Fractionation of membrane vesicles. II. A method for separation of membrane vesicles bearing different enzymes by free-flow electrophoresis.
Free-flow electrophoresis was used to subfractionate membrane vesicles from calf thymocyte plasma membranes. The fractionation resulted in a separation of vesicle populations bearing four different enzymes: alkaline nitrophenyl-phosphatase (orthophosphoric-monoester phosphohydrolase (alkalin optimum) EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.2), (Mg2+ + Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and acyl-CoA:lysophosphatidylcholine acyltransferase (acyl-CoA:1-acylglycero-3-phosphocholine-O-acyltransferase, EC 2.3.1.23). The specific content of cholesterol and total phospholipid coincided with the distribution of membrane-bound protein. However, vesicles migrating towards the cathode had a higher molar ratio of cholesterol to phospholipid (0.75) compared to those migrating to the anode (0.55). Sodium dodecyl sulphate-gel electrophoresis of pooled vesicle fractions also demonstrates distinct differences in their protein pattern. Electron-micrographic thin sections show that the vesicle populations have a similar morphology and size distribution. These results are discussed in terms of heterogeneity of the original thymocytes, contamination with intracellular membranes and a heterogeneous structure of the plasma membrane.
D008243:1-Acylglycerophosphocholine O-Acyltransferase / Q000032:analysis; D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D002784:Cholesterol / Q000032:analysis; D004586:Electrophoresis / Q000379:methods; D008854:Microscopy, Electron; D008970:Molecular Weight; D010743:Phospholipids / Q000032:analysis; D013347:Subcellular Fractions / Q000201:enzymology; D013385:Succinate Dehydrogenase / Q000032:analysis; D013950:Thymus Gland / Q000201:enzymology / Q000648:ultrastructure*; D005723:gamma-Glutamyltransferase / Q000032:analysis
D010743:Phospholipids; D002784:Cholesterol; D013385:Succinate Dehydrogenase; D008243:1-Acylglycerophosphocholine O-Acyltransferase; D005723:gamma-Glutamyltransferase; D000469:Alkaline Phosphatase