Abstract
Alkaline phosphatase of matrix vesicles isolated from fetal bovine epiphyseal cartilage was purified to apparent homogeneity using monoclonal antibody affinity chromatography. The enzyme from the butanol extract of matrix vesicles bound specifically to the immobilized antibody-Sepharose in the presence of 2% Tween 20 whereas the major portion of nonspecific protein was removed by this single step. Of various agents tested, 0.6 M 2-amino-2-methyl-1-propanol, pH 10.2, was the most effective in eluting 80-100% of the enzyme initially applied. Both Tween 20 and 2-amino-2-methyl-1-propanol associated with the eluted enzyme were effectively removed by the sequential application of DEAE-cellulose and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of a dominant band (using silver staining) corresponding to a molecular weight of 81,000. This molecular weight was nearer reported values for rat liver (Ohkubo, A., Langerman, N., and Kaplan, M. M. (1974) J. Biol Chem. 249, 7174-7180) and porcine kidney (Cathala, G., Brunel, C., Chapplet-Tordo, D., and Lazdunski, M. (1975) J. Biol. Chem. 250, 6040-6045) alkaline phosphatase, than to previously reported values for chicken (Cyboron, G. W., and Wuthier, R. E. (1981) J. Biol. Chem. 256, 7262-7268) and fetal calf (Fortuna, R., Anderson, H. C., Carty, R. P., and Sajdera, S. W. (1980) Calcif. Tissue Int. 30, 217-225) cartilage matrix vesicle alkaline phosphatase. The purified alkaline phosphatase was activated by micromolar Mg2+. The amino acid composition of cartilage alkaline phosphatase was found to be similar to that previously described for porcine kidney (Wachsmuth, E. D., and Hiwada, K. (1974) Biochem. J. 141, 273-282). Double immunoprecipitation data indicated that monoclonal antibody against cartilage alkaline phosphatase cross-reacted with fetal bovine liver or kidney enzyme but failed to react with calf intestinal or rat cartilage enzyme. Thus these observations suggest that alkaline phosphatase of matrix vesicles from calcifying epiphyseal cartilage is a liver-kidney-bone isozyme.
Highlights
Alkaline phosphatase of matrix vesicles isolated vesicles from calcifying epiphyseal cartilage is a liverfrom fetal bovine epiphyseal cartilage was purified to kidney-bone isozyme
To fully understand the role of matrix vesicles in zyme were effectively removed by the sequential ap- calcification, each relevant component of the matrix vesicle plication of DEAE-cellulose and Sepharose CL-GB must be identified, purified, and characterized
Sodium dodecyl sulfate-polyacryl- purification and characterization of alkaline phosphatase of amide gel electrophoresis of the enzyme preparation matrix vesicles from chick and bovine epiphyseal cartilage treated with sodium dodecyl sulfate and mercaptoeth- havebeen recently reported, several discrepancies with reanol showed the presence of a dominant band corresponding toa molecular weight of have emerged [3, 4]
Summary
Sodium dodecyl sulfate-polyacryl- purification and characterization of alkaline phosphatase of amide gel electrophoresis of the enzyme preparation matrix vesicles from chick and bovine epiphyseal cartilage treated with sodium dodecyl sulfate and mercaptoeth- havebeen recently reported, several discrepancies with reanol showed the presence of a dominant band (using spect to its response to Mg2+and its subunitmolecular weight silver staining) corresponding toa molecular weight of have emerged [3, 4].
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