The use of arylazido-beta-alanyl-ATP as a photoaffinity label for the isolated and membrane-bound mitochondrial ATPase complex.
The effects of a photoaffinity derivate of ATP, arylazido-beta-alanyl-ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl) adenosine 5'-triphosphate, on submitochondrial particles and the partially purified ATPase complex of beef heart mitochondria have been investigated. In the absence of light the ATP analogue has been found to be a substrate for the E132PA1P1-ATP exchange reaction of submitochondrial particles. When photoirradiated in the presence of arylazido-beta-alanyl-ATP, the ATPase activity and the the the [32P]Pi-ATP exchange reaction are inhibited maximally 80%. Arylazido-beta-alanyl-ATP following photolysis is a noncompetitive inhibitor with respect to ATP while arylazido-beta-alanine, the azido-containing adjunct of the ATP analogue, has no inhibitory effect under the same conditions. The inactivating effect of arylazido-beta-alanyl-ATP is prevented in part by the presence of ATP, or ADP and pyrophosphate. Photolabeling produces a covalent binding of the derivative with the F1ATPase being the major protein labeled. The binding of 0.22 mumol of arylazido-beta-alanyl-ATP/mg of mitochondrial protein is associated with a maximal inhibitory effect. The ATPase activity of the partially purified ATPase complex is also sensitive to photoirradiation in the presence of arylazido-beta-alanyl-ATP. When the ATPase complex is associated with liposomes there is an increase in the specific ATPase activity with a 10-fold increase in Vmax and a 4-fold decrease in KmATP associated with a parallel increase in the apparent affinity and maximal inhibitory effect of the arylazido-beta-alanyl-ATP. The photoinhibition of the ATPase complex in the presence of arylazido-beta-alanyl-ATP results in covalent binding of 1.6 mumol of arylazido-beta-alanyl-ATP/mg of protein. The alpha and beta subunits are the only components of the ATPase complex labeled by the [3H]arylazido-beta-alanyl-ATP. The relationship between the arylazido-beta-alanyl-ATP-labeled sites and the nucleotide binding sites on the mitochondrial ATPase is discussed.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D002417:Cattle; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008027:Light; D008929:Mitochondria, Heart / Q000201:enzymology*; D013367:Submitochondrial Particles / Q000201:enzymology
C012761:3'-O-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)adenosine-5'-triphosphate; D000345:Affinity Labels; D001386:Azides; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases