Photoaffinity labeling of the tight ADP binding site of the chloroplast coupling factor one (CF1): the effect on the CF1-ATPase activity.
Chloroplast thylakoid membranes contain tightly bound ADP which is intimately involved in the mechanism of photophosphorylation. The photoaffinity analog 2-azido-ADP binds tightly to spinach thylakoid membrane-bound coupling factor one (CF1) and, in a manner similar to ADP, inhibits the light-triggered ATPase activity (Czarnecki, J.J., Abbott, M.S. and Selman, B.R. (1983) Eur. J. Biochem. 136, 19-24). Ultraviolet irradiation of thylakoid membranes containing noncovalently, tightly bound 2-azido[beta-32P]ADP results in the inactivation of both the methanol-stimulated MgATPase activity of the membrane-bound CF1 and the octylglucoside-dependent MgATPase activity of the solubilized enzyme. There is a linear correlation between the loss of enzyme activity and the covalent incorporation of the photoaffinity analog. Full inactivation of catalytic activity is estimated to occur upon incorporation of 1.07 mol analog and 0.65 mol analog per mol enzyme for the methanol- and octylglucoside-stimulated activities, respectively. Since 2-azido-ADP modifies only the beta subunit of the CF1 and since there are probably three beta subunits per CF1, these results indicate strong cooperativity among beta subunits and between the site of tightly bound nucleotides and the catalytic sites.
Czarnecki
J J
JJ
Dunham
K R
KR
Selman
B R
BR
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase; D002736:Chloroplasts / Q000378:metabolism* / Q000528:radiation effects; D004789:Enzyme Activation / Q000187:drug effects; D005960:Glucosides / Q000494:pharmacology; D000432:Methanol / Q000494:pharmacology; D010785:Photophosphorylation; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; D005960:Glucosides; C018619:octyl-beta-D-glucoside; D000244:Adenosine Diphosphate; C022295:2-azidoadenosine 3',5'-diphosphate; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases; D000432:Methanol