Sachio Iida

Purification of nitric oxide synthase from bovine brain: Immunological characterization and tissue distribution

Nitric oxide (NO) synthase (EC 1.14.23) was purified to homogeneity from bovine cerebrum. The molecular weight of NO synthase was estimated to be 150 kDa by both SDS/PAGE and gel filtration at high salt concentration. For activity, the enzyme required NADPH, Ca2+, calmodulin and tetrahydrobiopterin as cofactors. Rabbit polyclonal antibody to bovine brain NO synthase reacted with 150 kDa NO synthase in various bovine and rat organs, including the brain, pituitary and adrenal glands, but not with that in stimulated macrophages, indicating that there are at least two immunologically distinct NO synthases.

Polyclonal antibody against an inducible form of nitric oxide synthase purified from the liver of rats treated with Propionibacterium acnes and lipopolysaccharide

A polyclonal antibody was raised in the rabbit against an inducible form of nitric oxide (NO) synthase (EC 1.14.23) purified from the liver of rats with acute liver necrosis induced by i.v. administration of Propionibacterium acnes and lipopolysaccharide. The antibody immunoprecipitated NO synthase activities in the soluble extract of the liver from treated rats. Western blot analysis showed that the cytosols of the liver, lung and spleen from the treated rats but not from non-treated rats, and that of murine macrophages cultured in the presence of lipopolysaccharide and interferon-gamma, contained immunoreactive protein with a molecular weight of 125 kDa. The antibody, however, does not cross-react with a 150 kDa constitutive form of NO synthase present in the brain of rats, indicating that the inducible and constitutive enzymes are immunologically distinguishable.

Induction of Ca2+/calmodulin-dependent NO synthase in various organs of rats by Propionibacterium acnes and lipopolysaccharide treatment

Ca2+/calmodulin‐dependent nitric oxide synthase was found to be induced during rat liver necrosis caused by administration of Propionibacterium acnes and E. coli lipopolysaccharide to rats. Examination of the specific induction of Ca2+/calmodulin‐dependent NO synthase showed that the enzyme was induced in the lung, spleen and colon as well as the liver. Northern blot analysis revealed that the induction occurred at the transcriptional level.

Nitric oxide synthase from human placenta

Summary Nitric Oxide Synthase (NOS) from both soluble and insoluble fractions of human placenta was purified completely to a single protein band having molecular weight of 135 kDa by using calmodulin affinity chromatography after 2′,5′-ADP agarose affinity chromatography. The Western Blot analyses using the three types of antibodies against NOSs clearly demonstrated that NOSs from human placenta were endothelial type of NOS, and not brain or macrophage type. Using immunohistochemistry at the light and electron microscopy level, NOS was localized in syncytiotrophoblast of human chorionic villi in both early and third trimester of pregnancy. NOS was localized in microvilli of syncytiotrophoblast. In placenta from third trimester of pregnancy, the localization of NOS was at the microvilli of syncytiotrophoblast from the maternal side and the cytoplasm of endothelial cell from the fetal side. These results demonstrated that NO is produced during pregnancy mainly by endothelial type of NOS in human placenta and may play an important role in maintaining pregnancy.