Photoaffinity labeling with 2-azidoadenosine diphosphate of a tight nucleotide binding site on chloroplast coupling factor 1

Abstract

An analog of ADP containing an azido group at the C-2 position of the purine ring was synthesized and used as an affinity probe of the membrane-bound coupling factor 1 [CF1] of spinach chloroplast thylakoid membranes. The 2-azido-ADP inhibited light-induced dark binding of ADP at the tight nucleotide binding site on the thylakoid membranes. The 2-azido-ADP itself bound tightly to the thylakoid membranes, with 1 .mu.M as the concentration giving 50% maximum binding. Tight binding of the analog required the thylakoid membranes to be energized, and the nucleotide remained bound after repeated washings of the membranes. The maximum extent of tight binding of the analog (1.2-1.3 nmol/mg of chlorophyll [Chl]) was stoichiometric with the known CF1 content of the thylakoid membranes but somewhat higher than that observed for ADP (0.5-0.9 nmol/mg Chl). Tight binding of 2-azido-ADP was decreased by the simultaneous addition of ADP. UV photolysis of washed thylakoid membranes containing tightly-bound 2-azido-[.beta.-32P]ADP resulted in the covalent incorporation of label into the membranes. Isolation of the chloroplast CF1 from these membranes followed by NaDod[dodecyl]SO4 gel electrophoresis demonstrated that the analog was covalently bound to the .beta. subunit of the CF complex.