Highly enriched, minimally disrupted plasma membrane vesicles from aortic myocytes grown in primary culture.
A plasma membrane-enriched fraction (fraction 1B) has been obtained from rat aortic myocytes grown in primary culture. Plasma membrane markers, 5'-nucleotidase and ouabain-sensitive (Na+ + K+)-ATPase, are enriched 4.1- and 8.7-fold, respectively, in this fraction. Although endoplasmic reticulum marker NADPH-cytochrome c reductase is the most enriched in mitochondrial and heavy sucrose density gradient fractions, substantial enrichment of this marker is also observed in membrane fraction 1. This membrane preparation therefore contains a certain quantity of endoplasmic reticulum. Cytochrome c oxidase is de-enriched by a factor of 0.04 in fraction 1, indicating that it is essentially clear of mitochondrial contamination. Homogenization of aortic media-intima layers using a whole-tissue technique induces greater disruption of mitochondria and subsequent contamination of membrane fractions than does the procedure for cell disruption. Analysis of electrophoretic gels, vesicle density distribution and electron micrographs of enriched membrane fractions provide evidence that plasma membrane enriched from cultured myocytes is less traumatized than comparable fractions obtained from intact tissue. The potential value of such a highly enriched, minimally disrupted plasma membrane preparation is discussed.
D015720:5'-Nucleotidase; D000818:Animals; D001013:Aorta, Thoracic / Q000648:ultrastructure*; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003576:Electron Transport Complex IV / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005260:Female; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology; D009131:Muscle, Smooth, Vascular / Q000648:ultrastructure*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009708:Nucleotidases / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D010042:Ouabain; D009251:NADPH-Ferrihemoprotein Reductase; D003576:Electron Transport Complex IV; D009708:Nucleotidases; D015720:5'-Nucleotidase; D000254:Sodium-Potassium-Exchanging ATPase