Kinetic properties of "soluble" adenylyl cyclase - Synergism between calcium and bicarbonate
Kinetic properties of "soluble" adenylyl cyclase - Synergism between calcium and bicarbonate
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DOI：
10.1074/jbc.m212475200
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发表时间：
2003-05-02
影响因子：
4.8
通讯作者：
Levin, LR
中科院分区：
文献类型：
--
作者：
Litvin, TN;Kamenetsky, M;Levin, LR
“可溶性”腺苷酸环化酶（sAC）是哺乳动物细胞中广泛表达的cAMP来源，在进化、结构和生化上与G蛋白应答的跨膜腺苷酸环化酶不同。与跨膜腺苷酸环化酶相比，sAC对异三聚体G蛋白调节和forskolin刺激不敏感，并且只受碳酸氢盐离子的调节。本文首次报道了纯化重组sAC的动力学分析和生化特性。我们证实，在哺乳动物sAC同源物中，碳酸氢盐的调控是保守的，并证明碳酸氢盐刺激与酶的V-max的增加是一致的，而对底物ATP-Mg2+的表观K-m几乎没有影响。碳酸氢盐可以通过解除底物抑制进一步提高sAC活性。我们还发现钙是sAC活性的直接调节剂。与碳酸氢盐相反，钙通过降低ATP-Mg2+的表观K-m来刺激sAC活性。由于其作用机制不同，钙和碳酸氢盐协同激活sAC，因此，钙或碳酸氢盐的微小变化都会导致细胞cAMP水平的显著变化。
"Soluble" adenylyl cyclase (sAC) is a widely expressed source of cAMP in mammalian cells that is evolutionarily, structurally, and biochemically distinct from the G protein-responsive transmembrane adenylyl cyclases. In contrast to transmembrane adenylyl cyclases, sAC is insensitive to heterotrimeric G protein regulation and forskolin stimulation and is uniquely modulated by bicarbonate ions. Here we present the first report detailing kinetic analysis and biochemical properties of purified recombinant sAC. We confirm that bicarbonate regulation is conserved among mammalian sAC orthologs and demonstrate that bicarbonate stimulation is consistent with an increase in the V-max of the enzyme with little effect on the apparent K-m for substrate, ATP-Mg2+. Bicarbonate can further increase sAC activity by relieving substrate inhibition. We also identify calcium as a direct modulator of sAC activity. In contrast to bicarbonate, calcium stimulates sAC activity by decreasing its apparent K-m for ATP-Mg2+. Because of their different mechanisms, calcium and bicarbonate synergistically activate sAC therefore, small changes of either calcium or bicarbonate will lead to significant changes in cellular cAMP levels.