Open AccessLocalization of the tight ADP‐binding site on the membrane‐bound chloroplast coupling factor one
Author(s) -
CZARNECKI Joseph J.,
ABBOTT Marilyn S.,
SELMAN Bruce R.
Publication year - 1983
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1983.tb07699.x
Subject(s) - thylakoid , photophosphorylation , membrane , f atpase , photoaffinity labeling , binding site , affinity label , biochemistry , biophysics , chemistry , atpase , nucleotide , chloroplast , biology , enzyme , gene
The photoaffinity analog 2‐azido‐ADP (2‐azidoadenosine 5′‐diphosphate) was used as a probe of the spinach chloroplast ATP synthase. The analog acted as a substrate for photophosphorylation. Several observations suggested that 2‐azido‐ADP and ADP bound to the same class of tight nucleotide binding sites: (a) 2‐azido‐ADP competitively inhibited ADP tight binding ( K i = 1.4 μM); (b) the concentration giving 50% maximum binding, K 0.5 for analog tight binding (1 μM) was similar to that observed for ADP (2 μM); (c) nucleotide tight binding required prior membrane energization and was completely reversed by re‐energization; (d) the tight binding of 2‐azido‐[β‐ 32 P]ADP was completely prevented by ADP; (e) the analog inhibited the light‐triggered ATPase activity at micromolar concentrations. Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2‐azido‐[β‐ 32 P]ADP resulted in the covalent incorporation of the label into the membranes. Denaturing polyacrylamide gel electrophoresis of the labeled membranes demonstrated that the β subunit of the coupling factor one complex was the only polypeptide in the thylakoid membranes which was labeled. These results identify the β subunit of the coupling factor as the location of the tightly bound ADP on the thylakoid membranes.