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 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 apparent K(m) for L-arginine was 15.72 microM. 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.
Nam
S W
SW
Department of Pharmacy, College of Pharmacy, Sungkyunkwan University, Suwon, Korea.
Seo
D W
DW
Sung
D S
DS
Han
J W
JW
Hong
S Y
SY
Lee
H W
HW
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D008970:Molecular Weight; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology*; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification
D011506:Proteins; D019001:Nitric Oxide Synthase