Purification of Insoluble Nitric Oxide Synthase from Rat Cerebellum1
- 1 May 1992
- journal article
- research article
- Published by Oxford University Press (OUP) in The Journal of Biochemistry
- Vol. 111 (5) , 556-558
- https://doi.org/10.1093/oxfordjournals.jbchem.a123795
Abstract
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×g supernatant after incubation with the detergent. The solubilized enzyme was then purified by sequential affinity chromatography using adeno-sine 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 160 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.Keywords
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