Difference in kinetic properties between hexokinase type I isoenzymes from various rat tissues with reference to the effect of a thiol inhibitor.
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ABSTRACT:
SUBMITTER: Kamikashi T
PROVIDER: S-EPMC1166094 | biostudies-other | 1974 Jan
REPOSITORIES: biostudies-other
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Project description: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.
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Project description:The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.
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Project description:We have constructed a relational data base containing the kinetic properties of the isoenzymes of hexokinase using the Knowledgeman data-base program on an IBM PC microcomputer. The natural subunit of this data base is the refereed publication, 165 of which were included. Reported values for the Mr (approx. 97,000) are in good agreement, but this agreement becomes progressively worse as one examines the Km values for glucose and ATP and the Ki for glucose 6-phosphate, where the reported values are spread over three orders of magnitude. Some quantities are very thinly or unreliably determined. Experimental conditions, especially free Mg2+ concentration, are rarely close to physiological. Reasons for the spread or uncertainty of numbers, and the distinctions that can be made between isoenzymes despite this spread, are discussed.
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Project description:The cellular distribution of hexokinase isoenzymes, N-acetylglucosamine Kinase and pyruvate kinases in rat liver was studied. Hepatocytes and non-parenchymal cells with high viability and almost no cross-contamination were obtained by perfusion in situ of the liver with collagenase, with the use of an enriched cell-culture medium in all steps of cell isolation. Separation of hexokinase isoenzymes was done by DEAE-cellulose chromatography, and enzyme activities were measured by a specific radioassay. Cytosol from isolated hepatocytes contained high-affinity hexokinases A, B and C, in addition to hexokinase D. The last-mentioned represented about 95% of total glucose-phosphorylating activity. Only hexokinase A was found associated t the particulate fraction. Isolated non-parenchymal cells contained only hexokinases A, B and C. N-Acetylglucosamine kinase was measured with a specific radioassay and was found as a single enzyme form in both hepatocytes and non-parenchymal cells, with higher activities in the former. Pyruvate kinase isoenzyme L was present only in the hepatocytes and isoenzyme K only in the non-parenchymal liver cells, confirming that they are good cellular markers.
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Project description:Two forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) present in Salmonella typhimurium were purified to homogeneity from the same cultures by (NH4)2SO4 fractionation and gel filtration, anion-exchange and affinity chromatography. Mr values, subunit structure, amino acid composition and activity and stability conditions were determined for the two forms. Kinetic and regulatory properties of the two purified isoenzymes were studied.
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Project description:A series of cycloplatinated(II) complexes with general formula of [PtMe(Vpy)(PR3)], Vpy = 2-vinylpyridine and PR3 = PPh3 (1a); PPh2Me (1b); PPhMe2 (1c), were synthesized and characterized by means of spectroscopic methods. These cycloplatinated(II) complexes were luminescent at room temperature in the yellow-orange region's structured bands. The PPhMe2 derivative was the strongest emissive among the complexes, and the complex with PPh3 was the weakest one. Similar to many luminescent cycloplatinated(II) complexes, the emission was mainly localized on the Vpy cyclometalated ligand as the main chromophoric moiety. The present cycloplatinated(II) complexes were oxidatively reacted with MeI to yield the corresponding cycloplatinated(IV) complexes. The kinetic studies of the reaction point out to an SN2 mechanism. The complex with PPhMe2 ligand exhibited the fastest oxidative addition reaction due to the most electron-rich Pt(II) center in its structure, whereas the PPh3 derivative showed the slowest one. Interestingly, for the PPhMe2 analog, the trans isomer was stable and could be isolated as both kinetic and thermodynamic product, while the other two underwent trans to cis isomerization.
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Project description:1. The substrate kinetic properties of cerebral hexokinases (mitochondrial and cytoplasmic) were studied at limiting concentrations of both glucose and MgATP(2-). Primary plots of the enzymic activity gave no evidence of a Ping Pong mechanism in three types of mitochondrial preparation tested (intact and osmotically disrupted mitochondria, and the purified mitochondrial enzyme), nor in the purified cytoplasmic preparation. 2. Secondary plots of intercepts from the primary plots (1/v versus 1/s) versus reciprocal of second substrate of the mitochondrial activity gave kinetic constants which differed from those obtained directly from the plots of 1/v versus 1/s or of s/v versus s, although the ratios of the derived constants were consistent. The kinetic constants obtained with the cytoplasmic enzyme from primary and secondary plots were consistent. 3. Deoxyglucose, as alternative substrate, inhibited cytoplasmic hexokinase by competition with glucose, but did not compete when MgATP(2-) was the substrate varied. The K(i) for deoxyglucose when glucose concentrations were varied was 0.25mm. 4. A range of ATP analogues was tested as potential substrates and inhibitors of hexokinase activity. GTP, ITP, CTP, UTP and betagamma-methylene-ATP did not act as substrates, nor did they cause significant inhibition. Deoxy-ATP proved to be almost as effective a substrate as ATP. AMP inhibited but did not act as substrate. 5. N-Acetyl-glucosamine inhibited all preparations competitively when glucose was varied and non-competitively when MgATP(2-) was varied. AMP inhibition was competitive when MgATP(2-) was the substrate varied and non-competitive when glucose was varied. 6. The results are interpreted as providing evidence for a random reaction mechanism in all preparations of brain hexokinase, cytoplasmic and mitochondrial. The kinetic properties and reaction mechanism do not change on extraction and purification of the particulate enzyme. 7. The results are discussed in terms of the participation of hexokinase in regulation of cerebral glycolysis.
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