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

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 ,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...

Photoaffinity labeling of ribulose-bisphosphate carboxylase/oxygenase with 8-azidoadenosine 5?-triphosphate

“…Most of these polypeptides appear to be chloroplastic in origin, which is not surprising considering the relative abundance of chloroplast proteins in plant extracts and the involvement of many of these in the synthesis or utilization of photochemical energy. Previous studies with chloroplast proteins have used photoaffinity labeling with adenine-nucleotide analogs to characterize the nucleotide-binding sites on the chloroplast couplingfactor 1 (Czarnecki et al 1982), and to identify thylakoid protein kinases (Lin et al 1982). The observation that the LSu of RuBPCase was capable of binding 8-N3ATP is consistent with its well-characterized propensity for binding phosphoesters at the substrate site (Badger and Lorimer 1981).…”

Investigation of nucleotide binding sites on chloroplast coupling factor 1 with 3'O-(4-benzoyl)benzoyl adenosine 5'-triphosphate.

“…Thus far, photoaffinity labeling of the site normally occupied by ADP has not been possible. Recently, however, photoreactive labeling with 2-azidoadenosine diphosphate bound to membrane-bound CF1 at a site normally containing ADP has resulted in labeling of the 3 polypeptide (10). A similar experiment with 3'-O-(4-benzoyl)benzoyl-ADP resulted in the labeling of both the a and 83 polypeptide chains (11).…”

ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

“…2-Azido-ADP photolabels ␤ subunits exclusively upon photoirradiation, in contrast to 8-azido-ADP or -ATP, which modify both ␣ and ␤ subunits (86,89,419,421). 2-Azido-ADP binds to F 1 with an affinity similar to the affinity of ADP (45), and upon irradiation it modifies ␤Leu342, ␤Ile344, ␤Tyr345, ␤Pro346, or ␤Tyr368 (bovine heart MF 1 ) (111, 132).…”