POTENTIATION OF AMP-AMINOHYDROLASE ACTIVITY OF BRAIN MITOCHONDRIA BY HEXOKINASE

Abstract— The addition of hexokinase (yeast and brain) to mitochondrial fractions of brain (rat) resulted in a considerable increase of formation of ammonia from AMP. The mechanisms underlying the activation of AMP-aminohydrolase of brain mitochondria by ATP and hexokinase are quite different. In the activation of AMP-aminohydrolase by hexokinase the SH-groups of both enzymes particularly of the latter are of importance. Brain mitochondria contain low-molecular dialysable substances of unknown nature which are necessary for the interaction of both enzymes.     

Hexokinases of Tobacco Leaves: Changes in the Cytosolic and Non-Cytosolic Isozyme Complexes Induced by Tobacco Mosaic Virus Infection

Changes in the cytosotic (soluble) and the non-cytosolic (particulate) isozyme composition of hexokinases and in their properties were studied by ion exchange chromatography on DEAE cellulose after the subcellular fractionation both in the healthy and the tobacco mosaic virus (TMV) infected tobacco leaves. Three main isozyme complexes were obtained: one particulate fraction (the particulate hexokinase phosphorylating both glucose and fructose, EC 2.7.1.1), and two soluble fractions (the soluble hexokinase phosphorylating both the glucose and the fructose, and the soluble fructokinase, which phosphorylates primarily fructose, EC 2.7.1.4). The total fructokinase activities were nearly twice higher than the total glucokinase activities (188.6 % of glucokinase activity in healthy plants and 181.3 % in infected plants). The total particulate glucokinase activity was increased to 120.6 % and the fructokinase to 118.9 % in TMV infected tissue when compared with healthy control. The similar pattern of activity was observed for soluble hexokinase isozymes - the sum of soluble glucokinase activity was increased to 175.4 % and of fructokinase activity to 131.2 % in TMV infected tissue. The isozymes isolated both from the healthy control and TMV-infected leaves had the similar elution profiles, displayed Michaelis-Menten kinetics, showed the identical profiles of pH optima and were Mg²⁺ dependent with the highest enzyme activity at equimolar Mg²⁺ and ATP concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Differences in the apparent molecular weight of soluble and mitochondrial hexokinases of rat brain

I n B rain tissue only 10–20 percent of the total hexokinase activity (EC 2.7.1.1) is present in the cytoplasm whereas the particulate activity is associated with mitochondria (B achelard , 1967; T eich-graber , B iesold and P igarewa , in press). Either small (W ilson , 1968) or insignificant kinetic differences (T hompson and B achelard , 1970; B igl , M üller and B iesold , 1971) between soluble and particulate enzyme activity have recently been reported. Though the DEAE cellulose column chromatography patterns of both hexokinases were reported to be similar (T hompson and B achelard , 1970; B igl , M üller and B iesold , 1971), differences were found between soluble and mitochondrial hexokinase after gel electrophoresis separation (B achelard , 1967; B igl , M uller and B iesold , 1971). Recently it has been shown in this laboratory that Triton X-100 also enhances the hexokinase activity of the cytoplasm as has already been demonstrated for the mitochondrial form (in preparation).
The purpose of the experiments described here was to elucidate whether the soluble cytoplasmic hexokinase differs from the mitochondrial-bound hexokinase in respect to its apparent molecular weight. To diminish the occurrence of experimental artefacts, different extraction procedures were employed. Furthermore, the effect of Triton X-100 on the molecular weight was investigated in an attempt to reveal an explanation for the increase in enzymic activity after incubation of hexokinase with this detergent.     

Some properties of soluble and solubilized particle-bound hexokinase

Abstract— Particle-bound hexokinase of rat brain homogenates was solubilized by successive treatment with 0-9 M-NaCl or 003 M-ATP (pH 8.0) and 0-5% (w/v) Triton X-100. This solubilized hexokinase and the soluble hexokinase present in cytoplasm of rat brain homogenates were chromatographed on DEAE-cellulose and some kinetic properties of the isolated hexokinase peaks were studied. The chromatographic separation was greatly influenced by the EDTA-concentration of the buffer used. No significant differences were observed in the chromatographic pattern and in the apparent K_m-values for ATP and glucose and the apparent K_t for glucose-6-phosphate (versus ATP) between the soluble and particulate hexokinase solubilized by different reagents. On agarose-electrophoresis the solubilized particulate enzyme migrates as one single band, the soluble hexokinase separates into one major and two minor bands.     

