[en] 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 ≤ 50 μM 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 ⁴⁵Ca²⁺ concentration (500 μM), monomeric Ca²⁺-ATPase was stable for several house. 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 10⁵-10⁶ 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 ⁴⁸V 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. The 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

[en] When high affinity Ca(2+)-binding proteins like calmodulin, or proteins with a high Ca(2+)-binding capacity like calsequestrin, underwent sodium dodecyl sulfate-gel electrophoresis in Laemmli systems, their electrophoretic migration rates were much higher in gels containing 1 mM Ca2+ than in gels containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Replacement of EGTA by Ca2+ in the gel, combined with the blotting of electrophoretically separated proteins on polyvinylidene difluoride membranes and subsequent 45Ca2+ overlay, proved a very effective means of detecting Ca(2+)-binding proteins. This combined approach is important since artifacts occur in both techniques when used separately. We found that the usual procedure of adding Ca2+ to the sample before electrophoresis without including it in the gel itself permitted the detection of only very high affinity Ca(2+)-binding proteins