Characterization of detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase by high-performance liquid chromatography.
Sarcoplasmic reticulum Ca2+-ATPase solubilized by the nonionic detergent octaethylene glycol monododecyl ether was studied by molecular sieve high-performance liquid chromatography (HPLC) and analytical ultracentrifugation. Significant irreversible aggregation of soluble Ca2+-ATPase occurred within a few hours in the presence of less than or equal to 50 microM Ca2+. The aggregates were inactive and were primarily held together by hydrophobic forces. In the absence of reducing agent, secondary formation of disulfide bonds occurred. The stability of the inactive dimer upon dilution permitted unambiguous assignment of its elution position and sedimentation coefficient. At high Ca2+ concentration (500 microM), monomeric Ca2+-ATPase was stable for several hours. Reversible self-association induced by variation in protein, detergent, and lipid concentrations was studied by large-zone HPLC. The association constant for dimerization of active Ca2+-ATPase was found to be 10(5)-10(6) M-1 depending on the detergent concentration. More detergent was bound to monomeric than to dimeric Ca2+-ATPase, even above the critical micellar concentration of the detergent. Binding of Ca2+ and vanadate as well as ATP-dependent phosphorylation was studied in monomeric and in reversibly associated dimeric preparations. In both forms, two high-affinity Ca2+ binding sites per phosphorylation site existed. The delipidated monomer purified by HPLC was able to form ADP-insensitive phosphoenzyme and to bind ATP and vanadate simultaneously. These results suggest that formation of Ca2+-ATPase oligomers in the membrane is governed by nonspecific forces (low affinity) and that each polypeptide chain constitutes a functional unit.
D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D009132:Muscles / Q000201:enzymology; D011092:Polyethylene Glycols; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012995:Solubility
D003902:Detergents; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D000252:Calcium-Transporting ATPases; D002118:Calcium