title: The functional unit of sarcoplasmic reticulum Ca2+-ATPase. Active site titration and fluorescence measurements.
abstract: The properties of sarcoplasmic reticulum Ca2+-ATPase have been studied after modification of the ATP high affinity binding site with fluorescein isothiocyanate, both in the membranous state and after solubilization with the nonionic detergent, octaethyleneglycol monododecyl ether. Total inactivation of both membrane-bound and solubilized Ca2+-ATPase requires covalent attachment of 1 mol of fluorescein/mol of enzyme (115,000 g of protein) or per binding site for ATP. Sedimentation velocity studies of soluble enzyme showed that both unlabeled and fluorescein-labeled Ca2+-ATPase were present in a predominantly monomeric form. The phosphorylation level of unlabeled Ca2+-ATPase was unchanged by solubilization. Dephosphorylation measurements at 0 degree C indicated that the phosphorylation is an intermediate in the ATPase reaction catalyzed by solubilized Ca2+-ATPase. Fluorescein labeling of half of the Ca2+-ATPase in the membrane did not influence the enzyme kinetics of the remaining unmodified Ca2+-ATPase. Measurements of both fluorescein and tryptophan fluorescence indicated that the soluble monomer of Ca2+-ATPase like the membrane-bound enzyme exists in a Ca2+-dependent equilibrium between two principal conformations (E and E). E (absence of Ca2+) is unstable in the soluble form, but the pCa dependence of the E - E equilibrium is identical with that of the membranous Ca2+-ATPase (pCa0.5 = 6.7 and Hill coefficient 2). These results suggest that the Ca2+-ATPase polypeptides function with a high degree of independence in the membrane.
mesh_terms: D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D009132:Muscles / Q000201:enzymology*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates / Q000494:pharmacology
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D013861:Thiocyanates; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate