Subfractionation of cardiac sarcolemma with wheat-germ agglutinin.
The properties of highly purified bovine cardiac sarcolemma subfractionated with the lectin, wheat-germ agglutinin (WGA) were studied. Two different membrane subfractions were isolated, one which was agglutinated in the presence of 1.0 mg of WGA/mg of protein (WGA+ vesicles) and a second fraction which failed to agglutinate (WGA- vesicles). These two membrane fractions had quantitatively different rates of Na+/K+-dependent, ouabain-sensitive ATPase and Na+/Ca2+ exchange activities, yet a similar protein composition, which suggests that they were both derived from the plasma membrane. WGA- vesicles had a decreased number of [3H]quinuclidinyl benzilate-binding sites and no detectable [3H]nitrendipine-binding sites. Electron-microscopic and freeze-fracture analysis showed that the WGA+ fraction was composed of typical spherical sarcolemmal vesicles, whereas the WGA- fraction primarily contained elongated tubular structures suggestive of the T-tubule vesicles which were previously isolated from skeletal muscle. Assays of marker enzymes revealed that these fractions were neither sarcoplasmic reticulum nor plasma membrane from endothelial cells. Moreover, WGA agglutination did not result in the separation of right-side-out and inside-out vesicles. On the basis of these findings we propose that the WGA+ fraction corresponds to highly purified sarcolemma, whereas the WGA- fraction may be derived from T-tubule membranes.
Charuk
J H
JH
Department of Pediatrics, University of Alberta, Edmonton, Canada.
Howlett
S
S
Michalak
M
M
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D005614:Freeze Fracturing; D007202:Indicators and Reagents; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000302:isolation & purification; D008854:Microscopy, Electron; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism / Q000648:ultrastructure*; D009568:Nitrendipine / Q000378:metabolism; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D012508:Sarcolemma / Q000378:metabolism / Q000648:ultrastructure*; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis; D014909:Wheat Germ Agglutinins
D007202:Indicators and Reagents; D008565:Membrane Proteins; D014909:Wheat Germ Agglutinins; D011813:Quinuclidinyl Benzilate; D009568:Nitrendipine; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium