Cloning and functional expression of human inducible nitric oxide synthase (NOS) cDNA from a glioblastoma cell line A-172.
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-alpha, and IFN-gamma. 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 immunoreactive band on Western blot analysis.
Hokari
A
A
Biochemistry Division, National Cancer Center Research Institute, Tokyo.
Zeniya
M
M
Esumi
H
H
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004790:Enzyme Induction; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases