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PubMed1985

Acidic phospholipids may inhibit rat brain hexokinase by interaction at the nucleotide binding site.

Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1) is inhibited by acidic phospholipids such as phosphatidylinositol, phosphatidylserine, and cardiolipin. Several aspects of this inhibition are atypical when compared to inhibition by established reversible inhibitors of this enzyme such as the product, Glc-6-P. Maximal inhibition is attained rather slowly (approximately 30 min at 22 degrees C), and is not reversed by simple dilution of the enzyme-lipid mixture. Ligands such as ATP or Glc-6-P can protect the enzyme against inhibition by acidic phospholipids; addition of protective ligands after mixing of enzyme and lipids does not, however, reverse inhibition that occurred prior to ligand addition. Inhibition can be prevented but not reversed by elevated (0.1-0.2 M) [NaCl], indicating a probable role for electrostatic forces in the interaction of lipid with enzyme. Greater inhibition is seen at 22 degrees C than at 3-4 degrees C, suggesting that hydrophobic interactions may also be involved. It is suggested that acidic phospholipids inhibit brain hexokinase by binding at the nucleotide-binding site of the enzyme. The effectiveness of ATP (or the ATP analog, Cibacron Blue) in protecting against inhibition by acidic phospholipids is attributed to direct competition between ATP and the phospholipid for a common binding site. The effectiveness of Glc-6-P (or analogs) in preventing the inhibition is attributed to a conformational change, induced by the binding of this ligand, which prevents binding of ATP or acidic phospholipids to the enzyme. The pH dependency of the inhibition has suggested involvement of the protonated form of a dissociable group (pK approximately 7) on the enzyme in the interaction with acidic phospholipids; this may be the histidyl residue implicated by Solheim and Fromm [Biochemistry 19, 6074-6080 (1984)] in the binding of ATP to brain hexokinase. Structural similarities in the nucleotide-binding sites of several nucleotide-binding enzymes suggest that similar inhibition by acidic phospholipids may be seen with other enzymes of this type; there are already some reports to this effect.

Adenosine Triphosphate↗

PubMed1985

Allosteric inhibition of brain hexokinase by glucose 6-phosphate in the reverse reaction.

A study of the reverse reaction of rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been performed using a photometric method based on a mutarotase-glucose oxidase-peroxidase-chromogen system to trap and visualize glucose, plus a glycerol kinase-glycerol system to trap ATP. Glucose 6-phosphate or 2-deoxyglucose 6-phosphate were used as phosphoryl donors at different concentrations of ADP. Variation of glucose 6-phosphate concentrations resulted in a biphasic curve from which apparent Km and Ki values of ca. 0.2 mM were calculated. In contrast, variation of 2-deoxyglucose 6-phosphate concentrations resulted in Michaelian kinetics with an apparent Km of 2 mM. The Km value for MgADP was 16 mM irrespective of the nature and concentration of the hexose 6-phosphate substrate. These results are fully consistent with an allosteric site for glucose 6-phosphate as an explanation for the inhibition of animal hexokinases by glucose 6-P and further indicate that the maximal rate is the parameter affected. From these observations and previous knowledge, the possible occurrence in animal hexokinases of a regulatory site for ATP to account for the competition between glucose 6-phosphate and ATP in the forward reaction is postulated.

Adenosine Diphosphate↗

PubMed1986

Rat brain hexokinase: location of the substrate nucleotide binding site in a structural domain at the C-terminus of the enzyme.

