The mode of activation and regulation of glutaminase in intact kidney mitochondria.
Experiments were carried out with isolated pig renal-cortex and rat kidney mitochondria. Respiration, glutaminase activity and swelling of the mitochondria were followed by an oxygen electrode, an NH+4-sensitive electrode and spectrophotometrically. The inhibitors of the transport of glutamate (avenaciolide) and phosphate (mersalyl) across the inner mitochondrial membrane were employed. On the basis of the experimental findings we made the following conclusions: 1. There are two mechanisms by which glutamate can leave the inner space of kidney mitochondria: (a) an electrogenic efflus coupled to the respiration driven proton translocation and the presence of a membrane potential (positive outside), and (b) an electroneutral glutamate-hydroxyl exchange which is inhibited by avenaciolide and which operates in both directions. 2. The activation of glutaminase in intact mitochondria by phosphate occurs only if the activator moves across the inner mitochondrial membrane. The enzyme can be activated even when the movement of phosphate is inhibited provided that the mitochondria are energized. 3. Glutaminase is intimely associated with the inner mitochondrial membrane so that the part of the enzyme with the binding site for phosphate is embedded in the membrane, whereas the part with the binding sites for the substrate and glutamate is exposed toward the matrix space. The mechanism of allosteric activation of glutaminase and of the efflux of glutamate from mitochondria are probably the crucial factors which regulate the enzyme activity, and, consequently, renal ammoniagenesis in vivo.
D000818:Animals; D004072:Digitonin; D003576:Electron Transport Complex IV / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D005972:Glutaminase / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D007672:Kidney Cortex / Q000201:enzymology; D007700:Kinetics; D007783:Lactones / Q000494:pharmacology; D008634:Mersalyl / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D008933:Mitochondrial Swelling / Q000187:drug effects; D008954:Models, Biological; D010101:Oxygen Consumption / Q000187:drug effects; D010710:Phosphates / Q000494:pharmacology; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D013552:Swine; D013696:Temperature
D007783:Lactones; D010710:Phosphates; D008634:Mersalyl; D005969:Glutamate Dehydrogenase; D003576:Electron Transport Complex IV; D005972:Glutaminase; D004072:Digitonin