Characterization of sarcolemma from calcium-tolerant canine cardiocytes

Large numbers of calcium-tolerant canine cardiocytes can be isolated from the collagenase-perfused canine myocardium. An average yield of 500 million cells provides abundant tissue for the preparation of subcellular fractions. Using nitrogen cavitation, along with extraction by 0.6 M potassium chloride/sucrose buffer, we have been able to prepare, after differential and sucrose gradient centrifugation, membrane fractions that are more than 100-fold enriched in sarcolemmal marker enzymes. This preparation of sarcolemma has the advantage of being essentially free of plasma membranes from endothelial, smooth muscle, and other cell types residing in the myocardium.