Interaction of CrATP with the phosphorylation site of the sarcoplasmic reticulum ATPase.
The chromium moiety of gamma,beta-bidentate CrATP slowly accepts a ligand from the sarcoplasmic reticulum Ca-ATPase to form an exchange inert coordination complex (k + 1 = 0.083 min-1; k - 2 = 0.003 min-1, 37 degrees C, 100 microM CaCl2). The stability of the Cr3+ coordinate bonds allowed the complex to be isolated by filtration techniques at neutral pH without acid precipitation. We found 4-5 nmol of [gamma-32P]CrATP to bind to 1 mg of sarcoplasmic reticulum protein with the subsequent occlusion of 7-8 nmol of 45Ca2+. At 37 degrees C, the CrATP.ATPase complex could be formed in the absence of Ca2+, although the reaction was 2-3 times slower than in the presence of Ca2+. Inhibition by Pi, by orthovanadate, and by fluorescein 5'-isothiocyanate verified that the bound CrATP was at the catalytic site. The site of CrATP attachment was found to be on the A tryptic fragment, possibly on the A2 subfragment. It was determined that Ca2+ binding to high affinity sites on the enzyme controls the rate by which the Cr3+ moiety accepts the ligand from the enzyme. The rate of change in the EPR spectrum of iodoacetamide spin-labeled ATPase was shown to follow the rate of ligand acceptance, rather than the binding of Ca2+ and substrate per se. This particular change has been attributed to the formation of an activated complex that is immediately precursory to phosphorylation and indicates here that this complex cannot be properly formed until the metal has been chelated by the enzyme. It is concluded that control over metal chelation (Cr3+ here, Mg2+ in the normal mechanism) is one means by which Ca2+ activates the enzyme.
Chen
Z D
ZD
Department of Physiology, School of Dentistry, University of the Pacific, San Francisco, California 94115.
Coan
C
C
Fielding
L
L
Cassafer
G
G
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002614:Chelating Agents; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D002614:Chelating Agents; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases