Calcium-dependent inactivation of the Ca2+-ATPase from sarcoplasmic reticulum by chemically reactive adenosine triphosphate.
1. ATP gamma P-imidazolidate, synthesized from ATP and carbonyldiimidazole, inhibits the Ca2+-ATPase of sarcoplasmic reticulum in a biphasic manner. A fast first phase is concentration-dependent while a slower second phase is independent of the inhibitor concentration. 2. The inhibition is calcium- and magnesium-dependent. No inhibition occurs in the absence of either cation. 3. Inhibition of the Ca2+-ATPase can be prevented by the protection with ATP. 4. The loss of ATPase activity is pH-dependent. Maximal inhibition coincides with maximal ATPase activity. It indicates a participation of the reacting amino acid side chain in the catalytic cycle. 5. The incorporation of radioactive inhibitor is reversed in a time-dependent fashion while the Ca2+-ATPase remains inhibited. 6. We conclude that ATP gamma P-imidazolidate initially reacts covalently with an amino acid side chain, probably Asp-351, but is subsequently expelled by a reaction with a second amino acid. 7. This two-step reaction induces an intramolecular cross-link which can be shown by the creation of a new protein band on SDS-PAGE which originates in the Ca2+-ATPase.
Bill
E
E
Institut für Biochemie der J-W Goethe-Universität, Universitäts Klinikum, Frankfurt am Main, Federal Republic of Germany.
Gutowski
Z
Z
Bäumert
H G
HG
D000227:Adenine Nucleotides / Q000138:chemical synthesis; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D008956:Models, Chemical; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000227:Adenine Nucleotides; D007093:Imidazoles; C057152:ATP gammaP-imidazolidate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium