Nitric oxide synthase from bovine pancreas: Purification and characterization

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...

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Bibliographic Details
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
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Summary: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.
ISSN:0253-6269
1976-3786
DOI:10.1007/BF02974016