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.     

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.     

Characterization of hexokinase isoenzyme types I and II in ascites tumor cells by an interaction with mitochondrial membrane

Summary A difference was observed in the intracellular distribution between type I and II hexokinases in Ehrlich-Lettre hyperdiploid ascites tumor cells (ELD cells). Experiment of the rebinding to the mitochondria for either each or mixture of the partially purified preparations of the two types of hexokinase indicated that the accepting site on the mitochondrial membrane was common for both types. Mild treatment of the two isoenzymes with chymotrypsin resulted in loss of the binding ability to mitochondria without change in the catalytic activity. It was deduced from these results that the essential region in the two types of hexokinase to interact with mitochondria, which was cleaved by chymotrypsin, was the same or near-similar.Secondly, rebinding to and releasing from mitochondria were examined for the two hexokinase isoenzymes in the presence of various factors affecting the interaction between hexokinase and mitochondria, such as divalent cations, glucose 6-phosphate, and Pi. In the absence of divalent cations, about a half of the type I isoenzyme was bound to mitochondria, whereas almost no type II was bound. A difference was also seen between the two types in the concentration of divalent cations required for the saturation of the binding. A more marked difference was observed in the effect of Pi either alone or in combination with glucose 6-phosphate on the activity and binding ability of the two hexokinases. For type I isoenzyme, Pi relieved both inhibitory and releasing effects of glucose 6-phosphate. On the contrary, for type II, Pi had no such a modulating effect on the releasing action of glucose 6-phosphate, and had the inhibitory effect for itself on the enzyme activity.From these results, it is likely that the difference in the intracellular distribution between type I and II hexokinases in ELD cells is due to the difference in their catalytic regions in the reaction with these ligands, which would induce the structural change in the region responsible for the binding to mitochondria.     

Polyamines stimulate the binding of hexokinase type II to mitochondria

Spermine and spermidine enhanced the binding of hexokinase isoenzyme type II to mitochondria, both of which were prepared from Ehrlich-Lettre hyperdiploid ascites tumor cells, at much lower concentrations than Mg2+. Chymotrypsin-treated hexokinase II could not bind to the mitochondrial membrane in the presence of either spermine or Mg2+, indicating that the effect of spermine is not a nonspecific action, since the treatment of chymotrypsin cleaves only the region essential for the binding without any significant effect of the catalytic activity. Both spermine and Mg2+ antagonized the glucose 6-phosphate-induced release of mitochondria-bound hexokinase, and promoted the binding of the solubilized hexokinase II even in the presence of glucose 6-phosphate. However, inhibition of the activity of soluble hexokinase by glucose 6-phosphate was not reversed by spermine and Mg2+. Hexokinase II rebound to mitochondria with spermine and Mg2+ produced glucose 6-phosphate using ATP generated inside the mitochondria, and no difference was observed between the spermine- and Mg2+-rebound systems. Significance of the binding of hexokinase to mitochondria, especially with polyamines, is discussed with reference to high glycolytic rate in tumor cells.     

Cloning of hexokinase isoenzyme PI from Saccharomyces cerevisiae: PI transformants confirm the unique role of hexokinase isoenzyme PII for glucose repression in yeasts

Summary Hexokinase isoenzyme PI was cloned using a gene pool obtained from a yeast strain having only one functional hexokinase, isoenzyme PI. The gene was characterized using 20 restriction enzymes and located within a region of 2.0 kbp. The PI plasmid strongly hybridized with the PII plasmids isolated previously (Fröhlich et al. 1984). Hence there was a close relationship between the two genes, one of which must have been derived from the other by gene duplication. In conrrast, glucose repression was restored only in hexokinase PII transformants; PI transformants remained non-repressible. This observation provided additional evidence for the hypothesis of Entian (1980) that only hexokinase PII is necessary for glucose repression. Furthermore, glucose phosphorylating activity in PI transformants exceeded that of wild-type cells, giving clear evidence that the phosphorylating capacity is not important for glucose repression.     

Isolation of hexokinase II isoenzyme from the hyaloplasm of rat skeletal muscle

A method for obtaining partially purified preparation of II isozyme of hexokinase from cytosol of the rat skeletal muscles has been suggested. According to the data of electrophoretic and kinetic analyses the preparation does not practically contain I isozyme of hexokinase and is characterized by high enzymatic activity. The obtained preparation of II isozyme of hexokinase may be successfully used for research of the adsorption mechanism controlling the enzyme activity.     

Suppression of apoptosis by cyclophilin D via stabilization of hexokinase II mitochondrial binding in cancer cells

The permeability transition pore is involved in the mitochondrial pathway of apoptosis. Cyclophilin D, a pore component, has catalytic activity as a peptidyl prolyl cis, trans-isomerase (PPIase), which is essential to the pore opening. It has been reported that cyclophilin D overexpression suppresses apoptosis in cancer cells. To clarify the mechanism of this effect, we generated glioma cells overexpressing wild-type or a PPIase-deficient mutant of cyclophilin D. Interestingly, we found that the PPIase-dependent apoptosis suppression by cyclophilin D correlated with the amounts of mitochondrial-bound hexokinase II, which has anti-apoptotic activity. Inactivation of endogenous cyclophilin D by small interference RNA or a cyclophilin inhibitor was found to release hexokinase II from mitochondria and to enhance Bax-mediated apoptosis. The anti-apoptotic effects of cyclophilin D were canceled out by the detachment of hexokinase II from mitochondria, demonstrating that mitochondrial binding of hexokinase II is essential to the apoptosis suppression by cyclophilin D. Furthermore, cyclophilin D dysfunction appears to abrogate hexokinase II-mediated apoptosis suppression, indicating that cyclophilin D is required for the anti-apoptotic activity of hexokinase II. Based on the above, we propose here that cyclophilin D suppresses apoptotic cell death via a mitochondrial hexokinase II-dependent mechanism in cancer cells.     

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.     

Further studies on the role of phospholipids in determining the characteristics of mitochondrial binding sites for type I hexokinase

Previous work has indicated that two types (A and B) of binding sites for hexokinase exist, but in different proportions, on brain mitochondria from various species. Hexokinase is readily solubilized from Type A sites by glucose 6-phosphate (Glc-6-P), while hexokinase bound to Type B sites remains bound even in the presence of Glc-6-P. Type A:Type B ratios are approximately 90:10, 60:40, 40:60, and 20:80 for brain mitochondria from rat, rabbit, bovine and human brain, respectively. The present study has indicated that MgCl2-dependent partitioning of mitochondrially bound hexokinase into a hydrophobic (Triton X-114) phase is generally correlated with the proportion of Type B sites. This partitioning behavior is sensitive to phospholipase C, implying that the factor(s) responsible for conferring hydrophobic character is(are) phospholipid(s). Substantial differences were also seen in the resistance of hexokinase, bound to brain mitochondria from various species, to solubilization by Triton X-100, Triton X-114, or digitonin. This resistance increased with proportion of Type B sites. Enrichment of bovine brain mitochondria in acidic phospholipids (phosphatidylserine or phosphatidylinositol), but not phosphatidylcholine or phosphatidylethanolamine, substantially increased solubilization of the enzyme after incubation at 37 degrees C. Collectively, the results imply that the Type A and Type B sites are located in membrane domains of different lipid composition, the Type A sites being in domains enriched in acidic phospholipids which lead to greater susceptibility to solubilisation by Glc-6-P.     

Interaction of hexokinase II isoenzyme from rat skeletal muscles with lecithin liposomes

It was found that in the presence of Mg2+ (pH 7.5) rat skeletal muscle hexokinase isozyme II is firmly adsorbed on mitochondrial and artificial phospholipid membranes (lecithin liposomes). In both cases the adsorption isotherm has similar quantitative and qualitative characteristics, which points to the absence of specific binding sites on the membranes. Under these conditions, immobilization of hexokinase on various membranes is concomitant with similar changes in the enzyme stability upon storage as well as with the pH-dependence of the enzyme activity. It was demonstrated that the bound hexokinase form has a greater value of V, an increased affinity for glucose and a decreased sensitivity to the inhibitory action of glucose-6-phosphate as compared to the free form. Besides, this form is in a greater degree subjected to the inhibitory influence of ADP with respect to glucose. In this case, the enzyme affinity for ATP and the Ki value for ADP with respect to ATP is practically the same both for the free and membrane-bound forms. The data obtained suggest that the phospholipid component of mitochondrial membranes participates in the enzyme binding in the presence of Mg2+. It was assumed that the model system used in the present study, i.e., hexokinase-Mg2+-liposomes, may be successfully used for the analysis of an adsorption mechanism of regulation of hexokinase activity in the cell.     

Regulation of mammalian hexokinase: regulatory differences between isoenzyme I and II

Mammalian hexokinase isoenzymes I and II have been shown to differ qualitatively in response to various modifiers. Although both enzymes are inhibited by glucose 6-phosphate, only isoenzyme II exhibits a slow response to the presence of this inhibitor. P_i decreases the affinity of glucose 6-phosphate for Sarcoma 37 hexokinase I, but has no effect on hexokinase II from the same cell. P_i overcomes all of the inhibition of red cell hexokinase by glucose-6-P and hence the two effectors act competitively. At pH 6.5, catecholamines increase the V of isoenzyme I of Sarcoma 37 and brain in the soluble and mitochondrial forms but do not activate these forms of tumor isoenzyme II. Citrate activates brain and tumor isoenzyme I when they are inhibited by tris(hydroxy-methyl)aminomethylethane sulfonate (TES) and ADP; however, tumor isoenzyme II is not activated.     

The binding of nucleotides and bivalent cations to the calcium-and-magnesium ion-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum.

The binding of MgATP to purified Ca2+Mg2+-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum was studied by using a flow-dialysis method. Phosphoryl-enzyme formation and catalytic activity were also measured, and all three processes demonstrated negative co-operativity, with half-saturation of all three parameters at a MgATP concentration of 40-50muM, and a Hill coefficient (h) of 0.8. The variation of the binding constant with with pH was measured and showed tighter binding of MgATP with increasing pH over the range 6.8-8.5. Binding parameters for ATP analogues were also measured. The binding of Ca2+ in the presence and absence of ATP analogues gave half saturation at a Ca2+ concentration of 1.2-1.3muM. Hill plots of Ca2+-binding data gave a slope of 0.8. These results show that the binding of MgATP and Ca2+ can occur in a random manner, with neither substrate influencing the affinity of the enzyme for the other.     

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.     

Hexose metabolism in pancreatic islets: regulation of mitochondrial hexokinase binding

A major fraction of hexokinase was found to be bound, presumably to mitochondria, in both normal and tumoral rat pancreatic islet cells examined after either mechanical disruption or digitonin treatment. Spermidine enhanced the binding and glucose 6-phosphate caused the release of hexokinase to and from islet mitochondria, in a manner comparable to that seen in parotid or brain homogenates. In hepatocytes, some hexokinase, but no glucokinase, was found in the bound form. In islet cells, however, the pattern of glucokinase binding was similar to that of hexokinase. It is speculated that the preferential location of both hexokinase and glucokinase on mitochondria may favor the maintenance of a high cytosolic ATP content in islet cells.