Purification of rat liver glycerate kinase and studies of its enzymatic and immunological properties.
Glycerate kinase was purified to near homogeneity from rat liver mitochondria. Sephadex G-100 chromatography and sodium dodecyl sulfate-gel electrophoresis showed that the enzyme is a monomer with a molecular weight of 51,000-56,000. Kinetic studies indicated that the reaction proceeds by the "rapid equilibrium random sequential" mechanism, and the Michaelis constants were found to be 0.032 mM and 0.091 mM for D-glycerate and ATP, respectively. The binding of D-glycerate specifically protected the enzyme from thermal inactivation. The dissociation constant of the enzyme.D-glycerate complex was similar to the Michaelis constant, suggesting that the protective effect of D-glycerate may be caused by the specific binding of the substrate to the active site of the enzyme. The antibody to the enzyme was prepared by immunizing a rabbit with the purified rat liver glycerate kinase. Immunological experiments indicated that the cytosol and mitochondrial enzymes are indistinguishable. It was also found by immunological studies that the increase in the cytosol and mitochondrial enzyme activity depending on dietary protein intake was proportional to the increase in enzyme protein. These results support our proposal (Kitagawa, Y., Katayama, H., & Sugimoto, E. (1979) Biochim. Biophys. Acta 582, 260-275) that cytosol and mitochondrial glycerate kinase arise from a common translation product and that dietary protein regulates the distribution of glycerate kinase to the cytosol and mitochondria.
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004044:Dietary Proteins / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D005988:Glyceric Acids / Q000302:isolation & purification / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D005779:Immunodiffusion; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats
D004044:Dietary Proteins; D005988:Glyceric Acids; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020670:glycerate kinase