The location of the calcium precipitating protein in the sarcoplasmic membrane

Fluram, a fast reacting reagent for primary amino groups does not label proteins enclosed in reconstituted sarcoplasmic vesicles (SR vesicles) when applied at low reagent/protein ratios. Hence, Fluram does not penetrate through SR membranes under these conditions. Likewise, the membrane of erythrocytes prevent the reagent from reacting with hemoglobin. However, at high reagent/protein ratios the membranes of the SR vesicles disintegrate. In contrast, liposomal membranes prepared from SR lipids are not affected even at a high degree of labeling. Disintegration of SR membranes starts to occur when the phosphatidylethanolamine fraction is completely substituted by Fluram and the transport protein is labeled with 3-4 mol of Fluram per 100000 d. When closed SR vesicles are labeled at low Fluram/protein ratios, the calcium precipitating protein is four times more intensely labeled than the transport protein. The labeling of the calcium transport protein in closed vesicles excludes its location in the intravesicular space. In SR vesicles solubilized with deoxycholate the relative degree of labeling of the calcium precipitating protein remains unchanged while the transport ATPase is more intensely labeled at the expense of the labeling of the amino lipids. The relative degree of labeling of the protein components depends not only on the number of labable groups but also on the rates with which these groups react with Fluram. Therefore, the experimental data do not give quantitative information concerning the distribution of the protein components in the SR membranes.