Changes in Ca2+ affinity related to conformational transitions in the phosphorylated state of soluble monomeric Ca2+-ATPase from sarcoplasmic reticulum

• Published in: The Journal of biological chemistry Vol. 260; no. 1; pp. 371 - 380
• Main Authors: Andersen, J P, Lassen, K, Møller, J V
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 10-01-1985; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphate - metabolism; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Calcium - metabolism; Calcium-Transporting ATPases - metabolism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Hydrolases; Kinetics; Magnesium - pharmacology; Mathematics; Models, Biological; Muscles - enzymology; Phosphorylation; Protein Conformation; Rabbits; Sarcoplasmic Reticulum - enzymology; Spectrometry, Fluorescence; Tryptophan - analysis; Ca-ATPase; Vertebrata; Phosphorylation; Mammalia; Enzyme; Sarcoplasmic reticulum; Rabbit; Lagomorpha; Striated muscle; Conformational transition
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)89742-3

Changes in Ca2+ binding after phosphorylation of membranous or detergent-solubilized preparations of sarcoplasmic reticulum Ca2+-ATPase with ATP were followed spectrophotometrically by the use of murexide. Distinct Ca2+ release from the two high-affinity translocation sites was observed, particularly at alkaline pH and at low Ca2+/Mg2+ concentration ratios. Phosphorylation also induced additional binding of Ca2+ at a third site in competition with Mg2+. Ca2+ release was increased after solubilization of Ca2+-ATPase in predominantly monomeric form with the nonionic detergent octaethyleneglycol monododecyl ether. At 0 degree C, chemical-quench studies with [32P]ATP indicated that release of Ca2+ is correlated with the level of ADP-insensitive phosphoenzyme (2 mol of Ca2+ released per mol of E2P formed), both for membranous and detergent solubilized Ca2+-ATPase. Ca2+ release was also found to be accompanied by changes in intrinsic fluorescence. Analysis of the data at 20 degrees C, pH 8.0, showed that binding of Ca2+ to transport sites on E2P occurs with a half-saturation constant of 0.7 mM and a Hill coefficient of 1.8. This is consistent with a drastic decrease in Ca2+ affinity following conversion of ADP-sensitive E1P to ADP-insensitive E2P. The similarity between membranous and detergent-solubilized Ca2+-ATPase supports the view that not more than a single Ca2+-ATPase polypeptide chain is required to complete the conformational transitions which are the basis for active transport of Ca2+.