title: Importance of the flux of phosphate across the inner membrane of kidney mitochondria for the activation of glutaminase and the transport of glutamine.
abstract: The effect of mersalyl, an inhibitor of phosphate transport across the inner mitochondrial membrane, was investigated on the uncoupled respiration of pig kidney mitochondria in the presence of glutamine as substrate and on the activity of the phosphate-dependent glutaminase in the intact organelles. In addition, the submitochondrial location of the enzyme was reinvestigated. (1) It was found that mersalyl completely inhibits uncoupled respiration of the mitochondria in the presence of glutamine as substrate, whereas respiration with glutamate was not affected. The same amount of mersalyl which inhibits coupled oxidation of glutamine also inhibits coupled oxidation of glutamate and some other substrates. (2) Mersalyl strongly inhibited the activation of glutaminase in intact mitochondria only in the presence of inhibitiors of electron transport or of an uncoupler. The addition of a detergent prevented or fully released the inhibition. The effect of mersalyl was observed even when the mitochondria were pre-incubated with phosphate or incubated in the phosphate-free medium. If mersalyl and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were added 3 min after pre-incubation with phosphate the same intramitochondrial concentration of the anion as in control experiments was found, whereas the activity of glutaminase was severely inhibited. These findings suggest that the activation of the enzyme by phosphate in intact nonenergized mitochondria occurs only if the activator moves across the inner mitochondrial membrane. (3) Mersalyl (plus CCCP) markedly decreased [14C]glutamine- and [32P]-phosphate-permeable mitochondrial spaces. A close correlation between the decrease of phosphate and glutamine permeable spaces and the inhibition of glutaminase activity was found. (4) If the activation energy of the enzyme was determined with frozen mitochondrial preparations, a discontinuity or break in the Arrhenius plot was observed, whereas the presence of a detergent completely abolished the break. Digitonin or ultrasonic treatment of the mitochondria followed by separation of the membrane and the soluble fraction revealed that glutaminase is a membrane-bound enzyme. On the basis of these findings it is concluded that there is an association between the transport on one side and the transport of glutamine and glutaminase activity on the other. It is possible that the movement of phosphate across the membrane activates the enzyme which facilitates diffusion of glutamine down a concentration gradient. However, the existance of a specific glutamine-phosphate carrier is not ruled out.
mesh_terms: D000818:Animals; D001693:Biological Transport, Active; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005972:Glutaminase / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D008634:Mersalyl / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D008933:Mitochondrial Swelling / Q000187:drug effects; D010101:Oxygen Consumption / Q000187:drug effects; D010710:Phosphates / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D013395:Sucrose / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D010710:Phosphates; D005973:Glutamine; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D013395:Sucrose; D008634:Mersalyl; D005972:Glutaminase