The purification and molecular characterization of alkaline phosphatases from chondrocytes and matrix vesicles of bovine fetal epiphyseal cartilage

Abstract

Intact matrix vesicles and chondrocytes were obtained from a crude coilagenase digest of bovine fetal epiphyseal cartilage. Alkaline phosphatase activity from the chondrocytes was purified 1800-fold to homogeneity. This was accomplished by solubilizing the chondrocytes in sodium deoxycholate, followed by subsequent gel filtration on Sepharose 6B and DE-52 cellulose chromatography. A 30-fold purification of the matrix vesicle alkaline phosphatase was obtained by solubilization of vesicles with sodium deoxycholate, extraction with n-butanol and gel filtration on Sepharose 6B. The purified chondrocyte alkaline phosphatase possessed a specific activity of 900 μmoles p-nitrophenyl phosphate hydrolyzed/minute/mg purified protein at pH 10.5, while the matrix vesicle alkaline phosphatase has a specific activity of 300 μmoles p-nitrophenyi phosphate hydrolyzed/minute/mg purified enzyme. The native molecular weight of the chondrocytic alkaline phosphatase and the matrix vesicle alkaline phosphatase were determined to be 141,000 and 310,00, respectively, by sucrose density gradient centrifugation. A functional subunit of the matrix vesicle alkaline phosphatase, identical to the isolated chondrocyte alkaline phosphatase, could be derived from the intact matrix vesicle enzyme by chromatography on DE-52 cellulose in the presence of 1 mM β-mercaptoethanol SDS polyacrylamide gel electrophoresis demonstrated that the chondrocytic alkaline phosphatase had one protein band of approximately 18,000 molecular weight and the matrix vesicle alkaline phosphatase had two bands, one of 18,000 and the other of 45,000 molecular weight.