Membranes of normal hamster lymphocytes and lymphoid cells neoplastically transformed by simian virus 40. I. High-yield purification of plasma membrane fragments.
In this first paper of a series comparing the membranes of normal lymphocyte populations from male outbred Syrian hamsters with those of neoplastic transformants (GD 248) induced by simian virus 40, a method is described for the isolation of representative plasma membrane (PM) fragments from both cell types. Multiple criteria were used to monitor the purity and yield of PM material after cell disruption by nitrogen cavitation and after membrane fractionation by a combination of differential centrifugation and isopyknic ultracentrifugation in dextran density gradients. Lactoperoxidase-catalyzed radioiodination before cell disruption was used as an extrinsic surface marker; Na+,K+-activated ATPase, as well as alkaline phosphatase, was used as intrinsic functional PM markers. The distribution of nuclei, mitochondria, lysosomes, and endoplasmic reticulum (ER) during fractionation was monitored by the measurement of DNA, succinate dehydrogenase and monoamine oxidase, beta-glucuronidase and glucose-6-phosphatase, and NADH:lipoamide oxidoreductase, respectively. According to the three PM markers employed, a 15- to 20-fold purification (over homogenate) and a PM yield of about 65% were obtained for both cell categories, with negligible contamination by DNA, mitochondria, lysosomes, and er. The procedure also allowed recovery of 60% of the mitochondria free of other cell elements.
D000251:Adenosine Triphosphatases / Q000032:analysis; D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000032:analysis; D002471:Cell Transformation, Neoplastic; D006224:Cricetinae; D004273:DNA, Neoplasm / Q000032:analysis; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis; D004721:Endoplasmic Reticulum / Q000032:analysis / Q000201:enzymology; D005952:Glucose-6-Phosphatase / Q000032:analysis; D007942:Leukemia, Experimental / Q000209:etiology / Q000648:ultrastructure*; D008214:Lymphocytes / Q000648:ultrastructure*; D008297:Male; D008563:Membrane Lipids / Q000378:metabolism; D008928:Mitochondria / Q000032:analysis; D010743:Phospholipids / Q000032:analysis; D013539:Simian virus 40
D004273:DNA, Neoplasm; D008563:Membrane Lipids; D010743:Phospholipids; D008058:Dihydrolipoamide Dehydrogenase; D000469:Alkaline Phosphatase; D005952:Glucose-6-Phosphatase; D000251:Adenosine Triphosphatases