Biochimica et Biophysica Acta 807 (1985) 293-299 293 Elsevier BBA 41743 Photoaffinity labeling of the TF1-ATPase from the thermophilic bacterium PS3 with 3'-O-(4-benzoyl)benzoyi ADP D. Bar-Zvi *, M. Yoshida ** and N. Shavit *** Department of Biology, Ben Gurion University of the Negev, Beer-Sheoa 84105 (Israel) (Received May 21st, 1984) (Revised manuscript received October 30th, 1984) Key words: ATPase; Photoaffinity labeling; Chemical modification; Nucleotide binding; Nucleotide analog; (Thermophilic bacterium PS3) (1) 3'-O-(4-Benzoyl)benzoyl ADP (BzADP) was used as a photoaffinity label for covalent binding of adenine nucleotide analogs to the nucleotide binding site(s) of the thermophilic bacterium PS3 ATPase (TF t ). (2) As with the CF1-ATPase (Bar-Zvi, D. and Shavit, N. (1984) Biochim. Biophys. Acta 765, 340-356) noncova- lently bound BzADP is a reversible inhibitor of the TFI-ATPase. BzADP changes the kinetics of ATP hydrolysis from noncooperative to cooperative in the same way as ADP does, but, in contrast to the effect on the CFi-ATPase, it has no effect on the Vma X. In the absence of Mg 2+ 1 mol BzADP binds noncovalently to TF1, while with Mg 2+ 3 moi are bound. (3) Photoactivation of BzADP results in the covalent binding of the analog to the nucleotide binding site(s) on TF~ and correlates with the inactivation of the ATPase. Complete inactivation of the TFi-ATPase occurs after covalent binding of 2 tool BzADP/mol TF v Photoinactivation of TF t by BzADP is prevented if excess of either ADP or ATP is present during irradiation. (4) Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the Bzl3H]ADP-labeled TFt-ATPase shows that all the radioactivity is incorporated into the fl subunit. Introduction Chemical modification of functional groups of Ft-ATPases from different sources has provided useful information on the participation of these * Present address: Department of Cellular and Developmen- tal Biology, The Biological Laboratories, Harvard Univer- sity, Cambridge, MA 02138, U.S.A. ** Present address: Department of Biochemistry, Jichi Medi- cal School, Minamikawachi-machi, Tochigi-Ken, Japan 329-04. *** To whom correspondence should be addressed. Abbreviations: CF 1 and TFI, coupling factors one ATPase from chloroplast and the thermophilic bacterium PS3, respec- tively; BzADP, Y-O-(4-benzoyl)benzoyl ADP; Hepes, 4-(2-hy- droxyethyl)-l-piperazineethanesulphonic acid; DCCD, N,N'- dicyclohexy!carbodiimide; Pi, inorganic phosphate. groups in the catalytic process. However, it is rather difficult to limit the covalent modification of different groups on a protein and to confine the modification only to the functional groups at the catalytic site. Derivatization of groups other than those at the catalytic site may result in the in- activation of the enzyme by interference with changes in the enzyme conformation necessary for catalysis. Affinity labeling may be more suitable for the specific modification of the enzyme's sub- strate binding sites, because such groups are ex- pected to interact with the reactive analog more readily than other amino acid groups that do not take part in the catalytic process. Indeed, photoaf- finity labeling of bacterial, mitochondrial and chloroplast Ft-ATPases with different photoreac- tive nucleotide analogs was described [1,2]. The 0005-2728/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)