Discrimination of antigenic site and thiol-inhibitor-sensitive site of hexokinase isoenzymes.

Rabbit antiserum was prepared against hexokinase isoenzyme type I which was purified from rat brain mitochondria. The antiserum inhibited the activity of the mitochondrial hexokinase type I as well as that of the cytosolic type I enzyme prepared from rat brain, kidney and spleen. It did not, however, inhibit the activity of type II hexokinase from muscle and spleen or that of the type III enzyme from spleen. The results suggest that all hexokinase type I isoenzymes may have a common antigenic site irrespective of their sources, though their responses to a thiol inhibitor are different.     

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.     

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.     

Hexokinase isoenzymes in tissues of the adult and developing guinea pig

Changes in the activities and isoenzyme distribution of hexokinase were determined in a number of tissues during the development of the guinea pig. The total activity in the fetal liver showed a large fall during the second half of gestation to reach adult values by term. With normal diet the fetal, neonatal, and adult livers had isoenzymes I and III but little or no detectable IV (glucokinase). The fetal liver had predominantly type I, but the proportion of type III increased during development. The kinetics of the guinea pig isoenzymes were similar to those reported for the rat. Two additional isoenzymes with mobility between I and II were detected in the fetal liver and blood. They appear to have kinetic properties similar to type I. Detectable liver glucokinase activity was induced by glucose administration to adult guinea pigs. The total activity in kidney, brain and skeletal muscle showed a postnatal rise while in the fetal heart it was high and declined after birth. These tissues contained predominantly type I with varying proportions of type III hexokinase. The ratio of particulate-bound to soluble hexokinase varied from tissue to tissue. All except the liver showed a significant increase in binding after birth. The changes are discussed in relation to the control of glucose utilization in the fetal and neonatal periods.     

Distribution of hexokinase isoenzymes depending on a carbon source in Saccharomyces cerevisiae.

DEAE cellulose chromatography and agar gel electrophoresis of glucose-phosphorylating enzymes in Saccharomyces cerevisiae showed the existence of glucokinase and two hexokinase isoenzymes ( designated as hexokinase I and II ). The distribution of hexokinase isoenzymes was dependent on a carbon source in the medium, while that of glucokinase was not dependent. The cells grown on 3 % ethanol as carbon source showed the isoenzyme pattern with predominant hexokinase I and a little hexokinase II. The isoenzyme pattern of the cells grown on 6 % glucose, which was differnt from that of the cells grown on ethanol, showed that hexokinase I and II were minor and major parts respectively. When the cells grown on 3 % ethanol were incubated on the medium containing 6 % glucose, hexokinase I was repressed and hexokinase II inducted. These facts suggest that two hexokinase isoenzymes, but not glucokinase, are adaptive enzyme.     

Interconversion of E and S isoenzymes of horse liver alcohol dehydrogenase. Several residues contribute indirectly to catalysis.

The E and S isoenzymes of horse liver alcohol dehydrogenase differ by 10 amino acid residues, but only the S isoenzyme is active on 3 beta-hydroxysteroids. This functional difference was correlated to the differences in structures of the isoenzymes by characterizing a series of chimeric enzymes, which could represent intermediates in the evolution of catalytic activity. Deletion of Asp-115 from the E isoenzyme created the E/D115 delta enzyme that is active on steroids. The deletion alters the substrate binding pocket by moving Leu-116, which sterically hinders binding of steroids in the E isoenzyme. A chimeric enzyme (ESE) that has four changes in or near the substrate binding pocket (T94I/R101S/F110L/D115 delta) was 15-30-fold more catalytically efficient (V/Km) on uncharged steroids than was the E/D115 delta enzyme. Molecular modeling suggests that the substitutions at residues 94 and 110 indirectly affect the activity on steroids. ESE enzyme was 6-fold more active than the S isoenzyme on neutral steroids, due to substitutions not in the substrate binding pocket. The K366E and the Q17E/A43T/A59T substitutions in the S isoenzyme gave 2-fold increases in V/Km on steroids, which together can account for the changes observed with the ESE enzyme. The enzymes that are active on steroids did not bind 2,2,2-trifluoroethanol as tightly and were catalytically less efficient than the E isoenzyme with small alcohols. However, these enzymes were two to three and four to five orders of magnitude more efficient with 1-hexanol and 5 beta-androstane-3 beta,17 beta-diol, respectively, than with ethanol. These results demonstrate that several residues not directly participating in substrate binding or chemical catalysis contribute to catalytic efficiency.     

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.     

Comparative binding of lactate dehydrogenase to mitochondrial fractions

Beef liver mitochondrial fraction showed LDH activity (1.76 +/- 0.25 U/g pellet). Sixty seven% of the initial mitochondrial pellet LDH activity (almost M4 isoenzyme) was released when suspended in NaCl 0.15 M. When the washed particles were sonicated in a 0.15 M NaCl medium, the solubilized LDH activity (all five isoenzymes as cytosoluble fraction) was 5-fold higher than the initial pellet activity. The different isoenzymatic composition of intramitochondrial and externally bound forms of the enzyme should be taken into account when investigating the physiological role of intramitochondrial LDH. Beef liver cytosoluble LDH (very little content of M4 isoenzyme) showed no affinity for the beef liver mitochondrial fraction but purified M4-LDH isoenzyme was able to bind to the particulate fraction from the same source. This suggests an isoenzyme specificity for the interaction. The maximum amount of cytosoluble LDH bound to the mitochondrial fraction depends on the enzyme and the particulate fraction source. Therefore, binding capacity to the mitochondrial fraction depends not only on the net charge of LDH isoenzymes, which play a predominant role in the binding, but also on individual characteristics of the LDH isoenzymes and mitochondrial fractions from different sources. This suggests that electrostatic forces are not the only ones involved in the binding process.     

The distribution of enzyme and isoenzyme activities between parenchymal and haematopoietic cells in the liver of the foetal guinea pig

The hepatocyte and haematopoietic cell contents of the liver of the foetal guinea pig were measured over the latter half of gestation. Hepatocytes represented about 30% of liver volume at mid-gestation and this increased to 70-80% by term; cell volume remained fairly constant until 5-7 days before term, then more than doubled. Haematopoietic cells represented about 5% of liver volume at mid-gestation and this progressively fell to <1% by term. At 75% of gestation hepatocytes and haematopoietic cells were prepared from perfused foetal livers by collagenase digestion. Enzyme activity of the hepatocyte was, without exception, similar to that of the whole liver. In general, enzyme activity in the haematopoietic cells was similar to that in erythrocytes, with relatively low values for aldolase, glycerol 3-phosphate dehydrogenase, phosphoglycerate mutase, enolase, lactate dehydrogenase, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, isocitrate dehydrogenase, ;malic' enzyme, glutamate dehydrogenase and aspartate aminotransferase. The haematopoietic cell contribution to total enzyme activity in the foetal liver was usually much less than 10% and could thus not account for the major changes in hepatic enzyme activity over the latter half of gestation. Hepatocytes contained hexokinase isoenzymes I and III, aldolase isoenzymes A and B and pyruvate kinase isoenzymes 1, 2 and 4. The haematopoietic cells contained hexokinase isoenzyme I and two additional bands of activity with slightly greater mobility, aldolase isoenzyme A and pyruvate kinase isoenzymes 2 and 4.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques. 3. The substrate specificity of isoenzymes of acid phosphatase in m.gastrocnemius of rabbits.

Three distinct isoenzymes of acid phosphatase have been separated from extracts of m.gastrocnemius of normal and of vitamin E deficient rabbits by gel filtration and polyacrylamide gel electrophoresis. These isoenzymes, termed I, II and III, have molecular weights of: 110,000--130,000, 60,000--78,000 and 12,500--14,500. Isoenzymes I and II split the substrates 4-methylumbelliferyl phosphate and naphthol AS-BI phosphate and the activity is strongly increased in the muscles of vitamin E deficient rabbits. Isoenzyme III splits only 4-methylumbelliferyl phosphate and the activity is not increased in the muscles of vitamin E deficient rabbits. The pH-optimum for isoenzymes I and II is 4.8 and for isoenzyme III 5.5. It has been shown that the histochemical semipermeable membrane technique, using substrate naphthol AS-BI phosphate, is a very reliable technique for demonstrating activity of the isoenzymes I and II in tissue sections. On the other hand, activity of isoenzyme III cannot be demonstrated with this histochemical technique. In pathologically altered muscles, the activity of the isoenzymes I and II is greatly increased whilst the activity of isoenzyme III is not significantly altered.     

Cytosolic and mitochondrial isoenzymes of branched-chain amino acid aminotransferase during development of the rat.

The isoenzymic forms of branched-chain amino acid aminotransferase in mitochondria of rat tissues were compared with the better-known cytosolic forms in order to find any regular pattern of expression of these isoenzymes during development. Mitochondria of all tissues examined except brain contained only a type-I isoenzyme differing from the cytosolic type-I isoenzyme in heat stability and activation by mercaptoethanol. Foetal and adult brain mitochondria contained isoenzymes type III as well as type I. The large excess of type-I isoenzyme in foetal liver was localized in mitochondria, apparently of haematopoietic cells. The activity of this isoenzyme declined precipitously (by 80%) from day 19 of gestation at the same period and rate as does the volume fraction of haematopoietic cells that are then leaving the liver. Cortisol treatment accelerated the loss of these cells, and proportionally accelerated loss of the mitochondrial isoenzyme I. A development succession of type-I isoenzyme by the unique type II of liver parenchymal cell cytosols could not be demonstrated, since small, about equal, amounts of types I and II were always present in cytosols of foetal and adult liver. Developmental succession of isoenzymes within tissues was limited to cytosols and was demonstrated by the presence of cytosolic isoenzyme III in foetal and newborn skeletal muscle and kidney, organs which contain only isoenzyme I in the adult.     

Lactate dehydrogenase binding to the mitochondrial fraction and to a mitochondrial inhibitor as a function of the isoenzymatic composition

Purified rabbit skeletal muscle LDH M4 isoenzyme, but not H4 isoenzyme, was observed to bind to either the crude mitochondrial fraction or a mitochondrial inhibitor. Several sources of LDH isoenzymes in which M-type subunits with an alkaline pI are predominant bind to this crude mitochondrial fraction and are inhibited by the mitochondrial inhibitor. Binding and inhibition have also been observed with H-type isoenzymes with a pI near 7. The binding and the inhibition processes did not occur with H-type isoenzymes with an acid pI or with M-type isoenzymes with pI near 6. The binding capacity of LDH to the mitochondrial fraction and to the mitochondrial inhibitor is very similar and depends on the net protein charge and not on whether the subunits are H- or M-type.     

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.     

EFFECT OF FATTY ACIDS ON RAT BRAIN PARTICULATE HEXOKINASE

Abstract— The effect of free fatty acids on rat brain particulate hexokinase was studied in vitro. Hexokinase bound with brain mitochondrial fraction was found to be sensitive to the action of free fatty acids, resulting in the solubilization of at least part of bound enzyme activity into the supernatant. The decrease of total enzyme activity observed at the highest free fatty acid concentration was probably due to the inhibition of hexokinase. The physiological consequence of hexokinase solubilization by low concentrations of free fatty acids, similar to that observed in vivo , is discussed in relation to activity changes of soluble and particulate enzyme forms demonstrated previously under hypoxic conditions.     

Competitive inhibition of hexokinase isoenzymes by mercurials.

A difference in the mode of inhibition of hexokinase [EC 2.7.1.1] isoenzymes by p-chloromercuribenzenesulfonate was confirmed with respect to glucose between two Type I isoenzyme preparations purified from the kidney and spleen of rat. Essentially the same difference was observed when galactose was used as the substrate in place of glucose, as the kidney Type I isoenzyme was inhibited in a competitive manner while the spleen counterpart was inhibited in a non-competitive manner by sulfhydryl inhibitor. Both the Type I isoenzymes, however, were competitively inhibited by other mercurial sulfhydryl inhibitors, methyl and butyl mercuric chlorides. On the other hand, the Type II hexokinase isoenzymes purified from the muscle, heart, and spleen were all inhibited competitively by p-chloromercuribenzenesulfonate with respect to glucose. The mechanism of competitive inhibition of the hexokinase isoenzymes by sulfhydryl inhibitors was discussed in view of the difference in the mode of action of the mercurials with different isoenzymes.     

Mitochondrial and cytosolic aspartate aminotransferase from chicken: Activity toward aromatic amino acids

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase from chicken heart accept as substrates L-phenylalanine, L-tyrosine and L-tryptophan. The specific activities of the mitochondrial isoenzyme toward these substrates are between 0.1 to 0.5% of that toward aspartate and two orders of magnitude higher than that toward alanine. The specific activities of the cytosolic isoenzyme toward the aromatic substrates are 10 to 70% of the respective values of the mitochondrial isoenzyme. The activities of both isoenzymes toward aromatic amino acids are increased two- to threefold by 1 M formate. Larger increases by formate were observed for the alanine aminotransferase activity of both isoenzymes whereas their aspartate aminotransferase activity was inhibited by formate. The opposite effects of formate on the activities toward the aromatic and aliphatic monocarboxylic substrates on the one hand and the dicarboxylic substrate on the other are consonant with the notion of formate occupying the binding site of the distal carboxylate group of the substrate (Morino Y., Osman A.M., and Okamoto M. (1974) J. Biol. Chem. $\text{249}$, 6684–6692). Apparently, in the ternary complex of aspartate aminotransferase with formate and aromatic amino acids, the aromatic rings of the latter bind to a site which does not overlap with the binding site for the distal carboxylate.     

Purification of the hexokinases by affinity chromatography on sepharose-N-aminoacylglucosamine derivates. Design of affinity matrices from free solution kinetics.

