Isolation of nitric oxide synthase in the rat uterus

Nitric oxide synthase (NOS) was isolated from the uterus of adult female rats by diethylaminoethyl (DEAE) column and further purified by 2',5'-ADP agarose. The chromatographic properties revealed two isoenzymes, NOS, and NOS2. The molecular weights of both isoenzymes was approximately 155 Kd by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) which was similar to NOS1 and NOS2 from rat brain. The enzymes required nicotinamide adenine dinucleotide phosphate (NADPH), Ca+2 and calmodulin as cofactors. However, in the absence of calmodulin and/or calcium NOS, was reduced by approximately 96%, while NOS2 was reduced by approximately 70%. This maximal enzyme activity was similar for brain. These results demonstrate that two isoforms of NOS are present in the rat uterus.