Comparative study of free and bound glycolytic enzymes from sea scorpion brain.

Free and bound forms of hexokinase, pyruvate kinase, and lactate dehydrogenase were prepared from the brain of the sea scorpion (Scorpaena porcus) in a low ionic strength medium. Properties of the free and bound forms were compared to determine whether binding to particulate matter could influence enzyme function or stability in vivo. Changes in pH differently affected the activity of the free and bound forms of all three enzymes. Furthermore, bound forms of hexokinase and pyruvate kinase were more stable than the free enzymes to heating at 45 degrees C. Bound hexokinase showed higher affinity for substrates (ATP, glucose) than the free form and bound lactate dehydrogenase had greater affinity for pyruvate and NADH. Although the affinities of the two forms of pyruvate kinase for substrates were similar, Hill coefficients for phosphoenolpyruvate as well as inhibition by ATP differed between the two enzyme forms. Free and bound lactate dehydrogenase also showed differences in Hill coefficients and bound lactate dehydrogenase was less sensitive to substrate inhibition by high pyruvate concentrations. The possible physiological role of the binding of these glycolytic enzymes to subcellular structures is discussed.     

The regulation of mitochondrial-bound hexokinases in the liver

A functional coupling between bound hexokinase and the inner mitochondrial compartment has been shown. It is based structurally on the binding of hexokinase to a pore protein which is present in zones of contact between the two boundary membranes. The latter was observed by electron microscopic localization of antiporin and hexokinase at the mitochondrial surface. The four isoenzymes present in liver differ considerably in their activity after binding to the mitochondrial surface. This was found by binding studies using the four isoenzymes isolated from the supernatant. Isoenzyme IV did not bind at all. Isoenzymes I-III did bind and became activated: I, 5.9-fold; II, 39-fold; and III, 1.3-fold. These results suggest that the in vivo activity of hexokinase in the mitochondrial fraction is much larger than so far observed. Furthermore the binding of isoenzymes was differently affected by metabolites. Glucose-6-phosphate exclusively desorbed isoenzyme I from the mitochondrial membrane whereas free fatty acids predominantly liberated isoenzymes II and III. A reciprocal change of the levels of free fatty acids and glucose 6-phosphate in livers of starved rats therefore, can explain why exclusively mitochondrial-bound isoenzymes II and III decreased 10-fold while at the same time isoenzyme I increased.     

Changes in enzymatic activities involved in glucose metabolism by acyl-CoAs in Trypanosoma cruzi

This study describes the effect of some saturated and unsaturated free fatty acids and acyl-CoA thioesters on Trypanosoma cruzi glucose 6-phosphate dehydrogenase and hexokinase activities. Glucose 6-phosphate dehydrogenase was sensitive to the destabilizing effect provoked by free fatty acids, while hexokinase remained unaltered. Glucose 6-phosphate dehydrogenase inhibition by free fatty acids was dependent on acid concentration and chain length. Both enzymes were inhibited when they were incubated with acyl-CoA thioesters. The acyl-CoA thioesters inhibited glucose 6-phosphate dehydrogenase at a lower concentration than the free fatty acids; the ligands glucose 6-phosphate and NADP+ afforded protection. The inhibition of hexokinase by acyl-CoAs was not reverted when the enzyme was incubated with ATP. The type of inhibition found with acyl-CoAs in relation to glucose 6-phosphate dehydrogenase and hexokinase suggests that this type inhibition may produce an in vivo modulation of these enzymatic activities.     

