Preparation and characterization of plasma membrane-enriched fractions from rat pancreatic islets.
Methods have been developed for the isolation on a semi-micro scale of a plasma membrane-enriched fraction from rat islets of Langerhans. An important feature of these experiments is the use of 125I-labeled wheat germ agglutinin as a specific probe for plasma membrane-containing fractions. The partly purified plasma membrane fraction had a density in sucrose of about 1.10 and was enriched in the activities of 5'-nucleotidase, alkaline phosphatase, sodium-potassium, and magnesium-dependent ATPase and adenylate cyclase. It contained only very low levels of acid phosphatase, cytochrome c oxidase, insulin, and RNA. Further purification was hampered by the relatively small amounts of fresh plasma membrane material that could be obtained from 16-24 rats in each experiment. When islets were prelabeled with radioactive fucose, the plasma membrane-enriched fraction contained radioactivity at a four- to fivefold higher specific acivity than the whole islet homogenate. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of plasma membrane-enriched fractions pooled from several experiments revealed a distinctive pattern of protein bands as compared with other less pure fractions. With respect to rapidity, apparent specificity, and easy reversibility of the labeling of the plasma membrane fraction, 125I-wheat germ agglutinin provides a highly useful tool for the detection of microgram quantities of plasma membrane components which should be applicable to many other systems as well.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000032:analysis / Q000201:enzymology / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D005643:Fucose; D007515:Islets of Langerhans / Q000648:ultrastructure*; D037102:Lectins; D051381:Rats; D013347:Subcellular Fractions; D014529:Uridine
D037102:Lectins; D005643:Fucose; D000262:Adenylyl Cyclases; D014529:Uridine