RRC ID 58226
Author Adachi H, Iida S, Oguchi S, Ohshima H, Suzuki H, Nagasaki K, Kawasaki H, Sugimura T, Esumi H.
Title Molecular cloning of a cDNA encoding an inducible calmodulin-dependent nitric-oxide synthase from rat liver and its expression in COS 1 cells.
Journal Eur J Biochem
Abstract Calmodulin-dependent nitric-oxide synthase, with an apparent molecular mass of 125 kDa, was induced in the liver of rats treated with Propionibacterium acnes and Escherichia coli lipopolysaccharide. Clones were isolated from a cDNA library obtained from induced rat liver using oligonucleotide probes which were synthesized based on the amino acid sequences of peptides of the purified enzyme. Four overlapping cDNA clones for a 3.8-kbp region were isolated and the nucleotide sequences were determined. These clones encompassed an open-reading frame of 3441 bases 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, consisting 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 structural comparison suggested that the liver nitric-oxide synthase is the same as the macrophage enzyme. Northern-blot analysis showed that the mRNA in the liver is approximately 4.2 kb long and is induced transcriptionally by treatment with P. acnes and lipopolysaccharide.
Volume 217(1)
Pages 37-43
Published 1993-10-1
DOI 10.1111/j.1432-1033.1993.tb18215.x
PMID 7693462
MeSH Amino Acid Oxidoreductases / biosynthesis Amino Acid Oxidoreductases / genetics* Amino Acid Sequence Animals Base Sequence Calmodulin / pharmacology* Cell Line Chlorocebus aethiops Cloning, Molecular* DNA Restriction Enzymes DNA, Complementary / chemistry DNA, Complementary / genetics* Enzyme Induction / drug effects Gene Expression* Kidney Liver / enzymology* Male Molecular Sequence Data Nitric Oxide Synthase Rats Rats, Sprague-Dawley Transfection
Times Cited 64
DNA material Inducible NOS cDNA (RDB01385)