Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of purified human platelet surface and intracellular membranes.
By using highly purified surface and intracellular membrane fractions prepared from human platelets by free-flow electrophoresis, the polypeptide and glycopeptides of these membranes have been characterized by high-resolution gel electrophoresis under reducing and non-reducing conditions. Silver staining and a variety of glycoprotein-staining procedures have been applied to identify the major components. The principal finding was the clear disparity between the distribution patterns for these two membrane fractions. There are proportionately more low-Mr acidic components present in the intracellular membrane than in the surface-derived membrane. Of the major platelet surface glycoproteins GPIb, IIb, IIIa and IIIb (or IV) well expressed in the surface membrane only, GPIIb and IIIa appear as trace components in the intracellular membrane. The cytoskeleton proteins, actin, myosin, tropomyosin, actin-binding protein and alpha-actinin are prominent features of the surface membrane and essentially absent from the intracellular membrane. Neuraminidase treatment at the whole-cell level, before homogenization, which is an essential requirement for good resolution of the two membrane subfractions, modifies a number of the glycoprotein subunits with respect to their pI characteristics, suggesting much molecular micro-heterogeneity with respect to sialic acid content. A comparison of the staining characteristics of the major glycoproteins with periodic acid/Schiff's reagent and concanavalin A/peroxidase detection and a combined procedure revealed significant differences in associated carbohydrate structures, and the major concanavalin A-binding component was shown to be GPIIIa. These observations are discussed in the context of functional activities of both membrane systems in the physiological behaviour of the platelet.
D001792:Blood Platelets / Q000032:analysis*; D001798:Blood Proteins / Q000032:analysis*; D002462:Cell Membrane / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D007425:Intracellular Membranes / Q000032:analysis; D007525:Isoelectric Focusing; D008565:Membrane Proteins / Q000097:blood*; D009439:Neuraminidase; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis
D001798:Blood Proteins; D006023:Glycoproteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D009439:Neuraminidase