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.     

Relative levels of hexokinase in isolated neuronal, astrocytic, and oligodendroglial fractions from rat brain

The levels of hexokinase, as well as those of the cytoplasmic glycolytic enzyme lactate dehydrogenase and the mitochondrial tricarboxylic acid cycle enzymes fumarase and citrate synthase, have been determined in whole rat brain and in neuronal, astrocytic, and oligodendroglial fractions isolated from rat brain. Compared with either whole brain or with isolated neurons or astrocytes, oligodendroglia are low in hexokinase content. This provides direct confirmation for the conclusion, based on an electron microscopic immunohistochemical method, that oligodendroglia, compared with other neural structures, contain relatively low levels of this key enzyme of glucose metabolism. Based on this confirmation, it is concluded that the electron-microscopic immunohistochemical procedure provides a valid indication of hexokinase content, and thus that other structures shown to stain weakly by the latter technique (e.g., dendritic terminals of cerebellar granule and Purkinje cells) are, indeed, low in hexokinase activity.     

Predominance of the Cytoplasmic Form of Brain Hexokinase in Cultured Astrocytes

Abstract: Astrocytes have been cultured from neonatal rat brain according to the flask culture procedure of Booher and Sensenbrenner. Approximately 80% of the hexokinase (ATP: d -hexose 6-phosphotransferase, EC 2.7.1.1) activity is found in the soluble fraction in homogenates of these cells, in contrast to only 20% of the total activity in the soluble fraction of whole brain homogenates. The hexokinase from the cultured astrocytes has been compared with the cytoplasmic and glucose-6-P-solubilized mitochondrial enzymes from whole brain. In kinetic properties and pH-activity relationships, the glial hexokinase was similar to the cytoplasmic enzyme but different from the mitochondrial enzyme of whole brain. Using immunohistochemical methods for detecting hexokinase localization at the electron microscopic level, most of the cells showed prominent staining of cytoplasmic areas. If the cultured astrocytes are accepted as valid models for astrocytes in situ , these results support the suggestion of Bigl and co-workers that the predominant form of hexokinase in glial cells is the cytoplasmic enzyme.     

DNA polymerases from non stimulated and phytohemagglutinin stimulated normal human lymphocytes

The presence and some properties of DNA polymerases isolated from normal human lymphocytes, non stimulated and stimulated by phytohemagglutinin, are described. In the non stimulated lymphocytes two cytoplasmic DNA polymerases are found, one eluting from DEAE cellulose at 0.07 M NaCl (CI_n) and the other at 0.13 M NaCl (CII_n). In the nuclear soluble fraction only one enzyme activity is found (NI_n) which does not adsorb to DEAE cellulose. In the cytoplasm of stimulated lymphocytes only one enzyme activity is detected (CI_s) which elutes from DEAE cellulose at 0.12 M NaCl. The nuclear soluble fraction contains two activities, NI_s, which does not adsorb to DEAE cellulose, and NII_s, which elutes from DEAE cellulose at 0.07 M NaCl. Some properties of the different enzymes are described which indicate that NI_n and NI_s enzymes are clearly different from the others.     

Changes in hexokinase isoenzymes in regions of rat brain during thyroid deficiency

In this work, activities of hexokinase isoenzymes Type I and Type II were measured in the soluble and particulate fractions from the brain regions (cerebral hemispheres (cerebrum), cerebellum and brain stem) of the thyroidectomized adult rats as well as of the thyroidectomized rats administered with triiodothyronine. Thyroidectomy generally decreased the hexokinase activity associated with particulate and soluble fractions. Hexokinase Type II isoenzyme was more affected than the Type I isoenzyme. Administration of triiodothyronine to the hypothyroid rats abolished the effect of thyroidectomy. Adult brain enzymes have been generally considered not be affected by thyroid hormones. The data obtained in this work are suggestive of an effect of thyroid hormones on hexokinase in the adult brain. Since the effects of thyroidectomy on the energy metabolism of the heart tissue are well known, the heart tissue was also studied for comparison.     

THE DISTRIBUTION OF CALCIUM-DEPENDENT PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHODIESTERASE IN RAT BRAIN

Abstract— The properties of Ca2⁺-dependent phosphatidylinositol-phosphodiesterase in membrane fractions and supernatants prepared from rat brain have been examined with the aim of providing firm evidence for the existence of a membrane-bound activity distinct from the soluble enzyme found in the cytosol (EC 3.1.4.10). The soluble enzyme is either stimulated or inhibited at pH 7.0 by deoxycholate depending on the ratio of detergent to substrate. The effects of deoxycholate are pH dependent and result in a shift of the enzyme optimum to a higher pH if the enzyme is assayed in the presence of deoxycholate. The soluble enzyme cannot hydrolgse membrane-bound phosphatidylinositol (in ₃₂P-labelled rat liver microsomes) unless deoxycholate is present. The pH optimum is 6.7 for this detergent-dependent hydrolysis and this is probably dependent on the ionization of deoxycholic acid. The lactate dehydrogenase (EC 1.1.1.27) content of rat brain membrane fractions has been measured to estimate the contamination of these fractions by supernatant phosphatidylinositol-phosphodiesterase. No evidence has been found for phosphatidylinositol-phosphodiesterase activities that cannot be explained by such contamination. It is concluded that all the properties of calcium-dependent phospha-tidylinositol-phosphodicsterase in rat brain can be explained by the existence of only the solublc cyto-plasmic enzyme: no evidence confirming a distinct membrane-bound activity has been obtained.     

Relationship between brain mitochondrial hexokinase and neuronal function

Mitochondrially bound brain hexokinase is solubilized by anesthetics and this effect has been suggested to contribute to anesthesia. In the present investigation the influence of the metabolic inhibitor 2-deoxy-D-glucose (2-DOG) was studied. An isolated rat brain preparation was used to avoid the contribution of peripheral reactions. Isolated rat brains were perfused for 45 min with media containing 4 mmol/l glucose, 10 mmol/l 2-DOG and/or 0.4 mmol/l thiopental. The EEG was monitored and acetylcholine, 2-DOG and its 6-phosphate, as well as the intracellular distribution of hexokinase activity were determined in brain tissue. Soluble hexokinase activity in brain cortex was enhanced by 2-DOG, as also by thiopental, and even more pronounced by both drugs used together. Results from in vitro experiments suggest that solubilization of mitochondrial hexokinase after 2-DOG is mediated by intracellularly accumulated 2-DOG-6-phosphate. 2-DOG produced a significant impairment of neuronal activity, revealing EEG patterns similar to those caused by thiopental anesthesia. Cortical acetylcholine levels were elevated by 2-DOG, as well as by thiopental, and again both drugs showed an additive effect when used in combination. This effect which may be the result of an inhibition of acetylcholine release, was also detectable in mice in vivo after 5 g 2-DOG/kg i.p., whereas the same dose of 3-O-methylglucose had no effect. The results provide further evidence that mitochondrial hexokinase may be involved in the relationship between cerebral metabolism and brain function.A preliminary report of these results has been made at the 22^nd spring meeting of the Deutsche Pharmakologische Gesellschaft at Mainz (38).     

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.     

