Abstract
A method of preparing highly purified plasma membrane from cardiac muscle is described using continuous ficoll-sucrose density gradient centrifugation in zonal rotors. 5′-Nucleotidase and ATPase (Mg 2+ dependent, Na + + K + stimulated and ouabain inhibited) were found to be concentrated in the plasma membrane fraction. At 11% ficollsucrose Ca 2+ and (Ca 2+ + K +) stimulation of the ATPase was very small (13%) and very low level of succinic dehydrogenase activity was present. Electron microscopic studies of the fraction revealed the presence of membrane vesicles and sheets. Composition of these membranes was analyzed by 7% polyacrylamide gel electrophoresis in the presence of 1% SDS. One protein band of 68 000 Daltons accounted for 18% of protein on the gel. Lipid and carbohydrate stains revealed six and two bands respectively. Studies on the conformation of proteins within membranes were achieved using circular dichroism (CD) and optical rotatory dispersion (ORD) techniques. Corrections were applied using the pseudo reference state approach.
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