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.
Highlights
From the $Department of Biochemistry and the §Institute for Enzyme Research, University of Wisconsin-Madison, Madison, Wisconsin 53706
Thylakoid membrane polypeptides (50 pgof chlorophyll) were separated by SDS-gel electrophoresis [36]and slices containing the @ subunit of the CF1 wereexcised from the gel.Gelslices containing the photolabeled @ subunit were subjected to electrophoresis and partial proteolysis according to the method of Cleveland et al [37] as described under "Experimental Procedures." Lanes 1,3,5, into thylakoid membranescontaining the slowly formed, and 7, photolabeled 0 subunit plus 0 (LaneI ), 15ng (Lane3), 100 ng tightly bound 2-N3-[32P]ATP(90 PM 2-N3-ADP, 90-s illumi- (Lane5),or 300 ng (Lane 7) of S. aureus protease V8; Lanes 2,4, 6, nation)
Our results imply that the analog is a suitable photoaffinity label for the ATP tight binding site(s)
Summary
From the $Department of Biochemistry and the §Institute for Enzyme Research, University of Wisconsin-Madison, Madison, Wisconsin 53706. 1-4 demonstrate thata rapidly labeled,tightly bound species of 2-N3-ATPis formed by brief illumination of thylakoid membranes in the presence of low concentrations of 2-N3-ADP and 32Pi.The rate, extent, and concentration dependence of the binding are identical to those observed with ADP.
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