Factors affecting the glucose 6-phosphate inhibition of hexokinase from cerebral cortex tissue of the guinea pig

1. The inhibition of hexokinase by glucose 6-phosphate has been investigated in crude homogenates of guinea-pig cerebral cortex by using a sensitive radio-chemical technique for the assay of hexokinase activity. 2. It was observed that 44% of cerebral-cortex hexokinase activity did not sediment with the microsomal or mitochondrial fractions (particulate fraction), and this is termed soluble hexokinase. The sensitivities of soluble and particulate hexokinase, and hexokinase in crude homogenates, to the inhibitory actions of glucose 6-phosphate were measured; 50% inhibition was produced by 0.023, 0.046 and 0.068mm-glucose 6-phosphate for soluble, particulate and crude homogenates respectively. 3. The optimum Mg(2+) concentration for the enzyme was about 10mm, and this appeared to be independent of the ATP concentration. In the presence of added glucose 6-phosphate, raising the Mg(2+) concentration to 5mm increased the activity of hexokinase, but above this concentration Mg(2+) potentiated the glucose 6-phosphate inhibition. When present at a concentration above 1mm, Ca(2+) ions inhibited the enzyme in the presence or absence of glucose 6-phosphate. 4. When the ATP/Mg(2+) ratio was 1.0 or below, variations in the ATP concentration had no effect on the glucose 6-phosphate inhibition; above this value ATP inhibited hexokinase in the presence of glucose 6-phosphate. ATP had an inhibitory effect on soluble hexokinase similar to that on a whole-homogenate hexokinase, so that the ATP inhibition could not be explained by a conversion of particulate into soluble hexokinase (which is more sensitive to inhibition by glucose 6-phosphate). It is concluded that ATP potentiates glucose 6-phosphate inhibition of cerebral-cortex hexokinase, whereas the ATP-Mg(2+) complex has no effect. Inorganic phosphate and l-alpha-glycerophosphate relieved glucose 6-phosphate inhibition of hexokinase; these effects could not be explained by changes in the concentration of glucose 6-phosphate during the assay. 5. The inhibition of hexokinase by ADP appeared to be independent of the glucose 6-phosphate effect and was not relieved by inorganic phosphate. 6. The physiological significance of the ATP, inorganic phosphate and alpha-glycerophosphate effects is discussed in relation to the control of glycolysis in cerebral-cortex tissue.     

The complete amino acid sequence of the catalytic domain of rat brain hexokinase, deduced from the cloned cDNA

The complete amino acid sequence of the catalytic domain of rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been deduced from the nucleotide sequence of cloned cDNA. Extensive similarity in sequence, taken to indicate similarity in secondary and tertiary structure, is seen between the mammalian enzyme and yeast hexokinase isozymes A and B. All residues critical for binding glucose to the yeast enzyme are conserved in brain hexokinase. A location for the substrate ATP binding site is proposed based on relation of structural features in the yeast enzyme to characteristics commonly observed in other nucleotide binding enzymes; sequences in regions proposed to be important for binding of ATP to the yeast enzyme are highly conserved in brain hexokinase.     

Distribution and properties of aldehyde dehydrogenase in regions of rat brain

Abstract: NAD-dependent aldehyde dehydrogenases (EC 1.2.1.3) were isolated from various subcellular organelles as well as from different regions of rat brain. The mitochondrial, microsomal, and cytosolic fractions were found to contain 40%, 28%, and 12%, respectively, of the total aldehyde dehydrogenase (5.28 ± 0.44 nmol NADH/min/g tissue) found in rat brain homogenate when assayed with 70 μ. M propionaldehyde at pH 7.5. The total activity increased to 17.3 ± 2.7 nmol NADH/min/g tissue when assayed with 5 m M propionaldehyde. Under these conditions the three organelles contained 49%, 23%, and 9%, respectively, of the activity. The enzyme isolated from cytosol possessed the lowest K _m. The molecular weight of the enzyme isolated from all three subcellular organelles was ∼100,000. Four activity bands were found by electrophoresis of crude homogenates, isolated mitochondria, or microsomes on cellulose acetate strips. Cytosol possessed just two of the forms. The total activity was essentially the same in homogenates obtained from cortex, subcortex, pons-medulla, or cerebellum. Further, the enzyme had the same molecular distribution and total activity in each of these four brain regions. Disulfiram was found to be an in vivo and in vitro inhibitor of the enzymes obtained from these brain regions. Mercaptoethanol, required for the stability of the enzyme, reversed the inhibition produced by disulfiram. The effect was greater for enzyme isolated from cytosol than from mitochondria. Calculations led to the prediction that aldehydes such as acetaldehyde are oxidized in cytosol.     

