EVIDENCE OF THE PRESENCE OF A SPECIFIC ATPASE RESPONSIBLE FOR ATP-INITIATED CALCIFICATION BY MATRIX VESICLES ISOLATED FROM CARTILAGE AND BONE

Accumulating evidence indicates that calcification by isolated mammali an matrix vesicles (MVs) can be initiated by ATP, Since ATP can be hyd rolyzed by either a specific ATPase or by nonspecific alkaline phospha tase (ALP), it remains to be established whether ATPase or ALP mediate s ATP-initiated Ca and P-i deposition. To support the hypothesis that specific ATPase is responsible for ATP-initiated calcification by MVs isolated from mammalian cartilage and bone, the effects of ATP analogs , ALP substrates, and specific inhibitors on ATP hydrolysis and ATP-in itiated calcification were compared between intact MVs and monoclonal antibody affinity-purified MV ALP, ATP analogs such as ADP and AMP exe rted marked inhibitory effects on both [gamma-P-32]ATP hydrolysis and ATP-initiated calcification by intact MVs, whereas phosphomonoesters s uch as beta-glycerophosphate or phosphoethanolamine had no effect, In contrast to intact MVs, purified MV ALP failed to calcify, and its [ga mma-P-32]ATP hydrolytic activity was readily inhibited by phosphomonoe sters. Additionally, [gamma-P-32]ATP hydrolysis by purified ALP in con trast to that by intact vesicles was completely inhibited by l-tetrami sole, a specific inhibitor of ALP, suggesting a loss of specific ATPas e during purification. Vanadate inhibition of ATP hydrolysis by purifi ed ALP can be decreased by increasing ATP concentrations, On the contr ary, ATP concentrations did not affect vanadate inhibition of ATP hydr olysis by intact MVs if ALP activity was blocked by l-tetramisole, The se observations, therefore, suggest that: 1) a portion of [gamma-P-32] ATP hydrolysis by MVs is attributable to a specific ATPase, whereas th e remaining activity is due to ALP; and 2) a specific ATPase, but not ALP, is responsible for ATP-dependent Ca- and P-i-depositing activity of MVs isolated from bone or cartilage.