Isolation of human platelet glycoproteins.
Human platelet glycoproteins were isolated from whole platelets by two methods. The first method, that of affinity chromatography on wheat germ agglutinin, is based on the known affinity of lectins for cell surface glycoproteins. When solubilized whole platelets are used as starting material for this procedure, elution with N-acetylglucosamine yields primarily a glycoprotein of Mr approximately 150 000 as estimated by sodium dodecyl sulfate-acrylamide gel electrophoresis. The second method is based on the ability of the chaotropic salt lithium diiodosalicylate to extract glycoprotein from particulate cell fractions in water-soluble form. This method yields three major glycopeptides with apparent molecular weights after sulfhydryl reduction of 145 000, 125 000, and 95 000 as estimated on 5.6% sodium dodecyl sulfate-acrylamide gels. Carboxymethylation of these preparations in the presence of sulfhydryl-reducing agent further resolves a glycoprotein of Mr approximately 165 000. Treatment of whole platelets by periodate oxidation and sodium[3H]-borohydride reduction labels the three major glycoproteins extracted by lithium diiodosalicylate and the glycoprotein of Mr approximately 150 000 isolated on wheat germ agglutinin confirming their surface orientation. However, glycoprotein with Mr approximately 165 000 resolved by carboxymethylation of the lithium diiodosalicylate extracted glycoprotein mixture was not labelled by this method, suggesting that it represents the granule protein with similar electrophoretic characteristics described by others. Phosphorylation of intact platelets with 32Pi also results in labelling of glycoproteins isolated by both methods, suggesting that these molecules traverse the bilipid layer of the platelet membrane, bearing reactive groups on both outer and cytoplasmic surfaces.
D001792:Blood Platelets / Q000032:analysis*; D002462:Cell Membrane / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000097:blood / Q000302:isolation & purification*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis; D008970:Molecular Weight; D011487:Protein Conformation
D006023:Glycoproteins; D008565:Membrane Proteins