Difference in hydrophobicity between mitochondria-bindable and non-bindable forms of hexokinase purified from rat brain

Hexokinase able to bind to mitochondria was purified to homogeneity from rat brain by two successive DEAE-cellulose chromatographic steps. The enzyme lost only the binding ability with almost undetectable change in molecular weight on mild chymotrypsin digestion. The bindable hexokinase was adsorbed to a Phenyl-Sepharose column and eluted with a Lubrol PX gradient, whereas non-bindable hexokinase and yeast hexokinase were not adsorbed under the similar conditions. These results suggest that mitochondria-bindable hexokinase has a hydrophobic region on its surface, which is responsible for the specific interaction with mitochondria.     

Studies on the Linkage of Energy Metabolism and Neuronal Activity in the Isolated Perfused Rat Brain

An isolated rat brain preparation was perfused using glucose-free (=aglycemic) media. The high-energy phosphates, substrates of the glycolytic pathway, free atnino acids, acetylcholine as well as the intracellular distribution of hexokinase activity were determined in brain tissues. The EEG was evaluated visually. The levels of glycolytic substrates, glutamate, and glutamine in cortical tissue decreased after aglycemic perfusion, whereas the aspartate level increased and the GABA level remained unchanged. The high-energy phosphate content seemed to be unaffected for about 15 min of aglycemic perfusion and fell significantly after 20 min. The EEG of the isolated brain changed rapidly after starting aglycemic perfusion and became isoelectric after 12–15 min. Hyperglycemic perfusion (35 mmol glucose per liter perfusion medium) did not alter the energy metabolism of the isolated brain. The breakdown of cerebral energy metabolism and of EEG activity was postponed when thiopental was added to the perfusion medium. The soluble hexokinase activity measured in cortical tissue was reduced after aglycemic perfusion and was enhanced after thiopental. Hyperglycemic perfusion did not influence the intracellular hexokinase distribution. The acetylcholine level in the striatum of the isolated rat brain was significantly decreased by aglycemia and was increased in hypothalamus by thiopental. It was suggested that hexokinase bound to the mitochondrial membrane may play an important role in the relationship of energy metabolism and neuronal activity.     

Glycogen metabolizing enzymes in brain

Three enzymes, glycogen phosphorylase, glycogen synthase, and phosphoglucomutase were evaluated in subcellular fractions and in brain regions. Also the development of each of these enzymes was evaluated in whole brain homogenates. Each enzyme increased during the first three weeks of post partum in a manner that is similar to the development of glycolytic enzymes during this period. The specific activity of each enzyme in various subcellular fractions indicated that the enzymes were primarily soluble. Also unlike the glycolytic enzyme phosphoglycerate kinase, the glycogen metabolizing enzymes had a lower specific activity in synaptosomes than in particle free supernatant fractions of homogenates. Regarding regional distribution small (less than twofold) but significant differences were seen between different brain areas. An inverse relationship between the glycogen metabolizing enzymes and hexokinase was observed, that is, regions highest in glycogen synthase and glycogen phosphorylase were lowest in hexokinase and regions highest in hexokinase were lowest in the glycogen metabolizing enzymes.     

Production and properties of polygalacturonate lyase by an alkalophilic microorganism Bacillus sp. RK9.

Bacillus sp. RK9 was isolated from soil and produced a constitutive polygalacturonate lyase. Production of the enzyme required the presence of complex nitrogen (peptone and yeast extract). Highest activity was obtained with an initial pH of 9.7. The organism was alkalophilic. No growth occurred below pH 7.5. The enzyme was purified by salt precipitation and diethylaminoethyl (DEAE) cellulose ion-exchange chromatography. The pH optimum for activity was 10.0 in 0.01 M glycine-NaOH buffer. Calcium alone, of divalent cations, activated the enzyme by 2.9-fold. Complete inhibition of enzyme activity was achieved by 1 mM ethylenediaminetetraacetic acid (EDTA). Hydrolysis of substrate occurred in a random fashion and the enzyme was 50% more active towards acid soluble pectic acid (ASPA) than towards sodium polypectate.