Azide as a probe of co-operative interactions in the mitochondrial F1-ATPase.
(1) The hydrolytic activity of the isolated mitochondrial ATPase (F1) is strongly inhibited by azide. However, at very low ATP concentration (1 microM or less), no inhibition by azide is observed. (2) The azide-insensitive ATPase activity represents a high-affinity, low-capacity mode of turnover of F1. This is identified with the low Km, low Vmax component seen in steady-state kinetic studies in the absence of azide. (3) The azide-insensitive ATPase activity shows simple Michaelis-Menten kinetics, with Km = 3.2 microM, and Vmax = 1.1 mumol/min per mg (6 s-1). It is unaffected by anions such as sulphite, or by increasing pH in the range 7 to 8, both of which stimulate the maximal activity of F1. (4) Both the azide-insensitive and azide-sensitive components of F1-ATPase activity are equally inhibited by labelling the enzyme with 7-chloro-4-nitrobenzofurazan, by binding the natural inhibitor protein, or by cold denaturation of the enzyme. (5) It is concluded that azide-insensitive ATP hydrolysis represents catalysis by F1 involving a single catalytic site, and that azide acts by abolishing intersubunit cooperativity between the three catalytic sites of F1. Azide-sensitivity is thus a useful probe for events which affect the active site of F1 directly.
Harris
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DA
Department of Biochemistry, University of Oxford, U.K.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000838:Anions; D001386:Azides / Q000494:pharmacology*; D001572:Benzofurans / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007293:Inosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013447:Sulfites / Q000494:pharmacology
D000838:Anions; D001386:Azides; D001572:Benzofurans; D013447:Sulfites; C043708:7-chloro-4-nitrobenzofuran; D007293:Inosine Triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; C025026:sodium sulfite