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PubMed1988

Inactivation of yeast hexokinase by o-phthalaldehyde: evidence for the presence of a cysteine and a lysine at or near the active site.

Yeast hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), a homodimer, was rapidly and irreversibly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics over a wide range of the inhibitor concentration. The second-order-rate constant for the inactivation of hexokinase was estimated to be 45 M-1.s-1. Hexokinase was protected more by sugar substrates than by nucleoside triphosphates during inactivation by o-phthalaldehyde. Absorption spectrum (lambda max 338 nm), and fluorescence excitation (lambda max 363 nm) and emission (lambda max 403 nm) spectra of the hexokinase-o-phthalaldehyde adduct were consistent with the formation of an isoindole derivative. These results also suggest that sulfhydryl and epsilon-amino functions of the cysteine and lysine residues, respectively, participating in the isoindole formation are about 3 A apart in the native enzyme. About 2 mol of the isoindole per mol of hexokinase dimer were formed following complete loss of the phosphotransferase activity. Chemical modification of hexokinase by iodoacetamide in the presence of mannose resulted in the modification of six sulfhydryl groups per mol of hexokinase with retention of the phosphotransferase activity. Subsequent reaction of the iodoacetamide modified hexokinase with o-phthalaldehyde resulted in complete loss of the phosphotransferase activity with concomitant modification of the remaining two sulfhydryl groups of hexokinase. Chemical modification of hexokinase by iodoacetamide in the absence of mannose resulted in complete inactivation of the enzyme. The iodoacetamide inactivated hexokinase failed to react with o-phthalaldehyde as evidenced by the absence of a fluorescence emission maximum characteristic of the isoindole derivative. The holoenzyme failed to react with [5'-(p-fluorosulfonyl)benzoyl]adenosine. The dissociated hexokinase could be inactivated by [5'-(p-fluorosulfonyl)benzoyl]adenosine; the degree of inactivation paralleled the extent of reaction between o-phthalaldehyde and the nucleotide-analog modified enzyme. Thus, it is concluded that two cysteines and lysines at or near the active site of the hexokinase were involved in reaction with o-phthalaldehyde following complete loss of the phosphotransferase activity. An important finding of this investigation is that the lysines, involved in isoindole formation, located at or near the active site are probably buried.(ABSTRACT TRUNCATED AT 400 WORDS)

Aldehydes↗

PubMed1996

Metabolic modulation of hexokinase association with mitochondria in living smooth muscle cells.

Hexokinase isoform I binds to mitochondria of many cell types. It has been hypothesized that this association is regulated by changes in the concentrations of specific cellular metabolites. To study the distribution of hexokinase in living cells, fluorophore-labeled functional hexokinase I was prepared. After microinjection into A7r5 smooth muscle cells, hexokinase localized to distinct structures identified as mitochondria. The endogenous hexokinase demonstrated a similar distribution with the use of immunocytochemistry. 2-Deoxyglucose elicited an increase in glucose 6-phosphate (G-6-P) and a decrease in ATP levels and diminished hexokinase binding to mitochondria in single cells. 3-O-methylglucose elicited slowly developing decreases in all three parameters. In contrast, cyanide elicited a rapid decrease in both ATP and hexokinase binding. Analyses of changes in metabolite levels and hexokinase binding indicate a positive correlation between binding and cell energy state as monitored by ATP. On the other hand, only in the presence of 2-deoxyglucose was the predicted inverse correlation between binding and G-6-P observed. Unlike the relatively large changes in distribution observed with the fluorescent-injected hexokinase, cyanide caused only a small decrease in the localization of endogenous hexokinase with mitochondria. These findings suggest that changes in the concentrations of specific metabolites can alter the binding of hexokinase I to specific sites on mitochondria. Moreover, the apparent difference in sensitivity of injected and endogenous hexokinase to changes in metabolites may reflect the presence of at least two classes of binding mechanisms for hexokinase, with differential sensitivity to metabolites.

3-O-Methylglucose↗

PubMed1982

Multiple forms of human red blood cell hexokinase. Preparation, characterization, and age dependence.

Human red blood cell hexokinase (EC 2.7.1.1) has been shown to exist in multiple molecular forms which are separable by ion exchange chromatography. Of the major forms, designated hexokinase Ia, Ib, and Ic, only hexokinase Ia corresponds to hexokinase type I from human liver, while the others differ from every other previously reported hexokinase isozyme. Hexokinase Ib is the predominant form in the fetal erythrocytes, while it is present at lower levels in the red blood cells of adults. Analysis of the hexokinase isozymic pattern in red cells of different mean age shows that the level of hexokinase Ib is also dependent on the age of the cell. The three major forms of hexokinase have the same molecular weight of 100,000, by sedimentation velocity on sucrose density gradients, the same Michaelis constants, substrate and coenzyme specificity, pH-dependent activity, and the same thermal stability. The only significant differences were found in the isoelectric points which were 5.7 pH units for hexokinase Ia, 5.5 pH units for hexokinase Ib, and 5.35 pH units for hexokinase Ic. These data, together with that previously reported for rabbit erythrocytes (Stocchi, V., Magnani, M., Canestrari, F., Dachà, M., and Fornaini, G. (1981) J. Biol. Chem. 256, 7856-7861) suggest that the presence of multiple forms of hexokinase is a common phenomenon in mammalian red blood cells.

Adult↗

PubMed1993

Hexokinase II mRNA and gene structure, regulation by insulin, and evolution.

A DNA segment that is highly conserved in glucokinase (hexokinase IV) and hexokinase I cDNA was used to identify specific cDNAs in a library prepared from rat adipose tissue mRNA. Some of these cDNAs were identified as being hexokinase I cDNA. Others, although similar to both the glucokinase and hexokinase I cDNAs, were unique. Two of these unique cDNAs overlapped and contained an open reading frame that encoded a protein of 103 kDa which, when expressed in Escherichia coli, had kinetic properties characteristic of hexokinase II. The entire hexokinase II mRNA sequence and the exon-intron structure of the hexokinase II gene were determined. A single transcription initiation site and two distinct termination sites account for the two observed hexokinase II RNA species of 5500 and 4400 nucleotides that were detected when either of the cDNAs was used as a hybridization probe against poly(A)+ RNA isolated from rat adipose tissue. Hexokinase II mRNA was decreased in adipose tissue from diabetic rats, but was restored by insulin treatment to levels found in nondiabetic control rats. Insulin also induced hexokinase II mRNA in two adipose cell lines (3T3-F442A and BFC-1B) and two skeletal muscle cell lines (C2C12 and L6). In L6 cells, this increase was accounted for by a corresponding increase of hexokinase II gene transcription. Comparison of the structures of the hexokinase II and glucokinase genes support the hypothesis that the 100-kDa hexokinase arose by gene duplication and tandem ligation of a 50-kDa glucokinase-like ancestral gene.

Adipose Tissue↗

PubMed1986

Effect of streptozotocin-induced diabetes and insulin treatment on the synthesis of hexokinase II in the skeletal muscle of the rat.

The relative rate of synthesis of hexokinase II in the skeletal muscle of the normal, streptozotocin-diabetic, and diabetic insulin-treated rat was determined by the rate of incorporation of [3H]leucine into hexokinase II and the total cytosolic proteins to determine if the rate of hexokinase II synthesis was altered relative to that of the average protein. This relative rate of synthesis of hexokinase II is approximately 1.9 times higher in the normal than in the diabetic rat. The administration of insulin to the diabetic animal increases the rate of hexokinase synthesis to approximately normal levels. An enzyme-linked immunosorbent assay procedure was developed to determine the amount of hexokinase II protein in the skeletal muscle extracts, and immunoprecipitation was utilized to determine the hexokinase II activity. The specific activity of hexokinase II was determined from these analyses. The specific activity of hexokinase II was the same in the skeletal muscle extracts from normal, streptozotocin-diabetic, and diabetic insulin-treated rats. These results suggest that the decrease in muscle hexokinase activity is not caused by the loss of an activator of the enzyme nor by the increased formation of a hexokinase inhibitor in streptozotocin-induced diabetes; rather the decrease in hexokinase II activity reported in diabetic rats relative to normal animals is a result of decreased synthesis coupled to increased degradation in the diabetic relative to the normal animal.

Animals↗

PubMed1971

Mitochondrial hexokinase from small-intestinal mucosa and brain.

