| Abstract: | The photoaffinity analog 2-azido-ADP has been used to investigate the high-affinity binding site(s) for ATP on the chloroplast thylakoid membrane. Photophosphorylation of 2-azido-ADP results in the rapid formation of 2-azido-ATP, which remains tightly bound to the membranes after extensive washing. The kinetic parameters of the tight binding of ATP and of 2-azido-ATP are similar (apparent Km = 1-2 microM; maximum extent = 0.2-0.4 nmol/mg of chlorophyll). Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2-azido-gamma-32P]ATP induces covalent incorporation of the label exclusively into the beta subunit of the chloroplast coupling factor one. Previous results have shown that the tight binding site for ADP is also located on the beta subunit of the ATP synthase (Czarnecki, J. J., Abbott, M. S., and Selman, B. R. (1983) Eur. J. Biochem. 136, 19-24). To further characterize the tight binding sites for ADP and ATP, the membrane-bound coupling factor has been covalently modified with either tightly bound 2-azido-gamma-32P]ATP or tightly bound 2-azido-beta-32P]ADP. The photolabeled beta subunits have been isolated and subjected to partial proteolytic digestion and SDS-gel electrophoresis. The results of these experiments demonstrate that the tight binding sites for ADP and ATP are located on identical portions of beta subunit polypeptide. |
