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.
Hsu
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HH
Department of Pathology and Oncology, University of Kansas Medical Center, Kansas City 66160-7410.
Morris
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DC
Davis
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Moylan
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Anderson
C H
CH
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005109:Extracellular Matrix / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D066298:In Vitro Techniques; D008081:Liposomes; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases; D051381:Rats; D012995:Solubility
D008081:Liposomes; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium