Characterization of human platelet surface and intracellular membranes isolated by free flow electrophoresis.
High voltage free flow electrophoresis has been applied to the separation of human platelet membranes. After short treatment with neuraminidase at the whole cell level, three membrane vesicle subpopulations have been isolated. Using a surface label (125I-labeled Lens culinaris lectin), the marker enzyme NADH-cytochrome c reductase, and lipid analysis, two of the fractions have been identified as of surface origin and the other consists of intracellular membrane elements. The distribution of adenylate cyclase, leucyl aminopeptidase, 5'-nucleotidase and Ca2+-ATPase has also been investigated, and their usefulness as markers for the different membrane fractions has been evaluated. All three fractions are vesicular but differ in size and character. Their phospholipid and cholesterol contents have been determined, and the cholesterol/phospholipid ratios of the two surface fractions are over twice that of the intracellular membrane, which also has a significantly lower microviscosity as determined by fluorescence polarization using diphenyl hexatriene. The polypeptide profiles from sodium dodecyl sulfate-polyacrylamide gel electrophoresis are particularly distinctive, with actin present in the two surface membrane fractions and absent from the intracellular membranes. Myosin, confirmed by its ATPase characteristics, is almost exclusively localized in one of the surface membrane fractions, and actin-binding protein is a prominent feature of the other.
D015720:5'-Nucleotidase; D000262:Adenylyl Cyclases / Q000097:blood; D001792:Blood Platelets / Q000201:enzymology*; D000252:Calcium-Transporting ATPases / Q000097:blood; D002462:Cell Membrane / Q000201:enzymology; D004586:Electrophoresis; D006801:Humans; D008565:Membrane Proteins / Q000097:blood*; D008970:Molecular Weight; D009245:NADH Dehydrogenase / Q000097:blood; D009708:Nucleotidases / Q000097:blood; D012794:Sialic Acids / Q000097:blood; D014783:Viscosity
D008565:Membrane Proteins; D012794:Sialic Acids; D009245:NADH Dehydrogenase; D009708:Nucleotidases; D015720:5'-Nucleotidase; D000262:Adenylyl Cyclases; D000252:Calcium-Transporting ATPases