The hydrolytic cycle of sarcoplasmic reticulum Ca2+-ATPase in the absence of calcium.
The hydrolytic cycle of sarcoplasmic reticulum Ca2+-ATPase in the absence of Ca2+ was studied. At pH 6.0, 10 degrees C and in the absence of K+, the enzyme displays a very low velocity of ATP hydrolysis. Addition of up to 15% dimethyl sulfoxide increased this velocity severalfold (from 5-18 nmol of Pi X mg of protein-1 X h-1) and then decreased at higher solvent concentrations. Dimethyl sulfoxide increased both enzyme phosphorylation from ATP and the affinity for this substrate. Maximal levels of 1.0-1.2 nmol of EP X mg of protein-1 and apparent KM for ATP of 5 X 10(-6) M were obtained at a concentration of 30% dimethyl sulfoxide. The same preparation under optimal conditions (pH 7.5, 10 microM CaCl2, 100 mM KCl and no dimethyl sulfoxide at 37 degrees C) displays a velocity of ATP hydrolysis between 8 and 12 X 10(5) nmol of Pi X mg of protein-1 X h-1 while the phosphoenzyme levels varied between 3.5 and 4.0 nmol of EP X mg of protein-1. Enzyme phosphorylation from ATP in the absence of Ca2+ always preceded Pi liberation into the assay media. Two different phosphoenzyme species were formed which were kinetically distinguished by their decomposition rates. The observed steady-state velocity of ATP hydrolysis could be accounted for either by the decay of the fast component or by the simultaneous decomposition of both phosphoenzyme species. The hydrolysis of the phosphoenzyme formed in the absence of Ca2+ was KCl-stimulated and ADP-independent. The rate constant of breakdown was equal to that observed for the phosphoenzyme formed in the presence of Ca2+. It is suggested that the rapidly decaying phosphoenzyme (and possibly both rapidly and slowly decaying species) are intermediates in the reaction cycle of Mg2+-dependent ATP hydrolysis of sarcoplasmic reticulum Ca2+-ATPase and may represent a bypass of Ca2+ activation by dimethyl sulfoxide.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D011189:Potassium Chloride / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000244:Adenosine Diphosphate; D011189:Potassium Chloride; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium; D004121:Dimethyl Sulfoxide