1. The submitochondrial localization of hexokinase activity in preparations of mitochondria from the small intestine of the guinea pig was studied by conventional methods. 2. Hexokinase activity in this tissue was predominantly associated with the outer mitochondrial membrane. 3. The inactivation of mitochondrial enzymes by trypsin in iso-osmotic and hypo-osmotic conditions was also used to determine the submitochondrial localization of hexokinase activity. 4. Hexokinase activity was found to be on the outside of the outer mitochondrial membrane. 5. It was shown that both type I and type II hexokinase activities are bound to the outside of the outer mitochondrial membrane. The types are present in the same ratio as that in which they occur in the cytosol of the cell. 6. Mitochondrial hexokinase from the small intestine did not show the latency phenomenon demonstrated by mitochondrial hexokinase from brain when subjected to a variety of treatments. However, hexokinase activity was solubilized from preparations of mitochondria from the small intestine by the same treatments as for mitochondrial hexokinase from brain. 7. The submitochondrial distribution of hexokinase activity in mitochondrial preparations from rat brain was determined by the trypsin inactivation method. 8. Hexokinase activity in preparations of mitochondria from rat brain was found on the outside of the outer membrane, between the mitochondrial membranes, and within the inner mitochondrial membrane. 9. Hexokinase from rat brain showed latency properties irrespective of its submitochondrial location.

Animals↗

PubMed1988

Inactivation of yeast hexokinase by 2-aminothiophenol. Evidence for a 'half-of-the-sites' mechanism.

Yeast hexokinase is a homodimer consisting of two identical subunits. Yeast hexokinase was inactivated by 2-aminothiophenol at 25 degrees C (pH 9.1). The reaction followed pseudo-first-order kinetics until about 70% of the phosphotransferase activity was lost. About 0.65 mol of 2-aminothiophenol/mol of hexokinase was found to be bound after the 70% loss of the enzyme activity. Completely inactivated hexokinase showed a stoichiometry of about 1 mol of 2-aminothiophenol bound/mol of the enzyme. The evidence obtained from kinetic experiments, stoichiometry of the inactivation reaction and fluorescence emission measurements suggested site-site interaction (weak negative co-operativity) during the inactivation reaction. The approximate rate constants for the reversible binding of 2-aminothiophenol to the first subunit (KI) and for the rate of covalent bond formation with only one site occupied (k3) were 150 microM and 0.046 min-1 respectively. The inactivation reaction was pH-dependent. Dithiothreitol, 2-mercaptoethanol and cysteine restored the phosphotransferase activity of the hexokinase after inactivation by 2-aminothiophenol. Sugar substrates protected the enzyme from inactivation more than did the nucleotides. Thus it is concluded that the inactivation of the hexokinase by 2-aminothiophenol was a consequence of a covalent disulphide bond formation between the aminothiol and thiol function at or near the active site of the enzyme. Hexokinase that had been completely inactivated by 2-aminothiophenol reacted with o-phthalaldehyde. Fluorescence emission intensity of the incubation mixture containing 2-aminothiophenol-modified hexokinase and o-phthalaldehyde was one-half of that obtained from an incubation mixture containing hexokinase and o-phthalaldehyde under similar experimental conditions. The intensity and position of the fluorescence emission maximum of the 2-aminothiophenol-modified hexokinase were different from those of the native enzyme, indicating conformational change following modification. Whereas aliphatic aminothiols were completely ineffective, aromatic aminothiols were good inhibitors of the hexokinase. Cyclohexyl mercaptan weakly inhibited the enzyme. Inhibition of the hexokinase by heteroaromatic thiols was dependent on the nature of the heterocyclic ring and position of the thiol-thione equilibrium. The inhibitory function of a thiol is associated with the following structural characteristics: (a) the presence of an aromatic ring, (b) the presence of a free thiol function and (c) the presence of a free amino function in the close proximity of the thiol function.(ABSTRACT TRUNCATED AT 400 WORDS)

Aniline Compounds↗

PubMed1998

Cloning and biochemical characterisation of Aspergillus niger hexokinase--the enzyme is strongly inhibited by physiological concentrations of trehalose 6-phosphate.

The Aspergillus niger hexokinase gene hxkA has been cloned by heterologous hybridisation using the Aspergillus nidulans hexokinase gene as a probe. The DNA sequence of the gene was determined, and the deduced amino acid sequence showed significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to those of the budding yeasts. The encoded protein was purified from a multicopy hxkA transformant, and extensively characterised. The hexokinase protein has a molecular mass of 54090, a pI of 4.9 and is a homodimer. D-Glucose, the glucose analogue 2-deoxy-D-glucose, D-fructose, D-mannose and D-glucosamine are phosphorylated by hexokinase, whereas the hexoses D-galactose, L-sorbose, methyl alpha-D-glucoside and the pentoses L-arabinose and D-xylose are not. The enzyme has high affinity for glucose (Km = 0.35 mM at pH 7.5) and for fructose (Km = 2.0 mM at pH 7.5) and is inhibited by ADP. The enzyme is strongly inhibited by physiological concentrations (0.1-0.2 mM) of trehalose 6-phosphate, which may be of importance for in vivo regulation of the enzyme. Inhibition of A. niger hexokinase by trehalose 6-phosphate is competitive towards the sugar substrate (Ki = 0.01 mM). Based on the kinetic constants of hexokinase and glucokinase their relative contribution to in vivo glucose phosphorylation was calculated and found to be strongly dependent on intracellular pH and glucose concentration. At pH 7.5 glucokinase is predominant, whereas at pH 6.5 hexokinase is predominant at glucose concentrations higher than 0.5 mM. Expression of the hexokinase and the glucokinase gene requires active carbon metabolism. Also on carbon sources which are not substrates for hexokinase or glucokinase, clear expression is observed. The hexokinase and glucokinase enzymes are quite stable in vivo. Even in the absence of transcription, active glucokinase and hexokinase remain present in the cells at almost the same level for at least 3-4 h after depletion of the carbon source.

Amino Acid Sequence↗

PubMed2000

[Identification and characterization of hexokinase isozyme predominantly expressed in malignant tumor cells].

Tumor cells show a higher glycolytic rate than normal cells. Of glycolytic enzymes, the activity of hexokinase, known as a rate limiting enzyme in glycolysis, is amazingly high in malignant tumor cells. In mammals, four isozymes of hexokinase are expressed but the question which isozyme is responsible for the high hexokinase activity observed in tumor cells was not yet clearly answered. By Northern blot analysis, we found that the type II isozyme, which is only slightly expressed in normal heart, muscle and adipose tissue, was remarkably expressed in malignant tumor cells. We next tried to understand how the expression of type II hexokinase gene is regulated in tumor cells. For this purpose, we first isolated the type II hexokinase gene and characterized its structural features. We further investigated the regulatory mechanisms of the expression of type II hexokinase in tumor cells. Results indicate the potential involvement of a serum responsive factor in the regulation of the expression of type II hexokinase in tumor cells. In addition to the remarkable expression, binding of the type II hexokinase to mitochondria is another characteristic of tumor cells, however, the physiological meaning of hexokinase binding to mitochondria was not yet fully understood. Our results clearly showed that the mitochondria-bound hexokinase utilize mitochondrially generated ATP more preferentially under normal conditions. However, when the rate of extramitochondrial ATP generating system (glycolysis) exceed that of mitochondrial ATP generating system (oxidative phosphorylation), the mitochondria-bound hexokinase utilize extramitochondrial ATP. This result indicates that the hexokinase binding enables a cross talk between oxidative phosphorylation and glycolysis.

Adenosine Triphosphate↗

PubMed1991

Human hexokinase type I microheterogeneity is due to different amino-terminal sequences.

Human placenta hexokinase type I was previously shown to be present in two subtypes with similar isoelectric points but different molecular masses of 112 and 103 kDa, respectively. In order to exclude that these subtypes arise by artifact(s) occurring during the protein purification, we have developed a single-step immunoaffinity chromatography for the isolation of microgram quantities of hexokinase. The results obtained confirmed the presence of both hexokinase subtypes in human placenta. By Northern blot analysis a single mRNA species that hybridized with a hexokinase-I cDNA was found to be present in human placenta. Furthermore, in vitro translation of placenta mRNA in a rabbit reticulocyte lysate followed by hexokinase immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that only one hexokinase with apparent molecular mass of about 112 kDa is expressed in this tissue and suggests a post-translational modification as a probable cause of hexokinase I microheterogeneity. To further investigate this point we have purified the high and low Mr hexokinase and determined their NH2-terminal sequences. The results obtained show that when compared with the amino acid sequence deduced from a cDNA the high Mr hexokinase starts at amino acid 11 while the low Mr hexokinase starts at amino acid 103. Since the first 10 amino acids are involved in the binding of hexokinase to mitochondrial porin these data provide an explanation both for the inability of these hexokinases to bind to mitochondria and for their differences in Mr.

