Purification of insoluble nitric oxide synthase from rat cerebellum.
Nitric oxide synthase [EC 1.14.23] from the particulate fraction of rat cerebella was purified and characterized. The homogenate of rat cerebella was centrifuged to obtain a pellet, which was washed and incubated with Triton X-100 containing buffer. The enzyme activity appeared in the 100,000 x g supernatant after incubation with the detergent. The solubilized enzyme was then purified by sequential affinity chromatography using adenosine 2',5'-diphosphate agarose and calmodulin Sepharose 4B, which gave a product that migrated as a single protein band on SDS/PAGE with a molecular mass of about 150 kDa. The purified enzyme exhibited an absolute requirement for FAD, in addition to NADPH and Ca2+/calmodulin. Thus, there is an insoluble nitric oxide synthase in rat cerebellum that has similar characteristics to the soluble type.
Hiki
K
K
Department of Internal Medicine, Faculty of Medicine, Kyoto University Hospital.
Hattori
R
R
Kawai
C
C
Yui
Y
Y
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002531:Cerebellum / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D002147:Calmodulin; D005182:Flavin-Adenine Dinucleotide; D011092:Polyethylene Glycols; D009249:NADP; D017830:Octoxynol; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases