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The distribution of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase (RbuP2Case; EC 4.1

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 ,uM 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-[13-32P]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 (3 subunit of the coupling factor complex.Energy-transducing membranes from mitochondria, chloroplasts, and bacteria contain a proton-translocating coupling factor or ATPase. These coupling factors are comprised of an intrinsic membrane portion, FO, which conducts protons, and an extrinsic complex, F1, which catalyzes the ATP synthesis reaction (1-3). The F1 coupling factors (MF1, CF1, and BF1 from mitochondria, chloroplasts, and bacteria, respectively) are readily released from the membrane, and the soluble enzymes have been studied extensively. The F1 ATPase consists of five types of subunits designated a through E in order ofdecreasing molecular weight. Although the stoichiometry of the subunits is still controversial, the two that are most frequently proposed are a3133ySE and a212yl-1281-2e12 (see ref. 4 (14) observed that extensive trypsin digestion of CF1, which leaves only the a and P subunits intact, resulted in the retention of bound nucleotides. In addition, by using purified individual subunits from bacterial coupling factors, nucleotides have been observed to bind only to the a and 13 subunits (15, 16). The use ofa variety ofalkylating (17-19) and photoaffinity (20-27) analogs of ADP and ATP has resulted in the labeling of the a and 13 subunits exclusively.Although the location of the tight nucleotide binding sites remains to be determined, the catalytic site is generally considered to be located on the , 1 subunit (1-3). Using 3'-arylazido-ATP, Hammes and co-workers (23,24) recently identified the location of a MgATP tight binding site on soluble spinach CF1 as primarily the 1 subunit. Their attempts to identify the nondissociable ADP site were unsuccessful.The difficulty in the Jocalization of the tight binding site stems largely from the...

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 (ZC, = 1.4 pM); (b) the concentration giving 50 % maximum binding, for analog tight binding (1 pM) was similar to that observed for ADP (2 pM); (c) nucleotide tight binding required prior membrane energization and was completely reversed by re-energization; (d) the tight binding of 2-azido-[P-"PIADP 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-a~ido-[P-~'PlADP resulted in the covalent incorporation of the label into the membranes. Denaturing polyacrylamide gel electrophoresis of the labeled membranes demonstrated that the j subunit of the coupling factor one complex was the only polypeptide in the thylakoid membranes which was labeled. These results identify the fi subunit of the coupling factor as the location of the tightly bound ADP on the thylakoid membranes.

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