Comparative analysis of glycopeptides derived from human platelet membrane glycoprotein Ib.
Although several purification procedures have been reported for platelet glycocalicin and macroglycopeptide, compositional data suggest that contamination with tightly associated peptide fragments is a continuing problem. This, together with the lack of a reliable estimate of molecular weights, has delayed a clear resolution of the relationship of intact platelet membrane glycoprotein Ib and these proteolytically derived glycopeptides. A new procedure was developed for purification of both macroglycopeptide and glycocalicin from human platelet plasma membranes. It consists of ion-exchange chromatography on diethylaminoethyl-Sephacel, lectin affinity chromatography on wheat germ agglutinin coupled to Sepharose, and gel filtration chromatography under denaturing conditions and avoids exposure of these sialylated glycoproteins to acidic conditions. Electrophoretic evidence for the purity of macroglycopeptide and glycocalicin prepared by this procedure was obtained by Laemmli (1970) [Laemmli, U.K. (1970) Nature (London) 227, 680-685] sodium dodecyl sulfate-polyacrylamide gel electrophoresis of samples radiolabeled by sequential sodium metaperiodate oxidation and borotritide reduction. Electrophoresis gave apparent molecular weights of 108 000 and 118 000 for macroglycopeptide and glycocalicin, respectively. However, sedimentation equilibrium centrifugation experiments using the meniscus-depletion method in 6 M guanidine hydrochloride established the weight-average molecular weights of macroglycopeptide and glycocalicin as 59 700 and 105 600, respectively. The molecular weight determinations are the first by a primary physical method for platelet macroglycopeptide and glycocalicin and, together with compositional analyses, permitted calculation of the compositions of the two glycopeptides in terms of residues per molecule, which is consistent with the derivation of macroglycopeptide from glycocalicin by proteolysis.
D000596:Amino Acids / Q000032:analysis; D001792:Blood Platelets / Q000032:analysis*; D002241:Carbohydrates / Q000032:analysis; D006020:Glycopeptides / Q000302:isolation & purification*; D006023:Glycoproteins / Q000032:analysis* / Q000302:isolation & purification*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification*; D008970:Molecular Weight; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins
D000596:Amino Acids; D002241:Carbohydrates; D006020:Glycopeptides; D006023:Glycoproteins; D008565:Membrane Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; C023592:glycocalicin