Glucose catabolism in brain. Intracellular localization of hexokinase

A major energy source in brain is glucose, which is committed to metabolism by hexokinase (Type I isozyme), an enzyme usually considered to be bound to the outer mitochondrial membrane. In this study, the subcellular location of hexokinase in brain has been rigorously investigated. Mitochondrial fractions containing hexokinase (greater than 500 milliunits/mg protein) were prepared by two different procedures, and then subjected to density gradient centrifugation before and after loading with barium phosphate, a technique designed to increase the density of the mitochondria. The gradient distribution patterns of both unloaded and loaded preparations show that brain hexokinase does not distribute exclusively with mitochondrial marker enzymes. This is particularly evident in the loaded preparations where there is a clear distinction between the peak activities of hexokinase and mitochondrial markers. The same observation was made when the mitochondrial fraction of either untreated or barium phosphate-loaded mitochondria was subjected to titration with digitonin. In fact, at concentrations of digitonin, which almost completely solubilize marker enzymes for both the inner and outer mitochondrial membranes, a significant fraction of the total hexokinase remains particulate bound. Electron microscopy confirmed that particulate material is still present under these conditions. Significantly, hexokinase is released from particulate material only at high concentrations of digitonin which solubilize the associated microsomal marker NADPH-cytochrome c reductase. Glucose 6-phosphate, which is known to release hexokinase from the brain "mitochondrial fraction" also releases hexokinase from this unidentified particulate component. These results on brain, a normal glucose utilizing tissue, differ from those obtained previously on highly glycolytic tumor cells where identical subfractionation procedures revealed a strictly outer mitochondrial membrane location for particulate hexokinase (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912). It is concluded that in brain, hexokinase has a greater propensity to localize at nonmitochondrial receptor sites than to those known to be associated with the outer mitochondrial membrane.     

Intracellular distribution of hexokinase in rabbit brain

Hexokinase in mammalian brain is particulate and usually considered to be bound to the outer mitochondrial membrane. Investigation of rabbit brain mitochondria prepared either by differential centrifugation and discontinuous density gradient centrifugation has provided evidence that this particulate fraction also contains endoplasmic vesicles and synaptosomes. Solubilization of the bound hexokinase by different combinations of detergents and metabolites has proved the existence of different hexokinase binding sites. Electron microscopic examination of hexokinase location by immuno-gold labelling techniques confirmed, that hexokinase is indeed predominantly bound to mitochondria but that a significant proportion is also bound to non-mitochondrial membranes. Attempts to quantify this distribution were unsuccessful since different figures were obtained using anti-hexokinase IgG affinity purified on immobilized native or denatured hexokinase. Binding studies of the purified rabbit brain mitochondrial hexokinase to rabbit liver mitochondria and microsomes confirmed that in addition to a binding site on mitochondria there is another binding site on microsomes. The N-terminal sequence of hexokinase has been shown to be important for mitochondria binding and also for microsome binding. These results suggest that the intracellular localization of hexokinase in rabbit brain is not exclusively mitochondrial and that the metabolic role of this enzyme should be reconsidered by including a binding site on the endoplasmic reticulum.     

Hexose metabolism in pancreatic islets: compartmentation of hexokinase in islet cells

Hexokinase activity was found in both soluble (cytosolic) and particulate subcellular fractions prepared from rat pancreatic islet homogenates. The bound enzyme was associated with mitochondria rather than secretory granules. Relative to the total hexokinase activity, the amount of bound enzyme was higher in islet homogenates prepared at pH 6.0 (72 +/- 7%) than in islets homogenized at pH 7.4 (38 +/- 1%). The affinity of hexokinase for equilibrated D-glucose was not different in the cytosolic and mitochondrial fractions. In both fractions, hexokinase displayed a greater affinity for alpha- than beta-D-glucose, but a higher maximal velocity with the beta- than alpha-anomer. Glucose 6-phosphate inhibited to a greater extent cytosolic than mitochondrial hexokinase. A high Km glucokinase-like enzymic activity was also present in both subcellular fractions. It is proposed that the ambiguity of hexokinase plays a propitious role in the glucose-sensing function of pancreatic islet cells.     

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 non-mitochondrial 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.     

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.     

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.     

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.     

Brain mitochondrial hexokinase: solubilization by adenine nucleotides and in coupled respiration

The relative potencies of ATP and ADP in debinding rat brain mitochondrial hexokinase were measured. At fixed total concentrations of adenine nucleotides, added exogenously, solubilization of the enzyme increased as the proportions of ATP to ADP were raised. The generation of physiological concentrations of ATP by the mitochondria during coupled respiration resulted in a 2-fold increase in solubilization. These findings support the hypothesis that the cytosol-mitochondrial compartmentation of hexokinase may be a factor in the regulation of hexokinase activity and glycolysis.     

