Studies on the hyaluronate binding properties of newly synthesized proteoglycans purified from articular chondrocyte cultures

Journal Article · Thu Jun 01 04:00:00 EDT 1989 · Arch. Biochem. Biophys.; (United States)

DOI:https://doi.org/10.1016/0003-9861(89)90280-4· OSTI ID:5768444

Sandy, J D; Plaas, A H

Primary cultures of rabbit articular chondrocytes have been maintained for 10 days and labeled with (35S)sulfate, (3H)leucine, and (35S)cysteine in pulse-chase protocols to study the structure and hyaluronate binding properties of newly synthesized proteoglycan monomers. Radiolabeled monomers were purified from medium and cell-layer fractions by dissociative CsCl gradient centrifugation with bovine carrier monomer, and analyzed for hyaluronate binding affinity on Sepharose CL-2B in 0.5 M Na acetate, 0.1% Triton X-100, pH 6.8. Detergent was necessary to prevent self-association of newly synthesized monomers during chromatography. Monomers secreted during a 30-min pulse labeling with (35S)sulfate had a low affinity relative to carrier. Those molecules released into the medium during the first 12 h of chase remained in the low affinity form whereas those retained by the cell layer rapidly acquired high affinity. In cultures where more than 90% of the preformed cell-layer proteoglycan was removed by hyaluronidase digestion before radiolabeling the newly synthesized low affinity monomers also rapidly acquired high affinity if retained in the cell layer. Cultures labeled with amino acid precursors were used to establish the purity of monomer preparations and to isolate core proteins for study. Leucine- or cysteine-labeled core proteins derived from either low or high affinity monomer preparations migrated as a single major species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with electrophoretic mobility very similar to that of core protein derived from extracted proteoglycan monomer. Purified low affinity monomers were converted to the high affinity form by treatment at pH 8.6; however, this change was prevented by guanidinium-HCl at concentrations above 0.8 M.

• Research Organization: Brown Univ., Providence (USA)
• OSTI ID: 5768444
• Journal Information: Arch. Biochem. Biophys.; (United States), Journal Name: Arch. Biochem. Biophys.; (United States) Vol. 271:2; ISSN ABBIA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
AMINES
AMINO ACIDS
ANIMAL TISSUES
ANIMALS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BODY
CARBOHYDRATES
CARBOXYLIC ACIDS
CARTILAGE
CELL CULTURES
CENTRIFUGATION
CHEMICAL PROPERTIES
CHONDROITIN
CHROMATOGRAPHY
CONNECTIVE TISSUE
CYSTEINE
DAYS LIVING RADIOISOTOPES
ELECTROPHORESIS
ENZYMES
EVEN-ODD NUCLEI
FRACTIONATION
GALACTOSIDASE
GLYCOPROTEINS
GLYCOSYL HYDROLASES
HYALURONIC ACID
HYALURONIDASE
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LEUCINE
LIGHT NUCLEI
LYASES
MAMMALS
MICROORGANISMS
MUCOPOLYSACCHARIDES
NUCLEI
O-GLYCOSYL HYDROLASES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXYGEN COMPOUNDS
POLYSACCHARIDES
PROTEIN STRUCTURE
PROTEINS
RABBITS
RADIOISOTOPES
REACTION KINETICS
SACCHARIDES
SEPARATION PROCESSES
STREPTOMYCES
SULFATES
SULFUR 35
SULFUR COMPOUNDS
SULFUR ISOTOPES
THIOLS
TISSUES
TRACER TECHNIQUES
VERTEBRATES