Nitric oxide synthase from bovine pancreas: Purification and characterization

• Published in: Archives of pharmacal research 1998-04, Vol.21 (2), p.128-134
• Main Authors: Nam, Suk Woo, Seo, Dong Wan, Sung, Dae Seok, Han, Jeung Whan, Hong, Sung Youl, Lee, Hyang Woo
• Format: Article
• Language: English
• Subjects: acinar cells; affinity chromatography; ammonium sulfate; Animals; antibodies; arginine; Blotting, Western; Bovines; brain; calcium; calmodulin; Cattle; Chromatography, Gel; citrulline; Electrophoresis, Polyacrylamide Gel; enzyme activity; Immunohistochemistry; Molecular Weight; NADP (coenzyme); nitric oxide synthase; Nitric Oxide Synthase - analysis; Nitric Oxide Synthase - isolation & purification; Nitric Oxide Synthase - metabolism; pancreas; Pancreas - enzymology; polyacrylamide gel electrophoresis; Proteins - analysis; Proteins - isolation & purification
• ISSN: 0253-6269; 1976-3786
• DOI: 10.1007/BF02974016

Nitric oxide synthase, NOS (EC.1.14.13.39), was purified from bovine pancreas over 5,500-fold with a 7.6% yield using 30% ammonium sulfate precipitation, and 2′,5′-ADP-agarose and calmodulin-agarose affinity chromatography. The purified bovine pancreatic NOS (bpNOS) showed a single band on SDS-PAGE corresponding to an apparent molecular mass of 160 kDa, whereas it was 320 kDa on non-denaturating gel-filtration. This indicated a homodimeric nature of the enzyme. The specific activity of the purified bpNOS was 31.67 nmol L-citrulline fored/mtn/mg protein and an apparentK ₘ for L-arginine was 15.72 μM. The enzyme activity was dependent on Ca²⁺ and calmodulin, and to a lesser extent on NADPH, FAD and FMN. H₄B was not required as a cofactor for the activity. In an inhibition experiment with L-arginine analogues, Nᴳ-nitro-L-arginine (NNA) had the most potent inhibitory effect on bpNOS, and Nᴳ, Nᴳ′-dimethyl-L-arginine (symmetric; sDMA) did not have any inhibitory effect. Immunohistochemical analysis of the bovine pancreas using brain type NOS antibody (anti-bNOS antibody) revealed that acinar cells showed strong immunoreactivity against the antibody.