Preparation of hexokinase isoenzyme II with expressed adsorption properties

The isolation of the native hexokinase isozyme II possessing a high adsorptive capacity is described. This property underlies the adsorption mechanism responsible for the control of the hexokinase activity in the cell and is realized only under conditions of the structural integrity of the enzyme. The latter is due, primarily, to the functional state of the specific adsorption domain which provides the specific interaction of hexokinase isozyme II with biological membranes. The criteria of nativity of skeletal muscle hexokinase were elaborated. A procedure for obtaining highly purified native hexokinase isozyme II from rat skeletal muscle was developed.     

Functional organization and evolution of mammalian hexokinases: mutations that caused the loss of catalytic activity in N-terminal halves of type I and type III isozymes.

Mammalian hexokinases are believed to have evolved from a 100-kDa hexokinase which itself is a product of duplication and fusion of an ancestral gene encoding a 50-kDa glucose 6-phosphate-sensitive hexokinase. Type II hexokinase has been shown to possess two distinct functional active sites, one in each half, which functionally resemble the original 100-kDa hexokinase, whereas type I and III isozymes possess only one active site in the C-terminal halves. This study was conducted to identify which mutations caused the loss of catalytic activity in the N-terminal halves of type I and III isozymes. Arg 174 and Ser 447 in type I isozyme and Asp 244 in type III isozyme are speculated to be the cause, because they reside adjacent to the "catalytic" site and corresponding residues, Gly 174, Asp 447, and Gly 231, are conserved in the N-terminal half of type II isozyme as well as all other 50-kDa units that possess catalytic activity. Mutations G174R and D447S in the N-terminal half of type II isozyme reduced specific activity by approximately 79 and 57%, respectively. Therefore, neither mutation alone can account for the inactivation of the N-terminal active site in type I isozyme. Either mutation, G174R or D447S, had moderate effects on Michaelis constants, K(m), for glucose and ATP. Mg(2+). Intriguingly, mutation D447S introduced a novel inhibition by unchelated ATP (K(i) = 68 microM ATP, competitive vs ATP. Mg(2+)) to the N-terminal active site of type II isozyme. Mutation G231D caused instability to type II hexokinase and near complete loss of catalytic activity (95%), suggesting that mutation G231D not only hinders catalysis at the N-terminal active site but also leads to structural instability in type II hexokinase.     

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.     

Regulation of glucose metabolism in rat lung: Subcellular distribution,isozyme pattern,and kinetic properties of hexokinase

The subcellular distribution and isozyme pattern of hexokinase in rat lung were studied. Of the total hexokinase activity of lung, one-third was bound to mitochondria and one-third of the mitochondrial activity was in a latent form. The overt-bound mitochondrial hexokinase was specifically solubilized by physiological concentrations of glucose 6-phosphate and ATP. Inorganic phosphate partially prevented the solubilization by glucose 6-phosphate (Glc 6-P), whereas Mg²⁺ ions promoted rebinding of the solubilized enzyme to mitochondria. Thus, the distribution of hexokinase between soluble and particulate forms in vivo is expected to be controlled by the relative concentrations of Glc 6-P, ATP, P_i, and Mg²⁺. Study of the isozyme pattern showed that hexokinase types I, II, and III constitute the cell-sap enzyme of lung. The overt and latent hexokinase activities could be separately isolated by successive treatments of mitochondria with Glc 6-P and Triton X-100. The overt-bound activity consisted primarily of hexokinase type I, with a small proportion of type II isozyme. The latent activity, on the other hand, exclusively consisted of type I isozyme. Type I hexokinase, the predominant isozyme in lung, was strongly inhibited by intracellular concentration of Glc 6-P and this inhibition was counteracted by P_i. The bound form of hexokinase exhibited a significantly higher apparent K_i for Glc 6-P inhibition and a lower apparent K_m for ATP as compared to the soluble form. Thus, the particulate form of hexokinase is expected to promote glycolysis and may provide a mechanism for the high rate of aerobic glycolysis in lung.     

The effect of insulin on the catalytic efficacy of rat skeletal muscle hexokinase isoenzyme II]

The effect of insulin on the intracellular localization of rat skeletal muscle hexokinase isozyme II (hexokinase II) was studied in vivo. It was found that after injection of the hormone the glucose concentration in the muscle gradually increases in parallel with the hexokinase II redistribution between the cytosol and the mitochondrial fraction in the direction of the bound form of the enzyme. This effect of insulin is due to glucose, an indispensable participant of the complex formation between the enzyme and the mitochondrial membrane. It was shown that the effect of glucose as a hexokinase II adsorbing reagent is a highly specific one. The hexokinase II binding to mitochondria in the presence of glucose is accompanied by changes in some kinetic properties of the enzyme. A kinetic analysis of catalytic efficiency of the free and bound hexokinase II forms revealed that the catalytic efficiency of hexokinase II within the composition of the enzyme-membrane complex exceeds by two orders of magnitude that of the free enzyme. The data obtained are discussed in the framework of an adsorption mechanism of hexokinase activity regulation in the cell.     

Hexokinases of tobacco leaves: influence of plant age on particulate and soluble isozyme composition

Changes in hexokinase particulate and soluble isozyme composition and activities in leaves of 65- and 115-d-old tobacco plants were determined by ion exchange chromatography on DEAE cellulose. During plant ageing, the activities of glucose and of fructose phosphorylating isozymes of particulate hexokinase decreased to 9.9 and 9.2 % of initial value, respectively. The activity of soluble hexokinase decreased to a lesser extent: that of glucose phosphorylating isozyme to 49.8 % and of fructose phosphorylating isozyme to 37.8 %. The activity of soluble fructokinase isozyme dropped to 34.8 %. Thus also the ratio of particulate and soluble isozymes was dependent on the age of leaf tissue. This revised version was published online in August 2006 with corrections to the Cover Date.     

Complete amino acid sequence of the type II isozyme of rat hexokinase, deduced from the cloned cDNA: comparison with a hexokinase from novikoff ascites tumor

The 917-residue amino acid sequence of the Type II isozyme of rat hexokinase has been deduced from the nucleotide sequence of cloned cDNA. The sequences of 197 nucleotides in the 5' untranslated region and 687 bases of the 3' untranslated region have also been determined. A region of overlap between two discrete cDNA clones was confirmed by isolation and sequencing of a genomic DNA clone that spanned the region. Within this region, the 634-nucleotide coding sequence was divided into three exons, each of 150-250 nucleotides; these results suggest that the gene encoding Type II hexokinase is likely to be quite complex. There is extensive similarity between the sequences of the N- and C-terminal halves of the Type II isozyme, as previously seen with the Type I and III isozymes; this is consistent with the view that these enzymes evolved by a process of gene duplication and fusion. A cDNA encoding the entire C-terminal half of a hexokinase from Novikoff ascites tumor cells was also isolated and found to be identical to a cDNA encoding the corresponding region of the Type II isozyme of skeletal muscle. Northern analysis indicated that a single mRNA, approx 5200 nucleotides in length, encoded both the skeletal muscle and the tumor enzymes. These results do not support previous speculation that the hexokinase isozymes of normal tissue are distinct from those of tumors, and suggest the possibility that post-translational modifications of a single protein species might account for apparent differences between the isozymes of normal and tumor tissues.     

Localization of the type III isozyme of hexokinase at the nuclear periphery.

The distribution of the type III isozyme of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) in rat kidney, liver, spleen, lung, and brain was determined immunohistochemically, using a monoclonal antibody generated against the enzyme purified from rat Novikoff hepatoma.In all tissues, specific cell types exhibited intense staining at the nuclear periphery, as confirmed by analysis using confocal microscopy. Isolated nuclei from kidney or liver were devoid of detectable type III hexokinase, although the enzyme was found in the "soluble" fraction from kidney or liver homogenates; these results suggest that the type III isozyme is associated in a labile manner with the external surface of the nucleus, with this association being disrupted by conventional homogenization and nuclear isolation procedures. The nuclear localization of the type III isozyme contrasts with previously demonstrated association of the type I and II isozymes with mitochondria. The physiological significance of a nuclear localization for the type III isozyme remains unclear. However, it was noted that many of the cells exhibiting prominent nuclear staining for type III hexokinase are endothelial or epithelial cells, suggesting a possible relationship between nuclear type III hexokinase and transport functions which are prominent in such cells.     

On hexokinase isozymes]

Electrophoretic study of hexokinase (HK) associated with the soluble fraction of mouse transplantable hepatoma 22a revealed that almost all bands of HK activities overlapped the bands of glucose-6-P dehydrogenase (G6PDH) activities in the gels. Similar results were obtained for liver, muscle and brain soluble fractions, as well as for various extracts from hepatoma 22a mitochondria and commercial preparation of yeast HK. A single type of HK, which does not overlap G6PDH activity, was located between types I and II (according to the Katzen classification) as a diffuse band of 1 hour manifestation. A possibility of structural organization of glycolytic enzymes in the cell essential for the quantitative estimation of the isozyme pattern is discussed.     

Hexokinase in developing rabbit erythroid cells

The activity and isozyme distribution of hexokinase were studied in bone marrow cells from normal and anemic rabbits seperated by density centrifugation or by unit-gravity sedimentation. The specific activity of the enzyme was found to be about 150-fold higher in the basophilic erythroblasts as compared with the mature circulating erythrocytes. Mos of the falls in hexokinase activity take place whent the cell completes its final division and matures from the polychromatic stage to the orthochromatic stage. Concomitant with this strong decrease in enzyme activity, qualitative as well as quantitative changes in the hexokinase isozymic pattern become apparent. While in the basophilic and polychromatic erythroblasts the only hexokinase isozyme present is hexokinase type I, the orthochromatic cells also contain hexokinase Ib. This last isozymic form, which increases further at the reticulocyte stage, is also present in the circulating reticulocytes but not in mature red blood cells.     

Changing patterns of placental hexokinase isozymes during the course of gestation.

Hexokinase, an enzyme that is capable of regulating the entry of glucose into metabolic sequences, is known to exist in four isozyme forms in a variety of mammalian tissues. Each of these isozymes possesses different kinetic properties, suggesting that they may serve in different regulatory capacities. In addition, the proportions of the four isozymes are characteristically different for various differentiated tissues, presumably reflecting different metabolic capabilities of the tissues.Extracts of rabbit and human placentas from early gestational age and term pregnancies were chromatographed and assayed for hexokinase activity. Four peaks of activity were observed. Elution positions of the four placental hexokinase isozymes were comparable to those from liver; however, the relative proportions were considerably different. In both rabbit and human placentas, the proportion of Type I hexokinase increased during gestation and Type III decreased, while Types II and IV remained essentially unchanged. Implications of the gestational changes for placental carbohydrate metabolism are discussed.     

Compartmentation of hexokinase in human blood cells characterization of soluble and particulate enzymes

The isozyme distribution, kinetic properties and intracellular localization of hexokinase (ADP: D-hexose-6-phosphotransferase, EC 2.7.1.1) were studied in erythrocytes, blood platelets, lymphocytes and granulocytes. Soluble and particulate fractions were separated by a rapid density centrifugation method after controlled digitonin-induced cell lysis. In lymphocytes and platelets the major part of total activity was particle-bound (78 and 88%, respectively). In granulocytes and erythrocytes most of the hexokinase activity was found in the cytosol. All cell types, except granulocytes, contain mainly the type I isozyme. Platelets contain only type I hexokinase, while in lyphocytes a minor amount of type III is present in the soluble fraction (less than 10% of total activity). The major constituent of granulocytes is type III hexokinase (70–80% of total activity), the remaining 20–30% is type I hexokinase. Erythrocytes contain a multibanded type I hexokinase. The substrate affinities of the type I hexokinase do not differ significantly between the different cell types or between soluble, bound and solubilized fractions. Only soluble hexokinase from lymphocytes shows a slightly decreased K_m apparent for glucose (P < 0.05).     

Hexokinase isozyme distribution and regulatory properties in lymphoid cells

The glycolytic enzyme hexokinase is studied in cultured leukemic lymphoblasts, in normal lymphocytes and in lymphoblasts obtained by stimulation of normal lymphocytes with phytohaemagglutinin.Hexokinase activity levels in cultured lymphoblasts and in normal lymphocytes are identical, but somewhat higher levels are found in stimulated lymphocytes. Cultured leukemic lymphoblasts differ in isozyme content in comparison to the other lymphoid cells. Besides hexokinase I, which is detected in all the lymphoid cells, they are characterized by the presence of hexokinase II. The concentration of type II increases during cell growth. Another difference between leukemic lymphoblasts and mature and stimulated lymphocytes is found in the regulatory properties of hexokinase I. Hexokinase I from both normal and stimulated lymphocytes is inhibited by glucose-1,6-diphosphate. This inhibition is decreased in part by addition of inorganic phosphate. Hexokinase I from leukemic lymphocytes, however, is inhibited to a lesser extent by glucose-1,6-diphosphate. Inorganic phosphate has no effect at all on this inhibition.In accordance with these findings a different pattern in the hexokinase I region was detected in electrophoresis with several cell types. The subisozyme hexokinase Ib, which appears to be the phosphate-regulated form, is predominant in lymphocytes, whereas it is present in a minor fraction in the cultured leukemic lymphoblasts. In these cells hexokinase Ic predominates.     

Transport and phosphorylation of hexoses in normal and rous sarcoma virus-transformed chick embryo fibroblasts

Effects of transformation by Rous sarcoma virus on sugar uptake and activity and the subcellular distribution of hexokinase isozymes in chick embryo fibroblasts were examined. Transformation caused a several-fold increase in the maximum velocity for uptake of 2-deoxyglucose without a significant change in K_m. Cytochalasin B (CB), was used to differentiate between the effects of transformation on facilitated diffusion and the nonsaturable (CB-insensitive) mode. Transformation was found to stimulate 2-deoxyglucose transport by both mechanisms, but the increase in transport by the CB-insensitive mode was greater. Transformation enhances the activity of hexokinase, the enhancement being confined to the particulate fraction of the enzyme. Heat-inactivation and electrophoretic mobility studies showed that although hexokinase Type I is the major form in both normal and transformed fibroblasts, there is a significant increase in the proportion of the Type II isozyme in the transformed cells.     

Effect of streptozotocin-induced diabetes on the turnover of hexokinase II in the rat

Skeletal muscle hexokinase II activity and turnover rates were measured in the normal and streptozotocin-induced diabetic rat. Enzyme activity decreases in the diabetic animal relative to the normal rat; however, the specific activity of hexokinase II is essentially the same for the two conditions. No alteration is observed in the relative rate of hexokinase II synthesis in the normal or diabetic rats, but there is a 3-fold increase in the rate of hexokinase II degradation in the latter group of animals. These results suggest that the primary cause of the well-established decrease in hexokinase II activity in skeletal muscle of the diabetic is an increase in the rate of enzyme degradation.     

Inactivation of G1ucose-6-Phosphate Dehydrogenase Isozymes from Human and Pig Brain by Aluminum

Prolonged intake of low levels of aluminum from the drinking water has been found to increase the aluminum content in rat brain homogenates and to reduce the activity of hexokinase and glucose-6-phosphate dehydrogenase (G6PD). To determine the interaction of G6PD with aluminum in the brain, we have recently purified two isozymes of G6PD (isozymes I and II) from human and pig brain. Unlike isozyme I, isozyme II also had 6-phosphogluconate dehydrogenase (6-PGD) activity. We report here that G6PD isozymes I and II from human and pig brain purified to apparent homogeneity are inactivated by aluminum. Aluminum did not affect the 6-PGD activity of isozyme II. The aluminum-inactivated enzyme contained 1 mol of aluminum/mol of enzyme subunit. The protein-bound metal ion was not dissociated by exhaustive dialysis at 4 degrees C against 10 mM Tris-HCl (pH 7.0) containing 0.2 mM EDTA. Preincubation of aluminum with citrate, NADP+, EDTA, NaF, ATP, and apotransferrin protected the G6PD isozymes against aluminum inactivation. However, when the G6PD isozymes were completely inactivated by aluminum, only citrate, NaF, and apotransferrin restored the enzyme activity. The dissociation constants for the enzyme-aluminum complex of the isozymes varied from 2 to 4 microM, as measured by using NaF, a known chelator for aluminum. Inhibition of G6PD by low levels of aluminum further strengthens the suggested role of aluminum toxicity in the energy metabolism of the brain.     

The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae.

Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase II is correlated with glucose repression.     

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

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

Rabbit red blood cell hexokinase:intracellular distribution during reticulocytes maturation

Summary The intracellular localization and isozyme distribution of hexokinase were studied during rabbit reticulocyte maturation and aging. In reticulocytes 50% of the enzyme was particulate while in the mature erythrocytes all the hexokinase activity was soluble. The bound enzyme co-sediments with mitochondria and by column chromatography it was found to be hexokinase Ia. The cytosol of reticulocytes contains hexokinase Ia (38%) and hexokinase Ib (62%) while the mature erythrocytes contain only hexokinase Ia. The amount of bound hexokinase decreases very quickly during cell maturation and aging as was shown by following in vivo reticulocyte maturation or by analysis of hexokinase compartmentation in cells of different ages, obtained by density gradient ultracentrifugations. A role for this intracellular distribution of hexokinase is suggested.