The location of membrane components in sarcoplasmic-reticulum membranes by using free and immobilized lactoperoxidase.

lzSI-labelling of external proteins with lactoperoxidase has been widely used as a probe of membrane structure. We have applied this technique to the sarcoplasmic reticulum from rabbit skeletal muscle isolated by differential centrifugation, and have compared the labelling of membrane components using free and immobilized lactoperoxidase. The major proteins in the sarcoplasmic reticulum are the (Ca2++ Mgz+)-activated ATPase* and the acidic protein calsequestrin. These have molecular weights of 1OOOOO and 60000 and account for 60-70% and 10-15 % of the total membrane protein respectively (Louis & Shooter, 1972). Lactoperoxidase was isolated from bovine milk and purified as described by Morrison & Hultquist (1963). It was coupled to Sepharose 4B (2mg of lactoperoxidase/ ml of beads) after CNBr activation as described by Cuatrecasas (1970), and was washed extensively before use. When assayed against L-Dopa (3,4-dihydroxyphenylalanine) the immobilized enzyme had 30% of the activity of an equivalent weight of free lactoperoxidase. * Abbreviations: ATPase, adenosine triphosphatase; Hepes, 2-(N-2-hydroxyethylpiperazinN'-y1)ethanesulphonic acid.