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Nitric oxide synthase from bovine pancreas: Purification and characterization

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Abstract

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.

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Correspondence to Suk Woo Nam or Hyang Woo Lee.

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Nam, S.W., Seo, D.W., Sung, D.S. et al. Nitric oxide synthase from bovine pancreas: Purification and characterization. Arch. Pharm. Res. 21, 128–134 (1998). https://doi.org/10.1007/BF02974016

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Key words

  • Nitric oxide
  • Nitric oxide synthase
  • Purification
  • Exocrine secretion
  • Pancreas