8-Azido-ATP serves as a substrate for rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1). Irradiation of hexokinase in the presence of this photoactivatable ATP analog results in inactivation of the enzyme. ATP and hexose 6-phosphates (Glc-6-P, 1,5-anhydroglucitol-6-P) previously shown to competitively inhibit nucleotide binding protect the enzyme from photoinactivation; other hexose 6-phosphates do not. Hexoses (Glc, Man) previously shown to enhance nucleotide binding also protect against photoinactivation; other hexoses do not. These effects of hexoses and hexose 6-phosphates can be interpreted in terms of the conformational changes previously shown to result from the binding of these ligands and to influence the characteristics of the nucleotide binding site (M. Baijal and J. E. Wilson (1982) Arch. Biochem. Biophys. 218, 513-524). Limited tryptic cleavage of the enzyme produces three major fragments having molecular weights of about 10K, 40K, and 50K, and thought to represent major structural domains within the enzyme (P. G. Polakis and J. E. Wilson (1984) Arch. Biochem. Biophys. 234, 341-352). Tryptic cleavage of the enzyme, photoinactivated in the presence of 14C-labeled azido-ATP, discloses prominent labeling of the 10K and 40K domains, which are known to originate from the N- and C-terminal regions, respectively. Labeling of the 40K domain is influenced by ligands in a manner that corresponds to the effectiveness of these ligands in protecting against photoinactivation whereas labeling of the 10K domain is not affected by these same ligands. It is concluded that the 40K domain includes the binding site for nucleotide substrates. More refined two-dimensional peptide mapping techniques demonstrate that the predominant site of labeling is a peptide segment, molecular weight approximately 20K, that is located in the central and/or C-terminal region of the 40K domain. Labeling of the 10K domain is attributed to nonspecific interaction of azido-ATP with the hydrophobic sequence shown to be located at the N-terminus of brain hexokinase (P. G. Polakis and J. E. Wilson (1985) Arch. Biochem. Biophys. 236, 328-337).

Adenosine Triphosphate↗

PubMed1987

Subfractionation of the outer membrane of rat brain mitochondria: evidence for the existence of a domain containing the porin-hexokinase complex.

Isolated and well-characterized rat brain nonsynaptic mitochondria were subfractionated by digitonin. Antibodies to a uniquely outer membrane protein, porin, have allowed us to use this protein for the first time as an outer membrane marker in brain. Hexokinase, which binds to porin, was also measured. Based upon the sequential release of these and other marker enzymes with increasing concentrations of digitonin, three outer membrane domains have been identified. Two populations of porin were found by this treatment. The most plausible interpretation of our results is that the two porin populations exist in different membrane environments with regard to cholesterol. One of these populations binds most of the hexokinase and appears to be associated with the inner membrane. It is proposed that the porin-hexokinase complex in brain mitochondria is located in a cholesterol-free membrane domain together with inner membrane components. This domain has the features of contact points which have been visualized by electron microscopy.

Animals↗

PubMed1988

Rat brain hexokinase: the hydrophobic N-terminus of the mitochondrially bound enzyme is inserted in the lipid bilayer.

Mitochondrially bound rat brain hexokinase was labeled with the photoactivatable reagent, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. This highly hydrophobic reagent is strongly partitioned into the hydrophobic environment of the membrane core, and thus selectively labels segments of a protein that penetrate this region of the membrane. Labeling of hexokinase was shown to be restricted to the N-terminal region of the molecule. Approximately 80% of the radiolabel was removed by treatment of the enzyme with chymotrypsin, which preferentially cleaves a hydrophobic 9-residue sequence at the extreme N-terminus of the enzyme, and it is considered likely that the remaining 20% was associated with two additional hydrophobic residues, immediately adjacent to this segment but not susceptible to cleavage by chymotrypsin. Labeling of the enzyme was shown to be dependent on maintenance of the association with the membrane. These results are consistent with a model in which binding of hexokinase involves insertion of an 11-residue hydrophobic N-terminal "tail," possibly existing in alpha-helical secondary structure, into the hydrophobic core of the membrane.

Affinity Labels↗

PubMed1989

Synthesis and targeting of hexokinase to mitochondria in hepatoma cells.

The synthesis and turnover of hexokinase has been measured in Zajdela hepatoma ascites cells labeled for short periods with [35S]methionine. Digitonin fractionation of the labeled cells into a soluble and a membrane fraction showed that only a small part of the newly labeled hexokinase is transferred to mitochondrial binding sites. The soluble enzyme disappears, however, with a half-life of less than 2 h. Glucose had no effect on the stability of the soluble enzyme in intact cells. Our experiments suggest that Zajdela cell hexokinase is synthesized in excess of binding sites and that the excess enzyme is not stable.

Animals↗

PubMed1989

Isolation and characterization of the discrete N- and C-terminal halves of rat brain hexokinase: retention of full catalytic activity in the isolated C-terminal half.

Selective stabilization of either the N- or C-terminal half (by ligands binding to these regions) of rat brain hexokinase against partial denaturation with guanidine hydrochloride and subsequent digestion with trypsin has provided a means for isolating these regions, referred to as N fragment and C fragment, respectively, in quantities adequate for characterization. The N fragment (mol wt 52 kDa) is devoid of catalytic activity. In contrast, the C fragment (mol wt 51 kDa) has a specific activity of about 110 U/mg, nearly twice that (60 U/mg) of the intact 100-kDa enzyme, indicating that the kappa cat is virtually identical for both species. Unlike the parent enzyme, the C fragment is quite sensitive to inhibition by Pi (competitive vs ATP, noncompetitive vs Glc); sulfate and arsenate, but not acetate, inhibit with effectiveness similar to that seen with Pi. The Glc-6-P analog, 1,5-anhydroglucitol-6-P, also inhibits the C fragment (competitive vs ATP, uncompetitive vs Glc). Both N and C fragments bind to Affi-Gel Blue, an affinity matrix bearing a covalently attached analog of ATP, and are eluted by hexose 6-phosphates competitive with nucleotide binding to the parent enzyme. Based on the ability of various hexoses and hexose 6-phosphates (and analogs) to protect against guanidine-induced denaturation and subsequent proteolysis it is concluded that both fragments contain discrete sites for hexoses and hexose 6-phosphates, with specificities resembling those seen for the binding of these ligands to the parent enzyme. Synergistic interactions between the hexose and hexose-6-P binding sites, previously seen with the parent enzyme, are also observed with the C fragment but not the N fragment. The existence of binding sites for hexoses and hexose 6-phosphates on both halves conflicts with previous binding studies demonstrating a single hexose binding site and a single hexose 6-phosphate binding site on the intact 100-kDa enzyme, leading to the conclusion that one of each pair of sites must be latent in the intact enzyme, becoming manifest only in the isolated discrete halves. Several investigators have previously suggested that the 100-kDa mammalian hexokinases evolved by duplication and fusion of a gene encoding an ancestral 50-kDa Glc-6-P-insensitive hexokinase, similar to the present-day yeast enzyme, with sensitivity to Glc-6-P resulting from evolution of a duplicated catalytic site into a regulatory site.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1990

Tetrameric structure of mitochondrially bound rat brain hexokinase: a crosslinking study.

Rat brain hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) was derivatized with sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3'-dithiopro pionate (SAND), a photosensitive and cleavable crosslinking agent. The catalytic activity and mitochondrial binding properties of the enzyme were only marginally affected by reaction with SAND. When the derivatized enzyme was bound to liver mitochondria, photolysis resulted in extensive formation of a single crosslinked species with estimated molecular mass 460 kDa. This was determined to contain only hexokinase and thus represents a tetramer of the 116 kDa (apparent molecular mass in gel system used) monomeric enzyme. Although small amounts of tetramer were detected after photolysis of relatively high concentrations of derivatized enzyme in free solution, tetramer formation was greatly enhanced when the enzyme was bound to mitochondria. No evidence of dimeric or trimeric structures was seen even when only a small fraction of the available binding sites on the mitochondrial membrane were occupied. It is thus concluded that tetramer formation is closely linked with binding of the enzyme to the outer mitochondrial membrane and, more specifically, to the pore structure through which metabolites traverse this membrane. It is speculated that a tetrameric structure surrounding the mitochondrial pores may facilitate interactions between the hexokinase reaction and oxidative phosphorylation, mediated by the adenine nucleotides which are common intermediates in these reactions.

Animals↗

PubMed1991

Disposition of mitochondrially bound hexokinase at the membrane surface, deduced from reactivity with monoclonal antibodies recognizing epitopes of defined location.

A panel of monoclonal antibodies against rat brain hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) has been employed to investigate the orientation of the mitochondrially bound enzyme on the mitochondrial surface. Based on their ability to immunoprecipitate truncated forms of the protein, obtained by in vitro translation of truncated versions of the mRNA, the epitopes for seven monoclonal antibodies were mapped to regions consisting of 20-50 amino acid residues within the sequence of the N-terminal half of the enzyme. There is extensive sequence similarity between the N- and C-terminal halves of this enzyme, which is thought to have evolved by a process of gene duplication and fusion. However, these antibodies react selectively with epitopes in the N-terminal half, and thus epitopic regions for several of these antibodies could be further defined by eliminating from consideration regions showing substantial sequence similarity with the C-terminal half. The epitope for one of the monoclonal antibodies, designated 4D4, was shown to involve the extreme N-terminus of the enzyme; selective proteolytic modification of this region resulted in loss of immunoreactivity. Relative location of epitopes for three other antibodies, designated 2B, 1C5, and 4C5, within a 20-residue segment was deduced from effects of modifying sulfhydryl residues within this segment on immunoreactivity. Thus, by a combination of sequence analysis and experimental methods, the epitopes for these seven antibodies could be localized to defined regions within the overall sequence. The ability of these antibodies to prevent binding of hexokinase to mitochondria, and their ability to recognize the mitochondrially bound enzyme, provided a basis for assessing the relative proximity of the corresponding epitopes to the mitochondrial surface when the enzyme was bound. The disposition of the bound enzyme on the mitochondrial surface was deduced by relating these results to the proposed structure for brain hexokinase.

Amino Acid Sequence↗

PubMed1992

Interaction of mitochondrially bound rat brain hexokinase with intramitochondrial compartments of ATP generated by oxidative phosphorylation and creatine kinase.

Previous work led to the conclusion that, during oxidative phosphorylation, mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from rat brain was dependent on intramitochondrially compartmented ATP as substrate. The present study demonstrated that, when oxidative phosphorylation was functioning concurrently, mitochondrial creatine kinase could also generate intramitochondrial ATP serving as substrate for hexokinase. In the absence of concurrent oxidative phosphorylation, the kinetics of glucose phosphorylation with ATP generated by creatine kinase were not consistent with the supply of ATP from a saturable intramitochondrial compartment as formed during oxidative phosphorylation. Evidence for intramitochondrially compartmented ATP, generated by creatine kinase, was obtained; this was distinct from compartmented ATP generated by oxidative phosphorylation in terms of kinetics of generation of the compartment and its capacity, sensitivity to release by carboxyatractyloside, and sensitivity to disruption by digitonin. That oxidative phosphorylation did induce a dependence on intramitochondrial ATP as a substrate was further indicated by the observation that, although the initial rate of glucose phosphorylation by mitochondrial hexokinase depended on the extramitochondrial concentration of ATP present at the time oxidative phosphorylation was initiated, a final steady state rate of glucose phosphorylation was attained that was independent of extramitochondrial ATP levels. These and previous results emphasize the probable importance of nucleotide compartmentation in regulation of cerebral glycolytic and oxidative metabolism.

Adenosine Diphosphate↗

PubMed1976

Glucose 6-phosphate-dependent binding of hexokinase to membranes of ascites tumor cells.

A pH-dependent, saturable binding of hexokinase isozyme I from Ehrlich ascites carcinoma to plasma membrane and microsome preparations from the same tissue is demonstrated. This binding is enhanced by glucose 6-phosphate and may be considered as the sum of a glucose 6-phosphate-dependent binding and an independent binding. The half saturation concentration of hexokinase is about 0.4 unit per ml for both types of binding, and a maximal binding of 0.5-2.0 units per mg membrane protein is observed for both, although the pH optimum of the independent binding (5.4) is lower than that of the dependent binding (5.9). The half saturation concentration of glucose 6-phosphate required for the dependent binding is 0.05 mM at pH 6.1. 2-Deoxyglucose 6-phosphate competatively reverses the effect of glucose 6-phosphate on binding but does not diminish its inhibition of hexokinase activity.

Animals↗

PubMed1975

Complementarity in the regulation of phosphoglucomutase, phosphofructokinase and hexokinase; the role of glucose 1,6-bisphosphate.

ATP and citrate, the well known inhibitors of phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), were found to inhibit the activities of the multiple forms of phosphoglucomutase (alpha-D-glucose 1,6-bisphosphate: alpha-D-glucose 1-phosphate phosphotransferase, EC 2.7.5.1) from rat muscle and adipose tissue. This inhibition could be reversed by an increase in the glucose 1,6-bisphosphate (Glc-1,6-P2) concentration. Other known activators (deinhibitors) of phosphofructokinase, viz. cyclic AMP, AMP, ADP or Pi, had no direct deinhibitory action on the ATP or citrate inhibited multiple phosphoglucomutases. Cyclic AMP and AMP, could however lead indirectly to deinhibition of the phosphoglucomutases, by activating phosphofructokinase which catalyzes the ATP-dependent phosphorylation of glucose 1-phosphate to form Glc-1,6-P2, the la-ter then released the multiple phosphoglucomutases from ATP or citrate inhibition. The Glc-1,6-P2 was also found to exert a selective inhibitory effect on hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) type II, the predominant form in skeletal muscle. This selective inhibition by Glc-1,6-P2 was demonstrated on the multiple hexokinases which were resolved by cellogel electrophoresis or isolated by chromatography on DEAE-cellulose. Based on the in vitro studies it is suggested that during periods of highly active epinephrine-induced glycogenolysis in muscle, the Glc-1,6-P2, produced by the cyclic AMP-stimulated reaction of phosphofructokinase with glucose 1-phosphate, will release the phosphoglucomutases from ATP or citrate inhibition, and will depress the activity of muscle type II hexokinase.

Adenosine Triphosphate↗

PubMed1979

Comparison of type I hexokinases from pig heart and kinetic evaluation of the effects of inhibitors.

Type I hexokinase (ATP:D-hexose 6-phospotransferase, EC 2.7.1.1) of porcine heart exists in two chromatographically distinct forms. These do not differ significantly in size, electrophoretic mobility at pH 8.6 or kinetic properties. Both forms obey a sequential mechanism and are potently inhibited by glucose 6-phosphate. In contrast to observations of type I hexokinase from brain, inhibition by glucose 6-phosphate is not relieved by inorganic phosphate. Under most conditions, low concentrations of phosphate (less than 10 mM) have little effect on the kinetic behaviour of the enzyme but at higher concentrations this ligand is an inhibitor. Mannose 6-phosphate inhibits in a manner analogous to glucose 6-phosphate but the Ki is much greater. In view of the similarity of the kinetic parameters governing phosphorylation of mannose and glucose, this difference in affinity for the inhibitor site is seen as consistent with the existence of a separate regulatory site on the enzyme. MgADP inhibits hexokinase but behaves as a normal product inhibitor and inhibition is competitive with respect to MgATP and non-competitive with respect to glucose.

Adenine Nucleotides↗

PubMed1980

Action of oxidized and reduced glutathione on rabbit red blood cell hexokinase.

