 and GLUT-3 mRNAs were not affected by glucose (0 or 11 mM glucose) over a period of 48 h. Immunoprecipitation of metabolically labeled beta TC3 cells with specific antibodies against GLUT-1 or GLUT-3 proteins revealed no effect of glucose on the biosynthesis of glucose transporters. Hexokinase [low Michaelis constant (Km) hexokinase] activity from cells incubated in 11 mM glucose for 48 h increased nearly twofold compared with cells maintained in 0 mM glucose, although the amount of cellular hexokinase protein detected by immunoblot analysis was unchanged between 0 and 11 mM glucose conditions. Glucokinase (high Km hexokinase) activity, in contrast, was not affected by glucose. Preincubation of beta TC3 cells with 2-deoxyglucose to inhibit hexokinase, thereby inhibiting all glycolysis, resulted in the decrease of glucose-stimulated insulin biosynthesis. Thus, in mouse beta TC3 cells that do not express GLUT-2, there is a close relationship between hexokinase activity and glucose-stimulated insulin biosynthesis, but not between the glucose transporter and glucose-stimulated insulin biosynthesis.

Animals↗
PubMed2006

A reevaluation of the roles of hexokinase I and II in the heart.

Hexokinase is responsible for glucose phosphorylation, a process fundamental to regulating glucose uptake. In some tissues, hexokinase translocates to the mitochondria, thereby increasing its efficiency and decreasing its susceptibility to product inhibition. It may also decrease free radical formation in the mitochondria and prevent apoptosis. Whether hexokinase translocation occurs in the heart is controversial; here, using immunogold labeling for the first time, we provide evidence for this process. Rat hearts (6 groups, n = 6/group), perfused with either glucose- or glucose + oleate (0.4 mmol/l)-containing buffer, were exposed to 30-min insulin stimulation, ischemia, or control perfusion. Hexokinase I (HK I) and hexokinase II (HK II) distributions were then determined. In glucose-perfused hearts, HK I-mitochondrial binding increased from 0.41 +/- 0.04 golds/mm in control hearts to 0.71 +/- 0.10 golds/mm after insulin and to 1.54 +/- 0.38 golds/mm after ischemia (P < 0.05). Similarly, HK II-mitochondrial binding increased from 0.16 +/- 0.02 to 0.53 +/- 0.08 golds/mm with insulin and 0.44 +/- 0.07 golds/mm after ischemia (P < 0.05). Under basal conditions, the fraction of HK I that was mitochondrial bound was five times greater than for HK II; insulin and ischemia caused a fourfold increase in HK II binding but only a doubling in HK I binding. Oleate decreased hexokinase-mitochondrial binding and abolished insulin-mediated translocation of HK I. Our data show that mitochondrial-hexokinase binding increases under insulin or ischemic stimulation and that this translocation is modified by oleate. These events are isoform specific, suggesting that HK I and HK II are independently regulated and implying that they perform different roles in cardiac glucose regulation.

Animals↗
PubMed2003

High glucose induces type 1 hexokinase gene expression in isolated glomeruli of diabetic rats and in mesangial cells.

BACKGROUND: Diabetic nephropathy, which is characterized by renal hypertrophy and accumulation of extracellular matrix, is one of the leading causes for end-stage renal disease. Pathophysiological changes, which finally lead to the development of diabetic nephropathy, act through an increase in the intracellular NADH/NAD ratio and the activation of the polyol and protein kinase C pathways. The first rate-limiting enzymes in intracellular glucose metabolism are the hexokinases, which catalyze the phosphorylation of glucose. Therefore, in order to examine a possible link between increased glucose metabolism and the development of diabetic nephropathy mRNA and protein expression as well as enzyme activity of type 1 hexokinase were examined in kidneys of control and diabetic rats and in mesangial cells. METHODS: Diabetes in rats was induced by intravenous injection of streptozotocin and animals were treated or not treated with insulin. RNA or protein was extracted from isolated glomeruli at different time intervals. In addition, glomerular mesangial cells were incubated in high glucose medium and hexokinase expression determined along with enzyme activity. RESULTS: The experiments demonstrate a significant increase in gene and protein expression of type 1 hexokinase in glomeruli of diabetic rats throughout a three week observation period. Insulin therapy reduced glomerular type 1 hexokinase mRNA expression. Gene expression and hexokinase enzyme activity were also increased in mesangial cells grown in high glucose medium. CONCLUSION: The present experiments demonstrate that the expression of type 1 hexokinase is increased in isolated glomeruli of diabetic animals and is regulated by high ambient glucose concentrations. These results add further evidence to the fact that the kidney is one of the tissues most sensitive to high glucose levels in diabetes.

Animals↗
PubMed1989

Human red blood cell loading with hexokinase-inactivating antibodies. An in vitro model for enzyme deficiencies.

The primary cause of red cell destruction in enzymopathies of anaerobic remains controversial and difficult to investigate especially because the erythrocyte population in enzymopenic patients is largely heterogeneous. We have shown that loading human erythrocytes with monospecific enzyme-inactivating antibodies could be useful in understanding the biochemical modifications occurring in enzymopenic erythrocytes and the mechanisms leading to red cell destruction. Hexokinase-inactivating antibodies were prepared and loaded in human erythrocytes using a procedure of encapsulation based on hypotonic hemolysis, isotonic resealing and reannealing. Red blood cells loaded with anti-hexokinase IgG showed 20 +/- 3% residual hexokinase activity while all other enzymes were normal. Lactate production by these cells was 30% of controls while the amount of glucose metabolized in the hexose monophosphate pathway (HMP) was unchanged under resting conditions. However, in the presence of methylene blue HMP rates were only 12% of controls. Determination of adenine nucleotide levels suggests that the antihexokinase-loaded red blood cells are not able to maintain, in vitro, their ATP level as well as their 2,3-diphosphoglycerate. Osmotic fragility, methemoglobin, and reduced glutathione content were near normal. These and other properties of the antihexokinase-loaded erythrocytes were similar to those found in cases of hexokinase deficiency. When the antibody-loaded erythrocytes were chromatrographed on immobilized Protein A columns 66-70% of cells were retained by the column against 0-10% of controls suggesting that hexokinase inactivation promotes autologous IgG binding. Since the phenomenon is known to be associated with red cell phagocytosis, it could be concluded that in hexokinase deficiency red cells are mainly removed by phagocytosis, and that hemolysis probably occurs in cases of oxidative stress when the production of a large amount of reducing equivalents (NADPH) is needed but not provided by the hexokinase-deficient erythrocytes.

Animals↗
PubMed1995

Upregulated hexokinase activity in isolated islets from diabetic 90% pancreatectomized rats.

Glucokinase is the beta-cell glucose sensor, i.e., the site in glucose metabolism that determines the glucose set-point (sensitivity) for insulin secretion. Hexokinase is also present, but it normally contributes little to glucose metabolism because of end-product inhibition by glucose 6-phosphate. There is a lowered glucose set-point for insulin secretion in 90% pancreatectomized (Px) diabetic rats. We investigated the mechanism by measuring hexokinase and glucokinase activity in islet extracts. Glucokinase activity was minimally raised in Px islets (Vmax 125% of sham-operated control rats). In contrast, hexokinase Vmax was 250% of the control value, suggesting that the increased hexokinase activity caused the beta-cell glucose hypersensitivity. Additional evidence was obtained with a 40-h fast that was performed because of a previous observation that the inhibitory effect of fasting on insulin secretion was impaired in Px rats. Glucokinase activity fell normally in the Px rats (32 +/- 4% reduction in sham vs. 37 +/- 4% in Px rats) as opposed to hexokinase activity, which was unaffected in either group. In summary, a feature of hyperglycemia is upregulated islet hexokinase activity. The result is that hexokinase assumes partial control over the glucose set-point for insulin secretion. As such, regulatory effects on insulin secretion, such as fasting, that are mediated through glucokinase activity may be altered.

Animals↗
PubMedDate not supplied

[Effect of L-thyroxine on the activity and isoenzymatic spectrum of liver hexokinase in intact and insulin-resistant rats].

Administration of L-thyroxine into intact rats caused a distinct increase in the activity of hexokinase in liver tissue. In the isoenzyme spectrum of hexokinase the activity of hexokinase II was increased significantly. After repeated administration of insulin, liver tissue cells lost their capacity to respond to the hormone administration by induction of hexokinase. Administration of L-thyroxine into animals caused a distinct increase in the hexokinase activity in liver tissue. In isoenzyme spectrum of hexokinase under effect of L-thyroxine a significant increase in the hexokinase II activity was also observed.

Agar↗
PubMed1990

Glucose phosphorylation in tumor cells. Cloning, sequencing, and overexpression in active form of a full-length cDNA encoding a mitochondrial bindable form of hexokinase.

In rapidly growing tumor cells exhibiting high glucose catabolic rates, the enzyme hexokinase is markedly elevated and bound in large amounts (50-80% of the total cell activity) to the outer mitochondrial membrane (Arora, K.K., and Pedersen, P.L. (1988) J. Biol. Chem. 263, 17422-17428; Parry, D.M., and Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912). In extending these studies, we have isolated a cDNA clone of hexokinase from a lambda gt11 library of the highly glycolytic, c37 mouse hepatoma cell line. This clone, comprising 4,198 base pairs, contains a single open reading frame of 2,754 nucleotides which encode a 918-amino acid hexokinase with a mass of 102,272 daltons. This enzyme exhibits, respectively, 68 and 32 amino acid differences, including several charge differences, from the recently sequenced human kidney and rat brain enzymes. The putative glucose and ATP binding domains present in the latter two enzymes and in rat liver glucokinase are conserved in the tumor enzyme. At its N-terminal region, tumor hexokinase has a 12-amino acid hydrophobic stretch which is present in the rat brain enzyme but absent in the rat liver glucokinase, a cytoplasmic enzyme. The mature tumor hexokinase protein has been overexpressed in active form in Escherichia coli and purified 9-fold. The overexpressed enzyme binds to rat liver mitochondria in the presence of MgCl2. This is the first report describing the cloning and sequencing of a tumor hexokinase, and the first report documenting the overexpression of any hexokinase type in E. coli. Questions pertinent to the enzyme's mechanism, regulation, binding to mitochondria, and its marked elevation in tumor cells can now be addressed.

Amino Acid Sequence↗
PubMed1990

Glucose phosphorylation. Interaction of a 50-amino acid peptide of yeast hexokinase with trinitrophenyl ATP.

A 50-amino acid peptide predicted by chemical modification studies of yeast hexokinase to contain an ATP-binding site has been synthesized and purified. The peptide, which includes residues from glutamate 78 at the NH2-terminal end to leucine 127 at the COOH-terminal, resides within the smaller of the two lobes found in the three-dimensional structure of yeast hexokinase. It is this region which has been reported recently to exhibit significant sequence homology with hexokinase types I and IV of higher eukaryotic cells and sequence homology with the active site of protein kinases. Similar to native yeast hexokinase, the 50-amino acid peptide interacts strongly with the fluorescent analog TNP-ATP [2',(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate]. A 5-fold enhancement is observed when 8 microM peptide interacts with 20 microM TNP-ATP. The stoichiometry of binding is very close to 1 mol of TNP-ATP/mol peptide. Also, similar to native yeast hexokinase, the fluorescent enhancement observed upon TNP-ATP binding to the synthetic peptide is greater than that observed upon TNP-ADP binding. Finally, TNP-AMP exhibits a much lower fluorescent enhancement in the presence of hexokinase or the synthetic peptide. The additional findings that ATP can readily prevent TNP-ATP binding and that TNP-ATP can substitute for ATP as a weak substrate for hexokinase in the phosphorylation of glucose indicate that the synthetic peptide described here comprises part of the catalytic site.

Adenosine Triphosphate↗
PubMed1986

[Specificity of interaction of hexokinase isozyme II with mitochondrial membranes].

Study on the mechanism of hexokinase isozyme II adsorption on mitochondrial membranes in the presence of 10 mM MgCl2 demonstrated that 0.16% of the total proteins of the soluble fraction and the total hexokinase pool are capable of reversible binding to the membrane. The plot for the dependence of the degree of enzyme adsorption on Mg2+ concentration is hyperbolic. Under these conditions, hexokinase competes favourably for the binding sites with lactate dehydrogenase and creatine kinase. Analysis of the adsorption capacity of natural and artificial phospholipid membranes showed that hexokinase isozyme II is adsorbed in much the same way on inner and outer mitochondrial membranes as well as on a mixture of membranes obtained from various sources and on lecithin liposomes. The adsorption properties of hexokinase isozyme II and of its functional analog--isozyme I--point to marked differences in the mechanism of their interaction with the membrane. In contrast with isozyme I, isozyme II of hexokinase undergoes kinetic alterations. Besides, it was found that mild autolysis of isozyme II is accompanied by a loss of the enzyme ability to bind to mitochondrial membranes. The data obtained suggest that the specificity of hexokinase isozyme II adsorption depends on the structural peculiarities of the protein but not on those of the mitochondrial membrane.

Adsorption↗
PubMed1979

Electrophoretic characterization and subcellular distribution of hexokinase isoenzymes in red blood cells of rabbits.

The isoenzyme pattern of hexokinase in rabbit red cells (erythrocytes, fetal erythrocytes and reticulocytes) were determined by means of agarose gel and disc electrophoresis. One duplicated hexokinase (4a and 4b according to the IUPAC-nomenclature) was detected in rabbit erythrocytes as also described for human erythrocytes. Besides the isoenzymes 4a and 4b reticulocytes also contain hexokinase 2 and 3 like rabbit and rat liver. The high KM glucose phosphorylating enzyme, hexokinase 1 could be demonstrated only under specific conditions in the reticulocytes during the initial stage of the anemia. After the fractionation of reticulocyte homogenates the total hexokinase activity was recovered in the mitochondria and cytosol to nearly equal amounts as revealed by the distribution of markers. Hexokinase 2 and 3 were detectable in reticulocytes and in isolated mitochondria only after the addition of certain dissociating agents. In contrast to the tightly bound mitochondrial hexokinases 2 and 3 the type 4a and 4b are more loosely bound and exhibit a bilocal distribution between mitochondria and cytosol of reticulocytes.

Animals↗
PubMed1984

Subcellular localization of hexokinase in pea leaves. Evidence for the predominance of a mitochondrially bound form.

Hexokinase (ATP: D-hexose-6-phosphotransferase, EC 2.7.1.1) activity was determined in subcellular fractions prepared from pea (Pisum sativum) leaf homogenates. About 60% of the total detectable activity of hexokinase was found associated with a particulate fraction consisting essentially of mitochondria and chloroplasts and free of cytosol contamination. The hexokinase specific activity of the particulate fraction was 2-fold higher than that of the homogenate and about 4-fold higher than that of the cytosol. Using a specially designed isokinetic-isopycnic sucrose density gradient centrifugation method, the distribution of hexokinase activity correlated with that of the mitochondrial marker (cytochrome oxidase) and not with that of the chloroplast membrane marker ( chlorophyll ) or that of the cytosol marker (phosphoenolpyruvate carboxylase). Thus, the hexokinase/mitochondria ratio was close to 1.0 along the entire gradient, while the hexokinase/chloroplast ratio varied over a 10-fold range. The results strongly suggest that hexokinase is predominantly bound to mitochondria of pea leaves, and that pea leaf chloroplasts are essentially devoid of any specifically associated hexokinase activity. This work provides the first demonstration of a specific association of hexokinase with mitochondria from photosynthetic tissues of higher plants.

Centrifugation, Isopycnic↗
PubMed1982

Genetic evidence for a role of hexokinase isozyme PII in carbon catabolite repression in Saccharomyces cerevisiae.

A mutant of Saccharomyces cerevisiae that was selected for resistance to carbon catabolite repression also had reduced hexokinase activity. Hexokinase isoenzymes were purified from mutant and wild type cells. The specific glucokinase and hexokinase isozyme PI were present at normal levels in mutant and wild type, but no hexokinase isozyme PII activity was detected in the mutant. Staining for enzyme activity after electrophoresis of crude extracts also indicated that hexokinase PII was absent in the mutant. Mutant and wild type segregants gained by tetrad analysis were investigated electrophoretically. Staining for enzyme activity confirmed that catalytically inactive hexokinase PII and the defect in carbon catabolite repression always co-segregated. The results support the hypothesis that hexokinase PII might mediate carbon catabolite repression.

Enzyme Repression↗
PubMed1995

Mitochondria-bound hexokinase from rabbit reticulocytes is resistant to the inactivation induced by Fe(II)/ascorbate.

Exposure of rabbit reticulocytes to Fe(II)/ascorbate induced a pronounced decay in hexokinase activity. In reticulocytes, this enzyme is present in at least three different molecular forms, Ia, Ia* and Ib, sub-types of hexokinase type I, which show different intracellular distribution. Hexokinase Ia and Ib are soluble, whereas hexokinase Ia* is almost entirely bound to the mitochondria. Anion exchange chromatography of hexokinase from intact reticulocytes exposed to Fe(II)/ascorbate revealed a selective inactivation of forms Ia and Ib, whereas the form Ia* did not show any decay. Binding to the mitochondrial membrane seems to be responsible for the observed resistance of the form Ia* to the inactivation elicited by Fe(II)/ascorbate. Indeed, by using a cell-free system in which hexokinase Ia* was solubilized using Triton X-100, the decay in hexokinase activity induced by iron/ascorbate involved all three enzymatic forms.

Animals↗
PubMed1993

Mitochondrial hexokinase in brain of various species: differences in sensitivity to solubilization by glucose 6-phosphate.

Approximately 90% of the hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) activity was solubilized by treatment of rat brain mitochondria with glucose 6-phosphate (Glc-6-P), while only about 20% of the hexokinase could be solubilized from human brain mitochondria. Intermediate amounts of solubilized activity were obtained with brain mitochondria from other species. In contrast, > or = 80% of the activity could be released by 0.5 M potassium thiocyanate (KSCN), regardless of the species from which the mitochondria were obtained. Hexokinase activities solubilized by treatment of bovine brain mitochondria with Glc-6-P (HKG6P) and by a subsequent treatment with KSCN (HKKSCN) were indistinguishable in their isoelectric focusing pattern and molecular weight, and both were inhibited by Glc-6-P with Ki approximately 20 microM. Both HKG6P and HKKSCN could bind to mitochondria from rat liver or brain, and both were again solubilized by a subsequent treatment with Glc-6-P. These results do not suggest any intrinsic molecular difference between HKG6P and HKKSCN. Rather, the difference in susceptibility to release by Glc-6-P is reasonably attributed to discrete types of binding sites for hexokinase on brain mitochondria, with the relative proportion of these varying with species. Bovine brain mitochondria bearing HKG6P and HKKSCN were not resolved by sucrose density gradient fractionation, suggesting that both forms may coexist on the same mitochondrion. Given the probable importance of mitochondrially bound hexokinase in regulating aerobic glycolysis in brain, these differences in hexokinase-mitochondrial interactions may be related to previously documented differences in cerebral energy metabolism of these various species.

Adolescent↗
PubMed1996

Functional organization of mammalian hexokinases: both N- and C-terminal halves of the rat type II isozyme possess catalytic sites.

Previous work has shown that catalytic function is associated exclusively with the C-terminal half of the Type I isozyme of mammalian hexokinase. In contrast, we now demonstrate that both halves of the Type II isozyme possess comparable catalytic activities. Mutation of a catalytically important Ser residue to Ala at analogous positions in either the N- or the C-terminal halves (S155A or S603A, respectively) of the rat Type II isozyme resulted in approximately 60% reduction in specific activity of the enzyme, with more than 90% reduction in the doubly mutated enzyme (S155A/S603A). Catalytic activity was retained in a chimeric hexokinase comprising the N-terminal half of Type II hexokinase and catalytically inactive (by site-directed mutation) C-terminal half of the Type I isozyme. The N- and C-terminal catalytic sites of Type II hexokinase are similar in V(max) and K(m) (approximately equal to 130 microM) for glucose; however, the N-terminal site has a lower (0.45 vs 1.1 mM) K(m) for ATP, is slightly more sensitive to inhibition by the product analog 1,5-anhydroglucitol-6-P, and is much more sensitive to inhibition by P(i). It is suggested that the Type II isozyme most closely resembles the 100-kDa hexokinase which resulted from duplication and fusion of a gene encoding an ancestral 50-kDa hexokinase and which was the precursor for the contemporary Type I, Type II, and Type III mammalian isozymes. Subsequent evolutionary changes could then have led to functional differentiation of the N- and C-terminal halves, as seen with the Type I (and possibly the Type III) isozyme.

Animals↗
PubMed1997

Functional organization of mammalian hexokinases: characterization of the rat type III isozyme and its chimeric forms, constructed with the N- and C-terminal halves of the type I and type II isozymes.

Previous studies have shown that catalytic function is associated with both halves of the Type II isozyme of mammalian hexokinase, while the Type I isozyme is functionally differentiated into a catalytic C-terminal half and regulatory N-terminal half. The Type III isozyme has now been shown to be similar to the Type I isozyme in its functional organization. Chimeras composed of the N-terminal half of Type III hexokinase and the C-terminal half of either Type I or Type II hexokinase have activities that can be attributed to the C-terminal half and are similar in activity to chimeras composed of the C-terminal half of Type III and the intrinsically inactive N-terminal domain of Type I or the inactivated (by site-directed mutation) N-terminal half of Type II hexokinase. Virtually no activity was seen with chimeras constructed with the N-terminal half of the Type III isozyme and catalytically inactive (by site-directed mutation) C-terminal halves of Type I or Type II hexokinase. Substrate inhibition by Glc is seen only with the Type III isozyme and with chimeric forms containing the C-terminal half of Type III hexokinase and the N-terminal half of Type I or Type II isozyme, the latter inactivated by site-directed mutation; this is attributed to conformational changes induced by binding of Glc to a low affinity site in the N-terminal half, with subsequent effect on catalytic activity of the C-terminal half. These results also provide further insight into the role of interactions (or lack of interactions) between the N- and C-terminal halves in the inhibition of the Type I-III isozymes by Glc-6-P, its antagonism by low concentrations of Pi, and the inhibition seen at higher concentrations of Pi.

Adenosine Triphosphate↗
PubMed1997

Hexokinase inactivation induced by ascorbic acid/Fe(II) in rabbit erythrocytes is independent of glutathione-reductive processes and appears to be mediated by dehydroascorbic acid.

Recent studies performed in our laboratory demonstrated that rabbit red blood cell hexokinase was remarkably inhibited by the cocktail ascorbic acid/Fe(II) (Stocchi et al., 1994, Arch. Biochem. Biophys. 311, 160-167) and that the formation of dehydroascorbic acid was a key event in this process (Fiorani et al., 1996, Arch. Biochem. Biophys, 334, 357-361). The present study was undertaken to determine the final hexokinase-inactivating species using cell-free extract as a model. Our results demonstrate superimposable kinetics of hexokinase decay promoted by either ascorbic acid/Fe(II) or dehydroascorbic acid in erythrocyte lysates in which the reduced glutathione (GSH) levels were variously manipulated. In particular, neither removal nor addition of this tripeptide was able to significantly alter the rate or extent of hexokinase inhibition. Thus, GSH-reductive processes are dispensable events in the process of hexokinase inhibition promoted by ascorbic acid/Fe(II) in red blood cells. As a consequence, dehydroascorbic acid appears to be the species which directly inhibits hexokinase. This inference is further supported by the observation that addition of dehydroascorbic acid to the purified enzyme leads to a remarkable inhibition in its activity.

Animals↗