Isolation and characterization of plasma membrane from lactating bovine mammary gland.
Plasma membranes were isolated from lactating bovine mammary gland. Two crude membrane fractions; medium/d 1.033 (light membrane) and 1.033/1.053 interfaces (heavy membrane), were obtained by Ficoll density gradient centrifugation of osmotically washed microsomal fraction. Two crude membranes were further purified separately by sucrose density gradient centrifugation. Both light and heavy membranes banded at a sucrose density of 1.14. The purified membranes appeared as heterogeneous smooth membrane vesicles on electron microscopy. The contaminating suborganelles were not detected. The yield of the purified membranes relative to the homogenate was 1.2%. The degree of purity of the membranes was shown by a great increase in the specific activity of 5'-nucleotidase over the homogenate of 20-fold for light membrane and of 16-fold for heavy membrane. The relative activities of Mg2+-ATPase, (Na+ + K+)-ATPase, gamma-glutamyl transpeptidase, phosphodiesterase I, alkaline phosphatase and xanthine oxidase were also high (12-18-times) and nearly 20% of these enzymes was recovered. The activity of marker enzyme for mitochondria, endoplasmic reticulum and Golgi apparatus was very low, while that of acid phosphatase for lysosome was relatively high (5-times). DNA and RNA contents were very low. The major polypeptides rich in other suborganelles were not detected profoundly in the membrane fraction and the polypeptide composition in both light and heavy membranes were similar upon SDS-polyacrylamide gel electorphoresis.
D000818:Animals; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007774:Lactation; D008321:Mammary Glands, Animal / Q000502:physiology / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000302:isolation & purification; D008854:Microscopy, Electron; D008970:Molecular Weight; D011247:Pregnancy
D008565:Membrane Proteins