大鼠小脑NOS纯化及其特性研究

PURIFICATION AND CHARACTERIZATION OF NITRIC OXIDE SYNTHASE FROM RAT CEREBELLUM

目的纯化大鼠小脑一氧化氮合酶并进行特性研究。方法采用改良的ＤＥ５２及２′，５′－ＡＤＰ－ａｇａｒｏｓｅ二步层析法。结果①大鼠小脑组织一氧化氮合酶（ＮＯＳ）纯化６０００倍，收率３０％，ＳＤＳ－ＰＡＧＥ成一条区带，分子量约为１５０ｋｕ；②纯化后的ＮＯＳ活性依赖Ｃａ２＋和ＣａＭ，其半激活浓度分别为２００ｎｍｏｌ／Ｌ和１０ｎｍｏｌ／Ｌ，并可被ＴＦＰ和ＮＯ２－Ａｒｇ抑制，其半抑制浓度分别为１０μｍｏｌ／Ｌ和０．６μｍｏｌ／Ｌ。结论ＮＯＳ经改良后的二步层析法纯化所得结果与文献报道结果符合，此方法可用于进一步的实验研究。

Objective To purify NOS from rat cerebellum and study the character.Methods Cytosolic nitric oxide synthase(NOS)from rat cerebellum was purified to hemogenity by improved sequential chromotographa steps on DE52 and 2′,5′-ADP-agarose.Results NOS was purified by 6000 fold,and migrated as a single band on SDS-PAGE.the molecular weight was about 150ku.the activity was dependent on Ca2+/CaM,and was inhibited by TFP and NO2-Arg.Conclusions The results from this improved two sequential chromotographa steps are consistant from reports.So this improved method may be practical for further experiments.