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. 1982 Dec;79(24):7744–7748. doi: 10.1073/pnas.79.24.7744

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

Joseph J Czarnecki *,†, Marilyn S Abbott †, Bruce R Selman †
PMCID: PMC347424  PMID: 16593263

Abstract

An analog of ADP containing an azido group at the C-2 position of the purine ring has been synthesized and used as an affinity probe of the membrane-bound coupling factor 1 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 μM 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) was stoichiometric with the known coupling factor 1 content of thylakoid membranes but somewhat higher than that observed for ADP (0.5-0.9 nmol per mg of chlorophyll). 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-[β-32P]ADP resulted in the covalent incorporation of label into the membranes. Isolation of the chloroplast coupling factor 1 from these membranes followed by NaDodSO4 gel electrophoresis demonstrated that the analog was covalently bound to the β subunit of the coupling factor complex.

Keywords: ATPase, adenine nucleotide analog

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Selected References

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