Evidence of the presence of a specific ATPase responsible for ATP-initiated calcification by matrix vesicles isolated from cartilage and bone
Evidence of the presence of a specific ATPase responsible for ATP-initiated calcification by matrix vesicles isolated from cartilage and bone
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DOI：
10.1074/jbc.271.42.26383
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发表时间：
1996-10-18
影响因子：
4.8
通讯作者：
Anderson, HC
中科院分区：
文献类型：
--
作者：
Hsu, HHT;Anderson, HC
越来越多的证据表明，分离的哺乳动物基质小泡(MVS)的钙化可以由ATP启动，因为ATP可以被特定的ATPase或非特异性的碱性磷酸酶(ALP)所水解，因此ATPase或ALP是否介导了ATP启动的Ca和P-I沉积仍有待确定。为了支持特定的ATPase导致哺乳动物软骨和骨骼MVS的ATP启动钙化的假设，比较了ATP类似物、ALP底物和特定的抑制剂对完整MVS和单抗亲和纯化的MV ALP的ATP水解和ATP启动的钙化的影响，结果表明，ADP和AMP等ATP类似物对完整MVS的[Gamma-P-32]ATP水解和ATP启动的钙化都有显著的抑制作用，而磷酸单酯如β-甘油磷酸或磷酸乙醇胺与完整的MVS相比没有作用，纯化的MV ALP不能钙化，其[Gamma-P-32]ATP水解酶活性很容易被磷酸单酯抑制。此外，与完整囊泡相比，纯化的碱性磷酸酶对[γ-P-32]ATP的水解作用可被碱性磷酸酶的特异性抑制剂L-四咪唑完全抑制，提示纯化过程中特异性ATPase的丢失。反之，如果用L-四咪唑阻断ALP的活性，这些观察结果提示：1)骨骼或软骨MVS的钙和磷沉积活性是由特定的ATPase引起的，而剩余的ATPase活性是由ALP引起的；2)特定的ATPase而不是ALP负责骨骼或软骨MVS的钙和磷沉积活性。
Accumulating evidence indicates that calcification by isolated mammalian matrix vesicles (MVs) can be initiated by ATP, Since ATP can be hydrolyzed by either a specific ATPase or by nonspecific alkaline phosphatase (ALP), it remains to be established whether ATPase or ALP mediates 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-initiated calcification were compared between intact MVs and monoclonal antibody affinity-purified MV ALP, ATP analogs such as ADP and AMP exerted marked inhibitory effects on both [gamma-P-32]ATP hydrolysis and ATP-initiated calcification by intact MVs, whereas phosphomonoesters such as beta-glycerophosphate or phosphoethanolamine had no effect, In contrast to intact MVs, purified MV ALP failed to calcify, and its [gamma-P-32]ATP hydrolytic activity was readily inhibited by phosphomonoesters. Additionally, [gamma-P-32]ATP hydrolysis by purified ALP in contrast to that by intact vesicles was completely inhibited by l-tetramisole, a specific inhibitor of ALP, suggesting a loss of specific ATPase during purification. Vanadate inhibition of ATP hydrolysis by purified ALP can be decreased by increasing ATP concentrations, On the contrary, ATP concentrations did not affect vanadate inhibition of ATP hydrolysis by intact MVs if ALP activity was blocked by l-tetramisole, These observations, therefore, suggest that: 1) a portion of [gamma-P-32]ATP hydrolysis by MVs is attributable to a specific ATPase, whereas the 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.