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Volume 271, Number 37, Issue of September 13, 1996 pp. 22679-22686
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Inducible Nitric Oxide Synthase Requires Both the Canonical Calmodulin-binding Domain and Additional Sequences in Order to Bind Calmodulin and Produce Nitric Oxide in the Absence of Free Ca²⁺

(Received for publication, February 12, 1996, and in revised form, July 1, 1996)

Jia Ruan , Qiao-wen Xie , Nancy Hutchinson , Hearn Cho , Gloria C. Wolfe and Carl Nathan

From the Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York, New York 10021 and the  Department of Immunology and Inflammation, Merck Research Laboratories, Rahway, New Jersey 07065

All three mammalian isoforms of nitric oxide synthase (NOS) must bind calmodulin (CaM) for enzymatic activity. Only NOS2 (the inducible isoform, iNOS) does so at the low levels of free Ca²⁺ in resting cells and when almost all Ca²⁺ is chelated in cell-free preparations. To test directly whether the predicted CaM-binding region of mouse NOS2 accounts for its Ca²⁺ independence, we prepared chimeric NOS's in which mouse NOS2 residues 503-532 were reciprocally exchanged with the corresponding residues 725-754 of rat NOS1 (neuronal NOS). Unlike either parent, both chimeras required an intermediate level of free Ca²⁺ to bind CaM and generate NO. In cell lysates, the concentration of Ca²⁺ necessary for half-maximal activity (EC₅₀) was ~0 for NOS2, 200-300 nM for NOS1, and 7-10 nM for the chimeras. Results were similar when the region exchanged was enlarged by 7-8 residues toward the amino terminus. In contrast, when the carboxyl-terminal half of NOS2 (residues 454-1144) was replaced with that of NOS1 (residues 675-1429), the resulting chimera resembled NOS1 (EC₅₀, 200-300 nM free Ca²⁺). Truncation analysis suggested that NOS2 residues within the sequence 484-726 were required for Ca²⁺-independent CaM-binding. Thus, both the canonical CaM-binding domain and additional residues within the region 484-726 are necessary for NOS2's ability to bind CaM and produce NO when Ca²⁺ levels approach zero.

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