Contamination of a cardiac sarcolemmal preparation with endothelial plasma membrane

Abstract

Preparation of sarcolemma from whole rabbit heart using the method of Jones et al. (Jones,L.R., Besch, H.R., Fleming, J.W., McConnaughey, M.M. and Watanabe, A.M. (1979) J. Biol. Chem. 254, 530-539) results in a 46-fold purification of the endothelial plasmalemma-specific marker angiotensin converting enzyme. This implies contamination of the sarcolemma with vascular endothelial plasmalemma. During preparation of sarcolemma from sheep heart, using the same method, angiotensin converting enzyme copurified with the general plasma membrane marker (Na+ + K+)-ATPase. The ratio of myocyte to endothelial plasma membrane in the final preparation is therefore similar to that in the whole heart homogenate. Ultrastructural analysis has shown that the myocyte/endothelial surface area is 70:30 in whole cardiac muscle. Comparison of angiotensin converting enzyme activity of an endothelial plasma membrane fraction with that of whole heart sarcolemma suggests an upper limit of 42% for endothelial contamination. Contamination by endothelial plasmalemma was dramatically reduced by preparing sarcolemma from myocytes produced by proteolytic disruption of whole hearts. Following disruption, myocytes were separated from non-muscle cells by sedimentation through 0.5 M sucrose. Sarcolemma prepared from sheep cardiac myocytes had approximately 15-fold less angiotensin converting enzyme activity than whole sheep heart sarcolemma but comparable ouabain-inhibitable (Na+ + K+)-ATPase activity.