Photoaffinity labeling of the TF1-ATPase from the thermophilic bacterium PS3 with 3′-O-(4-benzoyl)benzoyl ADP

D. Bar-Zvi, M. Yoshida, N. Shavit

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Abstract

(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 (TF1). (2) As with the CF1-ATPase (Bar-Zvi, D. and Shavit, N. (1984) Biochim. Biophys. Acta 765, 340-356) noncovalently bound BzADP is a reversible inhibitor of the TF1-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 CF1-ATPase, it has no effect on the Vmax. In the absence of Mg2+ 1 mol BzADP binds noncovalently to TF1, while with Mg2+ 3 mol are bound. (3) Photoactivation of BzADP results in the covalent binding of the analog to the nucleotide binding site(s) on TF1 and correlates with the inactivation of the ATPase. Complete inactivation of the TF1-ATPase occurs after covalent binding of 2 mol BzADP / mol TF1. Photoinactivation of TF1 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 Bz[3H]ADP-labeled TF1-ATPase shows that all the radioactivity is incorporated into the β subunit.

Original languageEnglish
Pages (from-to)293-299
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume807
Issue number3
DOIs
StatePublished - 31 May 1985

Keywords

  • (Thermophilic bacterium PS3)
  • ATPase
  • Chemical modification
  • Nucleotide analog
  • Nucleotide binding
  • Photoaffinity labeling

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