Interaction between F1-ATPase and its naturally occurring inhibitor protein. Studies using a specific anti-inhibitor antibody.
An antibody was raised to cross-linked ox-heart mitochondrial inhibitor protein, which cross-reacts with the free inhibitor but with no other mitochondrial membrane protein. This antibody yields an immunoprecipitate with the cross-linked inhibitor protein, but a soluble antibody-antigen complex with free inhibitor. The antibody binds well to inhibitor protein whether the latter is complexed with F1-ATPase or not. Antibody binding has no effect on the ability of the inhibitor protein to inhibit the ATPase activity of F1. These findings suggest that the antibody does not block the site of interaction between the inhibitor and F1. The inhibitor protein content of submitochondrial membrane preparations was determined by radioimmunoassay, activity measurements and an immunochemical 'back titration' technique. The inhibitor content of the membranes is shown to decrease after energisation, suggesting a loss of inhibitor from the membranes into solution. Binding antibody to the inhibitor protein on submitochondrial particles has no effect on the steady-state rate of phosphorylation, but it increases the lag phase preceding phosphorylation from 30 to 54 s. The rate constant for the approach to the steady state drops from 0.078 to 0.052 s-1. This effect confirms that the lag phase is due to inhibition of phosphorylation by the inhibitor protein. The increase in ATPase activity following energisation takes place by a fast phase (80% maximal activity reached within 90 s) and a slower phase (lasting about 10 min.). The rate constant of the rapid phase (0.017 s-1) is of the same order as that for the activation of phosphorylation. It is concluded that the rapid phase of ATPase induction is fast enough for this process to occur simultaneously with the activation of phosphorylation.
Husain
I
I
Jackson
P J
PJ
Harris
D A
DA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002417:Cattle; D007074:Immunoglobulin G / Q000276:immunology; D007163:Immunosorbent Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D010766:Phosphorylation; D011506:Proteins / Q000276:immunology / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000378:metabolism; D000097795:ATPase Inhibitory Protein
D000255:Adenosine Triphosphate; D000906:Antibodies; D007074:Immunoglobulin G; D011506:Proteins; D006180:Proton-Translocating ATPases; D000097795:ATPase Inhibitory Protein