JBC Invitrogen Ultrasensitive Cytokine Assays

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Originally published In Press as doi:10.1074/jbc.M212475200 on February 27, 2003

J. Biol. Chem., Vol. 278, Issue 18, 15922-15926, May 2, 2003
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Kinetic Properties of "Soluble" Adenylyl Cyclase
SYNERGISM BETWEEN CALCIUM AND BICARBONATE*

Tatiana N. Litvin, Margarita Kamenetsky, Alla Zarifyan, Jochen BuckDagger , and Lonny R. Levin

From the Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021

"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 Vmax of the enzyme with little effect on the apparent Km 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 Km 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.

* This work was supported in part by National Institutes of Health Training Grant DA07274 (to T. N. L.), NIH Grants GM62328 and HD42060 (to J. B.) and HD38722 (to L. R. L.), and by the Ellison Medical Foundation (to J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 212-746-6274; Fax: 212-746-6241; E-mail: jobuck@med.cornell.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.


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