Fluoride-inhibited calcium ATPase of sarcoplasmic reticulum. Magnesium and fluoride stoichiometry.
The sarcoplasmic reticulum (SR) CaATPase is inactivated by fluoride in the presence of magnesium (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 5229-5235). The inactive complex is very stable and can be isolated free of other components by 48 h of dialysis at 4 degrees C (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 16990-16994). In this study, we used a fluoride-specific electrode to determine that the amount of tightly bound fluoride in the complex was 9.4 +/- 2 nmol mg-1 SR protein. The rate constant of inactivation was very similar to the rate constant of fluoride incorporation and varied directly as the square of the fluoride concentration. Luminal Ca2+ accelerated reactivation of the inhibited enzyme, and the rate constants of activity regain and fluoride release were very similar. Although required for inhibition, added magnesium did not accelerate reactivation. Analysis for magnesium using antipyrylazo III of the inhibited enzyme showed 4.1 +/- 0.4 nmol mg-1 SR protein. As there is much evidence in the literature supportive of an estimate of calcium pumps equal to approximately 4-5 nmol mg-1 SR protein, our results indicate that each inhibited enzyme contains two tightly bound fluorides and one tightly bound magnesium.
Coll
R J
RJ
Department of Biochemistry, School of Dentistry, University of the Pacific, San Francisco, California 94115.
Murphy
A J
AJ
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004789:Enzyme Activation; D005459:Fluorides / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology*; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000252:Calcium-Transporting ATPases; D008274:Magnesium; D005459:Fluorides