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.     

Release of endothelial cell lipoprotein lipase by plasma lipoproteins and free fatty acids

Lipoprotein lipase (LPL) bound to the lumenal surface of vascular endothelial cells is responsible for the hydrolysis of triglycerides in plasma lipoproteins. Studies were performed to investigate whether human plasma lipoproteins and/or free fatty acids would release LPL which was bound to endothelial cells. Purified bovine milk LPL was incubated with cultured porcine aortic endothelial cells resulting in the association of enzyme activity with the cells. When the cells were then incubated with media containing chylomicrons or very low density lipoproteins (VLDL), a concentration-dependent decrease in the cell-associated LPL enzymatic activity was observed. In contrast, incubation with media containing low density lipoproteins or high density lipoproteins produced a much smaller decrease in the cell-associated enzymatic activity. The addition of increasing molar ratios of oleic acid:bovine serum albumin to the media also reduced enzyme activity associated with the endothelial cells. To determine whether the decrease in LPL activity was due to release of the enzyme from the cells or inactivation of the enzyme, studies were performed utilizing radioiodinated bovine LPL. Radiolabeled LPL protein was released from endothelial cells by chylomicrons, VLDL, and by free fatty acids (i.e. oleic acid bound to bovine serum albumin). The release of radiolabeled LPL by VLDL correlated with the generation of free fatty acids from the hydrolysis of VLDL triglyceride by LPL bound to the cells. Inhibition of LPL enzymatic activity by use of a specific monoclonal antibody, reduced the extent of release of 125I-LPL from the endothelial cells by the added VLDL. These results demonstrated that LPL enzymatic activity and protein were removed from endothelial cells by triglyceride-rich lipoproteins (chylomicrons and VLDL) and oleic acid. We postulate that similar mechanisms may be important in the regulation of LPL activity at the vascular endothelium.     

Changes in the subcellular distribution of brain and heart hexokinase isoenzymes during alloxan diabetes

Changes in the subcellular distribution of hexokinase activity from three brain regions and heart were studied during alloxan induced diabetes. There was an overall decrease in the particulate hexokinase with an increase in the soluble form, after different time intervals of the onset of diabetes. Administration of insulin to the diabetic rats showed a partial counteraction of the enzyme changes. A possible regulation of brain hexokinase by metabolite changes is proposed     

Binding of non-catalytic ATP to human hexokinase I highlights the structural components for enzyme-membrane association control

BACKGROUND: Hexokinase I sets the pace of glycolysis in the brain, catalyzing the ATP-dependent phosphorylation of glucose. The catalytic properties of hexokinase I are dependent on product inhibition as well as on the action of phosphate. In vivo, a large fraction of hexokinase I is bound to the mitochondrial outer membrane, where the enzyme adopts a tetrameric assembly. The mitochondrion-bound hexokinase I is believed to optimize the ATP/ADP exchange between glucose phosphorylation and the mitochondrial oxidative phosphorylation reactions. RESULTS: The crystal structure of human hexokinase I has been determined at 2.25 A resolution. The overall structure of the enzyme is in keeping with the closed conformation previously observed in yeast hexokinase. One molecule of the ATP analogue AMP-PNP is bound to each N-terminal domain of the dimeric enzyme in a surface cleft, showing specific interactions with the nucleotide, and localized positive electrostatic potential. The molecular symmetry brings the two bound AMP-PNP molecules, at the centre of two extended surface regions, to a common side of the dimeric hexokinase I molecule. CONCLUSIONS: The binding of AMP-PNP to a protein site separated from the catalytic centre of human hexokinase I can be related to the role played by some nucleotides in dissociating the enzyme from the mitochondrial membrane, and helps in defining the molecular regions of hexokinase I that are expected to be in contact with the mitochondrion. The structural information presented here is in keeping with monoclonal antibody mapping of the free and mitochondrion-bound forms of the enzyme, and with sequence analysis of hexokinases that differ in their mitochondria binding properties.     

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.     

Functional characteristics of hexokinase bound to the type a and type B sites of bovine brain mitochondria.

Hexokinase is released from Type A sites of brain mitochondria in the presence of glucose 6-phosphate (Glc-6-P); enzyme bound to Type B sites remains bound. Hexokinase of freshly isolated bovine brain mitochondria (Type A:Type B, approximately 40:60) selectively uses intramitochondrial ATP as substrate and is relatively insensitive to the competitive (vs ATP) inhibitor and Glc-6-P analog, 1,5-anhydroglucitol 6-phosphate (1,5-AnG-6-P). After removal of hexokinase bound at Type A sites, the remaining enzyme, bound at Type B sites, does not show selectivity for intramitochondrial ATP and has increased sensitivity to 1,5-AnG-6-P. Thus, the properties of the enzyme bound at Type B sites are modified by removal of hexokinase bound at Type A sites. It is suggested that mechanisms for regulation of mitochondrial hexokinase activity, and thereby cerebral glycolytic metabolism, may depend on the ratio of Type A:Type B sites, which varies in different species.     

Hexokinase isoenzymes in diabetes

Hexokinase is present in the tissues in four isoenzymic forms. Cerebral tissue contains predominantly Type I hexokinase which is believed to be insulin-insensitive. In cerebral tissue about 60 to 70% of the hexokinase is bound to the particulate fraction. The changes in the distribution of hexokinase Type I and Type II together with the bound and free hexokinase have been studied in control, diabetic and diabetic animals treated with insulin. The results indicate that the presence of insulin is essential for the normal binding of the hexokinase to the particulate fraction. In heart tissue, Type II hexokinase bound to the pellet shows a significant decrease in diabetes, which is reversed on insulin administration.     

Heart 6-phosphofructo-1-kinase. Subcellular distribution and binding to myofibrils

6-Phosphofructo-1-kinase (phosphofructokinase) (ATP:D-fructose-6-P 1-phosphotransferase, EC 2.7.1.11) can be identified in sheep heart homogenates in two forms, a soluble form and a form bound to the particulate fraction. Homogenates from immediately-dissected hearts have the enzyme in the soluble form, while those collected after a delay have the enzyme bound to the particulate fraction. Aldolase appears to show the same change in its location. Homogenization in a solution with concentrated macromolecular species (20% albumin) results in a greater association of phosphofructokinase and of aldolase to the particulate fraction in homogenates from immediately dissected hearts. Phosphofructokinase activity can be solubilized by two specific means: by high ionic strength, which is dependent upon specific salts; or by low ionic strength, which is dependent upon the presence of phosphofructokinase substrates or modifier ligands. These two means of solubilization are affected differently upon decreasing the pH below 6.9: the solubilization at low ionic strength is prevented, whereas phosphofructokinase is still solubilized by high ionic strength. Under the latter condition, the enzyme is in the inactive dimeric state, which can be activated at an alkaline pH. Myofibrils present in the particulate fraction can account for the binding of phosphofructokinase in heart homogenates. Purified myofibrils, when added to heart supernatant fluids, can bind phosphofructokinase at a slightly acidic pH. Conditions for phosphofructokinase binding to myofibrils, as well as its dissociation, follow what was observed with the binding of phosphofructokinase to the particulate fraction. At an acidic pH, and in the presence of a high concentration of ATP, phosphofructokinase exhibits low activity. However, if phosphofructokinase is assayed under these conditions while bound to myofibrils, the enzyme is activated.     

Changes in Hexokinase Isoenzymes in Regions of Rat Brain During Insulin-Induced Hypoglycemia

Activities of hexokinase isoenzymes were determined during insulin-induced hypoglycemia in soluble and total particulate fractions from three regions of rat brain. Type I hexokinase isoenzyme activity showed a small decrease in both soluble and particulate fractions from the cerebral hemispheres. In cerebellum and brain stem, however, Type I isoenzyme showed a decrease only in the soluble fraction. A significant increase was observed in hexokinase Type II isoenzyme from both the fractions, in all the three brain regions 1 h after insulin administration.