Inhibition of ion pump ATPase activity by 3'-O-(4-benzoyl)benzoyl-ATP (BzATP): assessment of BzATP as an active site-directed probe.
The interaction of 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) with the renal (Na+ + K+)-ATPase, the sarcoplasmic reticulum Ca-transport ATPase, and the gastric (H+ + K+)-ATPase has been investigated in order to determine whether BzATP is a suitable probe for the labeling and identification of a peptide from the ATP binding sites of these ion pumps. After ultraviolet irradiation BzATP inhibited the enzymatic hydrolysis of ATP by each of the ion pumps, and also was covalently incorporated into the 100 000 dalton polypeptides of each protein. The presence of excess ATP in the reaction solution did not prevent either the inactivation of ATPase activity or the labeling of the catalytic polypeptides by BzATP. Prior modification of the ATPases with fluorescein-5'-isothiocyanate (FITC), however, prevented much of the labeling of the 100 000 dalton polypeptides by BzATP. BzATP competitively inhibited the high-affinity binding of ATP to the ion pumps, but ATP did not block the high-affinity binding of BzATP by the enzymes. BzATP binds to the membrane-bound ATPases at a high-affinity site with a Kd of 0.8-1.2 microM and a Bmax of 2-3 nmol/mg, and also binds to at least one low-affinity, high-capacity site on the membranes. HPLC separation of the soluble peptides from a tryptic digest of BzATP-labeled (Na+ + K+)-ATPase revealed the presence of several labeled peptides, none of which was protected by either ATP or FITC. Although BzATP can displace ATP from a high-affinity binding site on the ion pumps, it appears, therefore, that inactivation of enzymatic activity is the result of reactions between BzATP and the proteins at locations outside this site. Thus, it is concluded from these experiments that BzATP is not likely to be a useful probe for the ATP binding sites on the ion transport ATPases.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D017506:H(+)-K(+)-Exchanging ATPase; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D010777:Photochemistry; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D013270:Stomach / Q000201:enzymology
D000345:Affinity Labels; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase