IN-VITRO CA DEPOSITION BY RAT MATRIX VESICLES - IS THE MEMBRANE ASSOCIATION OF ALKALINE-PHOSPHATASE ESSENTIAL FOR MATRIX VESICLE-MEDIATED CALCIUM DEPOSITION

1. Phosphatidylinositol phospholipase C (PI-PLC) treatment of rachitic rat matrix vesicles (MVs) released about 80% of membrane-bound alkaline phosphatase (ALP), AMPase, PPiase into the media. 2. About 20% hydrolytic activity was not released from MV membranes by PI-PLC treatment. 3. SDS-polyacrylamide gel electrophoresis and Western blot analysis showed only one immunoreactive protein corresponding to the molecular weight of ALP present in the soluble fraction after PI-PLC treatment. 4. The specific activity of the released ALP was at least 5-fold higher than the residual activity. 5. After PI-PLC treatment, MVs also demonstrated an 80% reduction of AMP- or beta GP-dependent calcium deposition. 6. The soluble fraction containing 80% of ALP activity was unable to support calcium deposition. The mixing of the soluble and insoluble fractions after PI-PLC treatment failed to fully restore calcium-depositing activity.