Purification and characterization of human platelet proteoglycan.
Freshly prepared platelets were shown to contain glycosaminoglycans equivalent to 530 micrograms of hexuronate/10(11) platelets. When the platelets were extracted with 4 M-guanidinium chloride containing proteinase inhibitors, and the extract was dialysed extensively against 7 M-urea solution, almost all of proteoglycan was recovered in the urea-soluble fraction. The proteoglycan was purified from the urea-soluble fraction with a yield of 47% by DEAE-Sephacel chromatography, CsCl-density-gradient centrifugation, Bio-Gel A-15m gel filtration and then rechromatography on DEAE-Sephacel. The purified proteoglycan contained 30% glucuronic acid, 32% N-acetylgalactosamine, 14% sulphate and 15% protein. Serine, glutamic acid, glycine, aspartic acid and leucine accounted for 64% of the total amino acids. The Mr of the proteoglycan was assessed to be approx. 136000 by sedimentation-equilibrium methods. The galactosaminoglycan released by alkaline-borohydride treatment of the proteoglycan was converted stoichiometrically into 4-sulphated unsaturated disaccharide by digestion with chondroitinase AC-II, indicating that the galactosaminoglycan was fully sulphated chondroitin 4-sulphate. The apparent Mr of the chondroitin sulphate was assessed to be 28000 by gel filtration on Bio-Gel A-0.5m (KD 0.18). On two-dimensional electrophoresis on a cellulose acetate membrane, the chondroitin sulphate gave a single compact spot co-migrating with a reference chondroitin sulphate, indicating that the chondroitin sulphate chains were homogeneous in both length and charge density. On the basis of these results, the proteoglycan in human platelets was concluded to be a macromolecule of Mr 136000 containing four chondroitin 4-sulphate chains each with the apparent Mr of 28000.
D000596:Amino Acids / Q000032:analysis; D001792:Blood Platelets / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004588:Electrophoresis, Cellulose Acetate; D006801:Humans; D008970:Molecular Weight; D011509:Proteoglycans / Q000097:blood* / Q000302:isolation & purification
D000596:Amino Acids; D011509:Proteoglycans