Hecker M, Walsh D T, Vane J R
William Harvey Research Institute, St. Bartholomew's Hospital Medical College, London, UK.
FEBS Lett. 1991 Dec 9;294(3):221-4. doi: 10.1016/0014-5793(91)81434-a.
Nitric oxide (.NO) synthase (NOS) activity in subcellular fractions from cultured endothelial cells (EC) and lipopolysaccharide-activated J774.2 monocyte/macrophages was investigated by monitoring the .NO-mediated increase in intracellular cyclic GMP in LLC-PK1 pig kidney epithelial cells. The constitutive NOS in EC (NOSc) was largely membrane-bound, whereas the inducible NOS in J774.2 cells (NOSi) was equally distributed among cytosol and membrane(s). Both the cytosolic NOSc in EC and the membrane-bound NOSi in J774.2 cells were strictly Ca(2+)-dependent, whereas the membrane-bound NOSc in EC and the cytosolic NOSi in J774.2 cells were not. L-Homoarginine and L-arginine-containing small peptides, such as L-arginyl-L-phenylalanine, replaced L-arginine as a substrate for the NOSc in EC and the Ca(2+)-independent NOSi in J774.2 cells, but not the Ca(2+)-dependent NOSi. Thus, irrespective of their intracellular localisation, at least three isoforms of NOS exist, which can be differentiated by their substrate specificity and Ca(2+)-dependency.
通过监测一氧化氮（·NO）介导的LLC-PK1猪肾上皮细胞内细胞内环鸟苷酸（cGMP）的增加，研究了培养的内皮细胞（EC）和脂多糖激活的J774.2单核细胞/巨噬细胞亚细胞组分中的一氧化氮合酶（NOS）活性。EC中的组成型NOS（NOSc）主要与膜结合，而J774.2细胞中的诱导型NOS（NOSi）在细胞质和膜之间均匀分布。EC中的胞质NOSc和J774.2细胞中与膜结合的NOSi都严格依赖Ca(2+)，而EC中与膜结合的NOSc和J774.2细胞中的胞质NOSi则不依赖Ca(2+)。L-高精氨酸和含L-精氨酸的小肽，如L-精氨酰-L-苯丙氨酸，可替代L-精氨酸作为EC中NOSc和J774.2细胞中不依赖Ca(2+)的NOSi的底物，但不能替代依赖Ca(2+)的NOSi。因此，无论其细胞内定位如何，至少存在三种NOS同工型，可通过其底物特异性和Ca(2+)依赖性进行区分。
