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KMID : 0043319980210020128
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Archives of Pharmacal Research
1998 Volume.21 No. 2 p.128 ~ p.134
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Purification and Characterization
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Nam Suk-Woo
Seo Dong-Wan
Sung Sae-Seok
Han Jeung-Whan
Hong Sung-Youl
Lee Hyang-Woo
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Abstract
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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 ,-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 apparent for L-arginine was 15.72 , The enzyme activity was dependent on and calmodulin, and to a lesser extent on NADPH, FAD and FMN. was not required as a cofactor for the activity. In an inhibition experiment with L-arginine analogues, -nitro-L-arginine (NNA) had the most potent inhibitory effect on bpNOS, and , -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.
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KEYWORD
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Nitric oxide, Nitric oxide synthase, Purification, Exocrine secretion, Pancreas
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