Purification of matrix-vesicle alkaline phosphatase from chicken epiphyseal cartilage and experiments on its ATP-hydrolyzing properties.

Matrix vesicles of epiphyseal growth cartilage are presumed to be the initial sites of mineralization. In order to discover whether matrix vesicle phosphatase could function as Ca2+-ATPase, we purified it from isolated matrix vesicles of chicken epiphyseal growth cartilage by n-butanol extraction, Sepharose 6B gel filtration, and DEAE-ion exchange chromatography. The enzyme protein hydrolyzes a number of phosphatase substrates, but showed one protein band when tested by polyacrylamide gel electrophoresis, and an antibody raised in a rabbit caused only one precipitation line in immunodiffusion and immunoelectrophoresis. The properties of the enzyme regarding the hydrolysis of ATP were studied at pH 7.6, which is close to the pH of the extracellular fluid of the cartilage. ATPase activity was stimulated by Mg2+ and Ca2+, but Ca2+ was a weaker activator and failed to stimulate the hydrolysis of ATP any further at the optimal Mg2+ concentration, indicating that the enzyme is not a "true" Ca2+-ATPase.