Plasma membranes from cardiac cells in culture. Enzymatic radio-iodination, evaluation of preparation and properties of the sarcolema.
Plasma membranes from heart (sarcolemma) were prepared by the method of Kidwai, A.M. (1975) Methods in Enzymology (Fleischer, S. and Packer, L., eds.), Vol XXXIA, pp. 134--144, Academic Press, New York). On many occasions the sarcolemmal fraction identified by the enzyme markers such as (Na+ + K+)-ATPase banded at heavier densities (d greater than 1.25 g/ml) than expected for plasma membrane (d less than 1.15 g/ml). Radio-iodination of the membrane was added as an independent marker and conditions for the reproducible preparation of the sarcolemma were studied. Cultured heart cells were enzymatically iodinated under conditions which did not affect viability and labeled primarily the sarcolemma. The distribution of radioactivity in homogenates of cultured cells on the density gradient corresponded to that of the enzymes' activity. The best sarcolemma preparation was obtained with 0.3 M KCl extraction of heart homogenates in the presence of 0.05 M pyrophosphate, especially if the salt was also present during the fractionation by density gradient centrifugation. Alterations in the density were also observed with erythrocytes and cultured liver cells' plasma membrane. The data suggests a meta-stable state of the plasma membranes due to handling or storage which could cause alterations of some of their physical properties (e.g. density).
D000251:Adenosine Triphosphatases / Q000032:analysis; D000818:Animals; D000831:Animals, Newborn; D002458:Cell Fractionation; D002478:Cells, Cultured; D003576:Electron Transport Complex IV / Q000032:analysis; D004910:Erythrocyte Membrane; D005947:Glucose; D005949:Glucose Oxidase; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods*; D007784:Lactoperoxidase; D009206:Myocardium; D009218:Myosins; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D012508:Sarcolemma / Q000201:enzymology; D012964:Sodium / Q000494:pharmacology
D007457:Iodine Radioisotopes; D012964:Sodium; D005949:Glucose Oxidase; D007784:Lactoperoxidase; D003576:Electron Transport Complex IV; D000251:Adenosine Triphosphatases; D009218:Myosins; D005947:Glucose; D011188:Potassium