Isolation and characterization of a 35,000 molecular weight subunit fetal cartilage matrix protein.

Abstract

A noncollagenous protein, which is a major component of the extracellular matrix of third trimester fetal epiphyseal cartilage, has been isolated and characterized.On sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the protein exists in the form of its 35,000 molecular weight subunit.Under nonreducing conditions, the protein exists as a 69,000 molecular weight dimer.In the studies reported here, the protein was usually identified as its 35,000 molecular weight subunit, demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions.Noncollagenous proteins and proteoglycans were extracted in 4 M guanidine hydrochloride containing protease inhibitors from the epiphyseal cartilage of bovine fetuses ranging in age from 106 to 270 days.The noncollagenous proteins were separated from proteoglycans by equilibrium density gradient centrifugation under associative conditions.The fraction containing the extracted proteins was examined by sodium dode-cy1 sulfate-polyacrylamide gel electrophoresis under reducing conditions, to identify the protein species extracted from fetal epiphyseal cartilages of different ages.During the third trimester the 35,000 molecular weight matrix protein was the protein species present in highest concentration in extracts of fetal epiphyseal cartilage.The matrix protein was isolated by chromatography on: (a) DEAE-Sephacel in 6 M urea; (b) Affi-Gel blue; (c) hydroxyapatite; and (d) Sephacryl S-200 in 4 M guanidine hydrochloride.The isolated protein was characterized in terms of amino acid composition, carbohydrate composition and molecular weights under denaturing and nondenaturing conditions.An antiserum was prepared against the 35,000 molecular weight protein and its homogeneity and immunological identity were examined by double immunodiffusion, crossed immunoelectrophoresis, and ELISA.Cartilate extracellular matrix is composed of collagen, proteoglycans, and a variety of noncollagenous proteins and glycoproteins, few of which have been isolated and characterized.