Reduced glutathione at 1 mM concentration is able to mantain rabbit red blood cell hexokinase (EC 2.7.1.1) in the reduced state with fully catalytic activity. At higher concentrations a marked inhibition is observed. In contrast, oxidized glutathione is a strong inhibitor of reduced erythrocyte hexokinase at all the concentration studied. Inactivation experiments show that some sulfhydryl groups reacting with oxidized glutathione are responsible for the enzyme inactivations. These findings suggest a cellular inter-relationship between redox and energetic metabolism coupled through glutathione at the hexokinase level.

Adenosine Triphosphate↗

PubMed1985

Evidence that transcription of the hexokinase gene is increased in a rapidly growing rat hepatoma.

The level of hexokinase-specific mRNA in rat liver and in rat ascites hepatoma (Zajdela) was assessed by translating free polysomes from both cell types in a reticulocyte lysate. Translation of hexokinase occurred with approximately a 10 fold higher frequency in tumor cells than in liver, suggesting that transcriptional activation is in part responsible for increased levels of hexokinase in tumor.

Animals↗

PubMed1988

Insulin effect on [14C]-valine incorporation and its relation to hexokinase activity in developing brain.

Using minced brain cortex from fetal and postnatal rats, we studied the incorporation of [14C]-valine into protein in the presence of insulin. We also assayed the "particle bound" and soluble hexokinase in these tissues. Insulin significantly stimulated the incorporation of [14C]-valine into brain proteins from fetal stage upto 2 days of life. After this period the insulin effect was minimal, with no effect by day 5. The "particle bound" (40,000g pellet) brain hexokinase, on the other hand, remained low till about 2 days of life and then increased to almost adult level by 5 days. Our results show that there is an inverse relation between this anabolic effect of insulin and the "particle bound" hexokinase activity in the cortex of developing rat brain.

Aging↗

PubMed1991

Glucose utilization by tumor cells: a post-translational modification of mitochondrial hexokinase may play a regulatory role.

In Northern blot analysis of a series of tumor cell lines a single hexokinase mRNA species of 4.3 Kb was detected. Detailed examination of one such line, the rat AS-30D hepatoma, revealed that two mitochondrial species of hexokinase are present with a molecular mass of 115 and 107 KDa. The smaller of the two species is 4-fold more active than the larger. Only the larger, less active species is detected in the well differentiated H-35 rat hepatoma cell line which exhibits a lower glucose catabolic rate. These results suggest that a post-translational proteolytic event may play a central role in regulating the glucose utilization capacity of tumor cells by modulating the relative levels of high and low activity forms of hexokinase.

Animals↗

PubMed1983

Age-dependent changes in glucose 1,6-bisphosphate levels and in the activities of glucose 1,6-bisphosphatase, and particulate hexokinase and 6-phosphogluconate dehydrogenase in rat skin.

The levels of glucose 1,6-bisphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, changed in rat skin during growth: Glc-1,6-P2 increased during the first week of age, and thereafter was dramatically reduced during maturation. The activity of glucose 1,6-bisphosphatase, the enzyme that degradates Glc-1,6-P2, changed with age in an invert manner as compared to the changes in Glc-1,6-P2. These findings suggest that the age dependent changes in this enzyme's activity may account for the changes in intracellular Glc-1,6-P2 concentration. The age-related changes in Glc-1,6-P2 were accompanied by concomitant changes in the activities of particulate (mitochondrial) hexokinase and 6-phosphogluconate dehydrogenase, the two enzymes known to be inhibited by Glc-1,6-P2. The activities of both these enzymes in the soluble fraction were not changed with age. The particulate enzymes were more susceptible to inhibition by Glc-1,6-P2 than the soluble activities, which may explain why only the particulate, but not the soluble activities, correlated with the age-dependent changes in tissue Glc-1,6-P2. These results suggest that the changes in particulate hexokinase and 6-phosphogluconate dehydrogenase resulted from changes in intracellular concentration of Glc-1,6-P2. The marked reduction in Glc-1,6-P2 during maturation, accompanied by activation of mitochondrial hexokinase and 6-phosphogluconate dehydrogenase, may reflect an enhancement in skin metabolism during growth.

Aging↗