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J. Biol. Chem., Vol. 280, Issue 10, 8678-8685, March 11, 2005

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Nitric Oxide Scavenging by the Cobalamin Precursor Cobinamide^*

Kate E. Broderick, Veena Singh, Shunhui Zhuang, Amanpreet Kambo, Jeffrey C. Chen, Vijay S. Sharma, Renate B. Pilz, and Gerry R. Boss¶

From the Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, California 92093-0652

Nitric oxide (NO) is an important signaling molecule, and a number of NO synthesis inhibitors and scavengers have been developed to allow study of NO functions and to reduce excess NO levels in disease states. We showed previously that cobinamide, a cobalamin (vitamin B₁₂) precursor, binds NO with high affinity, and we now evaluated the potential of cobinamide as a NO scavenger in biologic systems. We found that cobinamide reversed NO-stimulated fluid secretion in Drosophila Malpighian tubules, both when applied in the form of a NO donor and when produced intracellularly by nitricoxide synthase. Moreover, feeding flies cobinamide markedly attenuated subsequent NO-induced increases in tubular fluid secretion. Cobinamide was taken up efficiently by cultured rodent cells and prevented NO-induced phosphorylation of the vasodilator-stimulated phosphoprotein VASP both when NO was provided to the cells and when NO was generated intracellularly. Cobinamide appeared to act via scavenging NO because it reduced nitrite and nitrate concentrations in both the fly and mammalian cell systems, and it did not interfere with cGMP-induced phosphorylation of VASP. In rodent and human cells, cobinamide exhibited toxicity at concentrations 50 µM with toxicity completely prevented by providing equimolar amounts of cobalamin. Combining cobalamin with cobinamide had no effect on the ability of cobinamide to scavenge NO. Cobinamide did not inhibit the in vitro activity of either of the two mammalian cobalamin-dependent enzymes, methionine synthase or methylmalonyl-coenzyme A mutase; however, it did inhibit the in vivo activities of the enzymes in the absence, but not presence, of cobalamin, suggesting that cobinamide toxicity was secondary to interference with cobalamin metabolism. As part of these studies, we developed a facile method for producing and purifying cobinamide. We conclude that cobinamide is an effective intra- and extracellular NO scavenger whose modest toxicity can be eliminated by cobalamin.

Received for publication, September 13, 2004 , and in revised form, December 27, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

Supported by Institutional National Research Service Award 5T32 CA81211.

^¶ Supported by National Institutes of Health Award 1R01 CA90932. To whom correspondence should be addressed: Dept. of Medicine, University of Calif., San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0652. Tel.: 858-534-8805; Fax: 858-534-1421; E-mail: gboss{at}ucsd.edu.

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