The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Cloning and Functional Expression of Human Inducible Nitric Oxide Synthase (NOS) cDNA from a Glioblastoma Cell Line A-172
Atsushi HokariMikio ZeniyaHiroyasu Esumi
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1994 Volume 116 Issue 3 Pages 575-581

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Abstract

Nitric oxide (NO) is a messenger molecule with diverse functions throughout the body. The inducible type of nitric oxide synthase (NOS) is considered to be a key molecule in the immune responses to bacteria, parasites, and tumors, and its gene expression is regulated by cytokines. We isolated 3 overlapping partial inducible NOS cDNA clones from a human glioblastoma cell line A-172 induced by IL-1, TNF-α, and IFN-γ. The 3, 963-bp human glioblastoma inducible NOS cDNA contained the longest open reading frame of 3, 459 bp, which encoded a polypeptide of 1, 153 amino acids with a calculated molecular mass of 131 kDa. This human inducible NOS possessed consensus recognition sites for the cofactors FMN, FAD, and NADPH and calmodulin recognition sites, and displayed 48.1% sequence identity with the endothelial type, 43.1% with the neuronal type, and 99.3% with the inducible type from hepatocytes, and 99.9% with the inducible type from chondrocytes and adenocarcinoma. An expression plasmid consisting of pSG5 expression vector and cDNA containing the entire putative coding sequence was constructed and transfected into COS-1 cells. COS-1 cells showed nitric oxide synthase activity together with a 130 kDa immunore-active band on Western blot analysis.

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© The Japanese Biochemical Society
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