The purification is described of rat hepatic hexokinase type III and kidney hexokinase type I on a large scale by using a combination of conventional and affinity techniques similar to those previously used for the purification of rat hepatic glucokinase [Holroyde, Allen, Storer, Warsy, Chesher, Trayer, Cornish-Bowden & Walker (1976) Biochem. J. 153, 363-373] and muscle hexokinase type II [Holroyde & Trayer (1976) FEBS Lett. 62, 215-219]. The key to each purification was the use of a Sepharose-N-aminoacylglucosamine affinity matrix in which a high degree of specificity for a particular hexokinase isoenzyme could be introduced by either varying the length of the aminoacyl spacer and/or varying the ligand concentration coupled to the gel. This was predicted from a study of the free solution kinetic properties of the various N-aminoacylglucosamine derivatives used (N-aminopropionyl, N-aminobutyryl, N-aminohexanoyl and N-aminooctanoyl), synthesized as described by Holroyde, Chesher, Trayer & Walker [(1976) Biochem. J. 153, 351-361]. All derivatives were competitive inhibitors, with respect to glucose, of the hexokinase reaction, and there was a direct correlation between the Ki for a particular derivative and its ability to act as an affinity matrix when immobilized to CNBr-activated Sepharose 4B. Muscle hexokinase type II could be chromatographed on the Sepharose conjugates of all four N-aminoacylglucosamine derivatives, although the N-aminohexanoylglucosamine derivative proved best. This same derivative was readily able to bind hepatic glucokinase and hexokinase type III, but Sepharose-N-amino-octanoyl-glucosamine was better for these enzymes and was the only derivative capable of binding kidney hexokinase type I efficiently. Separate studies with yeast hexokinase showed that again only the Sepharose-N-amino-octanoylglucosamine was capable of acting as an efficient affinity matrix for this enzyme. Implications of these studies in our understanding of affinity-chromatography operation are discussed.     

Subcellular distribution and properties of aldehyde dehydrogenase from 2-acetylaminofluorine-induced rat hepatomas

The subcellular distribution and properties of four aldehyde dehydrogenase isoenzymes (I-IV) identified in 2-acetylaminofluorene-induced rat hepatomas and three aldehyde dehydrogenases (I-III) identified in normal rat liver are compared. In normal liver, mitochondria (50%) and microsomal fraction (27%) possess the majority of the aldehyde dehydrogenase, with cytosol possessing little, if any, activity. Isoenzymes I-III can be identified in both fractions and differ from each other on the basis of substrate and coenzyme specificity, substrate K(m), inhibition by disulfiram and anti-(hepatoma aldehyde dehydrogenase) sera, and/or isoelectric point. Hepatomas possess considerable cytosolic aldehyde dehydrogenase (20%), in addition to mitochondrial (23%) and microsomal (35%) activity. Although isoenzymes I-III are present in tumour mitochondrial and microsomal fractions, little isoenzyme I or II is found in cytosol. Of hepatoma cytosolic aldehyde dehydrogenase activity, 50% is a hepatoma-specific isoenzyme (IV), differing in several properties from isoenzymes I-III; the remainder of the tumour cytosolic activity is due to isoenzyme III (48%). The data indicate that the tumour-specific aldehyde dehydrogenase phenotype is explainable by qualitative and quantitative changes involving primarily cytosolic and microsomal aldehyde dehydrogenase. The qualitative change requires the derepression of a gene for an aldehyde dehydrogenase expressed in normal liver only after exposure to potentially harmful xenobiotics. The quantitative change involves both an increase in activity and a change in subcellular location of a basal normal-liver aldehyde dehydrogenase isoenzyme.     

Differential molecular sieving permits visualization of hexokinase isoenzymes following electrophoretic separation in high resolution acrylamide gels

1. Differential molecular sieving is the concept applied to bring together isoenzymes of ATP:D-hexose-6-phosphotransferase (hexokinases) with glucose 6-phosphate dehydrogenase in acrylamide gels by utilization of their dissimilar electrophoretic mobilities. 2. The hexokinase isoenzymes migrate and separate in gels with pore sizes selected to entrap glucose 6-phosphate dehydrogenase in their interstices. The locations of the bands of specific activity are visualized by fluorescence of NADPH under long wave, ultraviolet radiation. 3. A new discontinuous electrochemical system has been devised to deliver protective thiol groups into the gel. Cysteine (trailing ion) and SO4(2-) (leading ion) form a sharp moving boundary. 4. The high resolution of the system has permitted visualization of a rapidly migrating, high Km hexokinase in murine spleen, fat, kidney and lymph nodes. Hexokinase Types I and II, were observed in all tissues tested, but Type IV was seen only in the liver. 5. The importance of glucose concentration effects on hexokinase activity is emphasized by inactivation of slowly migrating low Km hexokinase Types I and II following exposure to 200 mM glucose during preparation of extracts.