The significance of the attachment of rat kidney glutaminase to the inner mitochondrial membrane.
The inner mitochondrial membrane of rat kidney mitochondria was altered by 0.03% Triton X-100 treatment in such a way as to render it permeable to NAD and CoA molecules without release of phosphate-dependent glutaminase. A break of linearity in the Arrhenius plot of the enzyme activity was characteristic for a conformational change of a membrane-bound enzyme. The activity of phosphate-dependent glutaminase immobilized in the inner mitochondrial membrane, as studied in 0.03% Triton X-100-treated mitochondria, and solubilized, as in the supernatant of sonicated mitochondria, was hyperbolic with respect to glutamine concentration. Under optimal conditions (pH 8.6 and 100 mM phosphate) the Vmax and Km were 216 +/- 12 nmol/mg per min and 2.7 +/- 0.4 mM, respectively, for Triton X-100-treated mitochondria, and 121 +/- 8 nmol/mg per min and 15.9 +/- 1.8 mM for sonicated mitochondria. Under near physiological conditions (pH 7.8 and 20 mM phosphate), distinct differences in phosphate-dependent glutaminase kinetics were observed. The Vmax as 29.8 +/- 0.4 and 2.6 /- 0.3 nmol/mg per min and the apparent Km 1.55 +/- 0.06 and 24.5 +/- 6.6 mM for Triton X-100 and sonicated mitochondria, respectively. The sigmoidal activation by phosphate at pH 7.8 was significantly shifted to the left in Triton X-100-treated as compared to sonicated mitochondria. As opposed to the data obtained in sonicated mitochondria, the kinetics of phosphate-dependent glutaminase in 0.03% Triton X-100-treated mitochondria agreed quite well with those obtained in intact, rotenone-inhibited and metabolically active mitochondria. These results suggest that an attachment of phosphate-dependent glutaminase to the inner membrane of kidney mitochondria has a profound effect on its kinetics, particularly under near physiological conditions.
D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D005972:Glutaminase / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D017830:Octoxynol; D010710:Phosphates / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013010:Sonication
D010710:Phosphates; D005973:Glutamine; D011092:Polyethylene Glycols; D017830:Octoxynol; D005972:Glutaminase