Synthesis of low buoyant density proteoglycans by human chondrocytes in culture.
Human chondrocyte strains were derived from explant outgrowth of nonarthritic or osteoarthritic human cartilage. Chondrocytes radiolabeled with [35SO4] or [35S]-methionine were used to measure the biosynthesis of proteoglycans recovered from the most buoyant fraction (A4) of a CsCl density gradient centrifugation performed under associative conditions. The proteoglycans isolated from the A4 fraction (rho < 1.47 g/ml) were hydrodynamically small and contained both large and small glycosaminoglycan chains. When assessed by SDS/PAGE using 3-16% gradient gels, two subpopulations of small proteoglycans (smPG) were identified. The larger of the two species (smPG-I) migrated slower than the 200 kDa marker protein; when reassessed on 3-5% acrylamide gels, its apparent molecular mass was larger than the 480 kDa and 440 kDa alpha and beta heavy chains of dynein. We estimated the apparent molecular size of this smPG to be approximately 520 kDa. The smaller smPG (smPG-II) had an apparent average molecular mass of 180 kDa (range 170-210 kDa) after 3-16% SDS/PAGE. Three monoclonal antibodies, 1C6, 5D4, and S103L, reactive with the hyaluronic acid binding region of the aggregating proteoglycan core protein, keratan sulfate, and a core protein domain in the chondroitin sulfate attachment region, respectively, reacted with a single protein (apparent molecular mass, 180 kDa) that was similar in size to smPG-II.
Malemud
C J
CJ
Department of Medicine, Case Western Reserve University, Cleveland, OH 44106.
Papay
R S
RS
D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002809:Chondroitin Sulfates / Q000276:immunology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D007632:Keratan Sulfate / Q000276:immunology; D008970:Molecular Weight; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D011509:Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D011509:Proteoglycans; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D007632:Keratan Sulfate