Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca 2+-pump and Ca 2+-binding proteins

Abstract

A procedure for the isolation of highly purified sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been described using sucrose gradient centrifugation in zonal rotors. The yield of our purest fraction was 300 mg of sarcoplasmic reticulum protein using 1 kg muscle. The sarcoplasmic reticulum vesicles were relatively simple in composition. The Ca 2+-pump protein accounted for most (approx. two-thirds) of the sarcoplasmic reticulum protein. Two other protein components, a Ca 2+-binding protein and a M 55 protein (approx. 55 000 daltons) each accounted for about 5–10% of the protein. Enrichment in the level of phosphoenzyme by the Ca 2+-pump protein was regarded as an important index of the purification of sarcoplasmic reticulum vesicles. The sarcoplasmic reticulum vesicles were capable of forming 6.4 nmoles of 32P-labelled phosphoenzyme per mg protein and had a high capacity of energized Ca 2+ uptake. The Ca 2+-dependent formation of phosphoenzyme has been used to estimate the sarcoplasmic reticulum protein content in rabbit skeletal muscle and found to be about 2.5% of the total muscle protein. The Ca 2+-pump and Ca 2+-binding proteins were isolated with a purity of 90% or more by treating the purified sarcoplasmic reticulum vesicles with bile acids in the presence of salt. The solubilized Ca 2+-pump protein reaggregated during dialysis together with phospholipid to form membranous vesicles which were capable of forming approx. 9 nmoles 32P-labelled phosphoenzyme per mg protein. The Ca 2+-binding protein was water soluble and contained a high percentage of acidic amino acids (35% of total residues). Ca 2+ binding by sarcoplasmic reticulum vesicles and by the Ca 2+-pump and Ca 2+-binding proteins was studied by equilibrium dialysis. Sarcoplasmic reticulum vesicles and Ca 2+-pump protein contained nonspecific high-affinity Ca 2+ binding sites with a capacity of 90–100 and 55–70 nmoles Ca 2+ per mg protein, respectively. Both of them specifically bound 10–15 nmoles Ca 2+ per mg protein. The binding constants for nonspecific and specific Ca 2+ binding by both preparations were approx. 1 μM −1. The Ca 2+-binding protein nonspecifically bound 900–1000 nmoles Ca 2+ per mg protein with a binding constant of about 0.25 μM −1.