Amino Acid Sequence↗

PubMed1989

Stabilization of hexokinases I and II of ELD cells by binding to mitochondria.

Significance of the binding of hexokinase to mitochondria was examined with respect to stabilization of the enzyme by the binding. Stability during the incubation of the mitochondria-bound forms of hexokinases I and II, both prepared from Ehrlich-Lettre ascites hyperdiploid tumor cells (ELD cells), were compared with that of the corresponding free forms. During the incubation at pH 7.4 and 37 degrees C up to 60 min, hexokinase activities decreased gradually, and the decrease in the activity of the free form was much more marked than that of the bound form for both hexokinases. Hexokinase II was much less stable than I, and the activity of the free form of the former was almost lost by the incubation for 15 min. But, more than a half of the original activity of hexokinase II was retained even after 60 min of the incubation when the enzyme was bound to mitochondria. Addition of 50 mM glucose increased the stability of hexokinase II, but the stabilizing effect was less marked for hexokinase I. On the other hand, addition of 28 mg/ml of bovine serum albumin markedly stabilized hexokinase I to almost the same extent as was observed with mitochondria. On the contrary, the serum albumin had little stabilizing effect on hexokinase II. These findings indicate that the binding to mitochondria stabilizes the hexokinases of ELD cells, though the stability is different by nature between hexokinases I and II.

Animals↗

PubMed1994

The 50-kDa glucose 6-phosphate-sensitive hexokinase of Schistosoma mansoni.

Hexokinase has been purified from adult Schistosoma mansoni worms and the activity shown to be associated with a single protein species having an M(r) about 50,000. This protein is recognized on Western blots probed with antisera against rat Type I hexokinase or against a recombinant S. mansoni hexokinase that had been expressed in Escherichia coli using a previously cloned cDNA. An 18-residue N-terminal sequence determined for the purified S. mansoni hexokinase is identical to that deduced from the nucleotide sequence of the cDNA, consistent with the view that the cloned cDNA encodes the hexokinase characterized in the present study. The S. mansoni enzyme has a relatively low Km (approximately 60 microM) for glucose and is sensitive to inhibition (competitive versus ATP, Ki approximately 50 microM) by its product, glucose 6-phosphate (Glc-6-P). With these kinetic properties and 50 kDa molecular mass, S. mansoni hexokinase resembles the ancestral hexokinase predicted to have given rise, by gene duplication and fusion, to the present day 100-kDa Glc-6-P-sensitive mammalian hexokinases. The schistosomal hexokinase represents the first 50-kDa Glc-6-P-sensitive hexokinase whose sequence has been obtained. The schistosomal hexokinase does not bind to mitochondria, consistent with its lack of a hydrophobic segment at the N terminus which is required for binding of the mammalian Type I and II isoenzymes to mitochondria. The marked Crabtree effect exhibited by S. mansoni cercariae may be at least partly attributed to the expression of rather high levels of a hexokinase having a high affinity for glucose but only a moderate sensitivity to product inhibition by Glc-6-P.

Amino Acid Sequence↗

PubMed1992

Hexokinase receptors: preferential enzyme binding in normal cells to nonmitochondrial sites and in transformed cells to mitochondrial sites.

Hexokinase plays an important role in normal glucose-utilizing tissues like brain and kidney, and an even more important role in highly malignant cancer cells where it is markedly overexpressed. In both cell types, normal and transformed, a significant portion of the total hexokinase activity is bound to particulate material that sediments upon differential centrifugation with the crude "mitochondrial" fraction. In the case of brain, particulate binding may constitute most of the total hexokinase activity of the cell, and in highly malignant tumor cells as much as 80 percent of the total. When a variety of techniques are rigorously applied to better define the particulate location of hexokinase within the crude "mitochondrial fraction," a striking difference is observed between the distribution of hexokinase in normal and transformed cells. Significantly, particulate hexokinase found in rat brain, kidney, or liver consistently distributes with nonmitochondrial membrane markers whereas the particulate hexokinase of highly glycolytic hepatoma cells distributes with outer mitochondrial membrane markers. These studies indicate that within normal tissues hexokinase binds preferentially to nonmitochondrial receptor sites but upon transformation of such cells to yield poorly differentiated, highly malignant tumors, the overexpressed enzyme binds preferentially to outer mitochondrial membrane receptors. These studies, taken together with the well-known observation that, once solubilized, the particulate hexokinase from a normal tissue can bind to isolated mitochondria, are consistent with the presence in normal tissues of at least two different types of particulate receptors for hexokinase with different subcellular locations. A model which explains this unique transformation-dependent shift in the intracellular location of hexokinase is proposed.

Animals↗

PubMed2005

Involvement of porin N,N-dicyclohexylcarbodiimide-reactive domain in hexokinase binding to the outer mitochondrial membrane.

The proportion of hexokinase that is bound to the outer mitochondrial membrane is tissue specific and metabolically regulated. This study examined the role of the N,N-dicyclohexylcarbodiimide-binding domain of mitochondrial porin in binding to hexokinase 1. Selective proteolytic cleavage of porin protein was performed and peptides were assayed for their, effect on hexokinase I binding to isolated mitochondria. Specificity of DCCD-reactive domain binding to hexokinase I was demonstrated by competition of the peptides for porin binding sites on hexokinase as well as by blockage hexokinase binding by N,N-dicyclohexylcarbodiimide. One of the peptides, designated as 5 kDa (the smallest of the porin peptides, which contains a DCCD-reactive site), totally blocked binding of the enzyme to the mitochondrial membrane, and significantly enhanced the release of the mitochondrially bound enzyme. These experiments demonstrate that there exists a direct and specific interaction between the DCCD-reactive domain of VDAC and hexokinase I. The peptides were further characterized with respect to their effects on certain functional properties of hexokinase I. None had any detectable effect on catalytic properties, including inhibition by glucose 6-phosphate. To evaluate further the outer mitochondrial membrane's role in the hexokinase binding, insertion of VDAC was examined using isolated rat mitochondria. Preincubation of mitochondria with purified porin strongly increases hexokinase I binding to rat liver mitochondria. Collectively, the results imply that the high hexokinase-binding capability of porin-enriched mitochondria was due to a quantitative difference in binding sites.

Animals↗

PubMed1988

Purification, properties, and evidence for two subtypes of human placenta hexokinase type I.

In human placenta 85% of total hexokinase activity (EC 2.7.1.1) was found in a soluble form. Of this, 70% is hexokinase type I while the remaining 30% is hexokinase type II. All the bound hexokinase is type I. Soluble hexokinase I was purified 11,000-fold by a combination of ion-exchange chromatography, affinity chromatography, and dye-ligand chromatography. The specific activity was 190 units/mg protein with a 75% yield. The enzyme shows only one band in nondenaturing polyacrylamide gel electrophoresis that stains for protein and enzymatic activity; however, two components (with Mr 112,000 and 103,000) were constantly seen in sodium dodecyl sulfate-gel electrophoresis. Many attempts were made to separate these two proteins under native conditions; however, only one peak of activity was obtained when the enzyme was submitted to gel filtration (Mr 118,000), preparative isoelectric focusing (pI 5.9), anion-exchange chromatography, hydroxylapatite chromatography, and affinity chromatography on immobilized dyes and immobilized glucosamine. The high and low molecular weight hexokinases show the same isoelectric point under denaturing conditions as determined by two-dimensional gel electrophoresis. Each hexokinase subtype was obtained by preparative sodium dodecyl sulfate electrophoresis followed by electroelution. Monospecific antibodies raised in rabbits against electroeluted high and low molecular weight hexokinases were not able to recognize the native enzymes but each of them detected both hexokinases on immunoblots. Amino acid compositions and peptide mapping by limited proteolysis of the high and low molecular weight hexokinases were also performed and suggested a strong homology between these two subtypes of human hexokinase I.

Centrifugation, Density Gradient↗

PubMed1985

In vitro synthesis of rat brain hexokinase.

Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been synthesized in the rabbit reticulocyte lysate system directed by poly(A)+ mRNA isolated from rat brain. Identification of the in vitro synthesis product as hexokinase was based on its immunoprecipitation with anti-hexokinase serum as well as the generation of identical peptide maps after partial cleavage of the in vitro product and authentic hexokinase with Staphylococcus aureus V8 proteinase or chymotrypsin. The in vitro product and authentic hexokinase were indistinguishable in molecular weight (SDS-gel electrophoresis); thus, despite the fact that, in situ, much of the hexokinase in brain is found in association with mitochondria, it is not synthesized in the form of a higher molecular weight precursor as is characteristic of other mitochondrial proteins. This is in accord with the view that hexokinase is best considered as a classical 'soluble' enzyme which is capable of exhibiting reversible association with mitochondria. The in vitro product cochromatographs (during anion-exchange HPLC) with authentic hexokinase previously shown to have a blocked (presumably acetylated) N-terminus; this procedure is capable of resolving the N-terminally blocked form of the enzyme from a partially proteolyzed form having a free N-terminal amino group. Thus the in vitro product is apparently N-acetylated by an enzyme system previously shown to be present in reticulocyte lysates. A significant fraction of the in vitro synthesized hexokinase attained a conformation characteristic of the native enzyme as judged by the observations that it could be immunoprecipitated by monoclonal antibodies recognizing the native enzyme but not by antibodies recognizing denatured hexokinase, and limited tryptic cleavage of the in vitro product gave fragments identical to those seen with the native enzyme and thought to reflect the organization of structural domains in that enzyme. However, based on these same criteria, the majority of the hexokinase synthesized in vitro appears to exist in a folding state that is not identical to that of either the fully denatured or native enzyme.

Animals↗

PubMed1997

Molecular bases of hexokinase deficiency.

Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1; HK) deficiency is a rare disease where the predominant clinical effect is nonspherocytic hemolytic anemia. We have previously shown that the only patient for which hexokinase deficiency has been so far investigated at molecular level is a double heterozygote carrying a T1667 --> C substitution on one HK type I allele and a 96 bp deletion (concerning nucleotides 577 to 672 in the HK cDNA sequence) in the other allele. To investigate whether these mutations found in the patient with the hexokinase variant referred to as 'HK-Melzo' could be associated with hexokinase deficiency, we have expressed in E. coli the wild-type human hexokinase type I and two different mutants carrying the T --> C nucleotide substitution at position 1667 and the nt 577-672 deletion, respectively. Wild-type human recombinant hexokinase is expressed in bacterial cells as a soluble catalytically active enzyme that, upon purification to homogeneity, exhibited the same kinetic properties of human placenta hexokinase type I. Both mutant hexokinases were also expressed as soluble recombinant proteins under the same conditions, but they showed an impaired catalytic activity with respect to the wild-type enzyme. In particular, the T1667 --> C substitution, causing the amino acid change from Leu529 to Ser, is responsible for the complete loss of the hexokinase catalytic activity, while the 96 bp deletion causes a drastic reduction of the hexokinase activity. Taken together, both mutations explain the hexokinase deficiency found in the patient with the 'HK-Melzo' variant.

Amino Acid Sequence↗

PubMed1970

Cerebral-cortex hexokinase. Comparison of properties of solubilized mitochondrial and cytoplasmic activities.

1. Cerebral-cortex mitochondria, after purification by using high-density sucrose solutions, were extracted with Triton X-100. The total hexokinase activity of the intact mitochondria was increased by 50-80% in the Triton extracts. 2. Triton X-100 was removed from mitochondrial extracts by a combination of ammonium sulphate fractionation and DEAE-cellulose chromatography. Mitochondrial hexokinase remained soluble after removal of extractant. 3. The behaviour of solubilized mitochondrial hexokinase was compared with soluble cytoplasmic hexokinase from the same samples of cerebral cortex on identical columns of DEAE-cellulose. Two peaks were eluted from each source of hexokinase. The distribution between hexokinase peaks was similar for the two sources. Peak I (approx. 80% of the total hexokinase) from each was eluted at identical concentrations of potassium chloride and slight differences were observed in the elution profiles for peak II. 4. The purified mitochondrial hexokinase showed the following kinetic properties: peak I, K(m)(ATP) 0.60mm, K(m)(glucose) 0.042mm; peak II, K(m)(ATP) 0.66mm, K(m)(glucose) 0.043mm. The purified cytoplasmic hexokinase Michaelis constants were: peak I, K(m)(ATP) 0.56mm, K(m)(glucose) 0.048mm; peak II, K(m)(ATP) 0.68mm, K(m)(glucose) 0.062mm. 5. Although no significant differences between mitochondrial and cytoplasmic hexokinases were noted in chromatographic behaviour or in the kinetic properties studied, the purified mitochondrial enzyme was activated slightly (approx. 20%) by Triton X-100, in contrast with the cytoplasmic enzyme, which was not affected. 6. The results, taken to indicate basic similarity between mitochondrial and cytoplasmic hexokinases, are discussed in relation to the role of the two sources of enzyme in the metabolism of the tissue.

Animals↗