MOLECULAR-CLONING OF A CDNA-ENCODING AN INDUCIBLE CALMODULIN-DEPENDENT NITRIC-OXIDE SYNTHASE FROM RAT-LIVER AND ITS EXPRESSION IN COS-1 CELLS

Calmodulin-dependent nitric-oxide synthase, with an apparent molecular mass of 125 kDa, was induced in the liver of rats treated with Propio nibacterium acnes and Escherichia coli lipopolysaccharide. Clones were isolated from a cDNA library obtained from induced rat liver using ol igonucleotide probes which were synthesized based on the amino acid se quences of peptides of the purified enzyme. Four overlapping cDNA clon es for a 3.8-kbp region were isolated and the nucleotide sequences wer e determined. These clones encompassed an open-reading frame of 3441 b ases encoding 1147 amino acids. The deduced amino acid sequence of the cDNA suggested that the protein contains binding sites for NADPH, FAD and FMN. The structure of the possible calmodulin-binding site, consi sting of a strongly hydrophobic region surrounded by basic amino acids , is present. The full-length cDNA was expressed in COS 1 cells under the control of a cytomegalovirus promoter and the expressed enzyme was found to be a calmodulin-dependent nitric-oxide synthase. A structura l comparison suggested that the liver nitric-oxide synthase is the sam e as the macrophage enzyme. Northern-blot analysis showed that the mRN A in the liver is approximately 4.2 kb long and is induced transcripti onally by treatment with P. acnes and lipopolysaccharide.