Schmidt H H, Pollock J S, Nakane M, Gorsky L D, Förstermann U, Murad F
Abbott Laboratories, Abbott Park, IL 60064-3500.
Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):365-9. doi: 10.1073/pnas.88.2.365.
The soluble form of guanylyl cyclase-activating-factor (GAF) synthase from rat cerebellum was purified to homogeneity by sequential affinity chromatographic steps on adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and calmodulin-agarose. Enzyme activity during purification was bioassayed by the L-arginine-, NADPH-, and Ca2+/calmodulin-dependent formation of a plasma membrane-permeable nitric oxide-like factor that stimulated soluble guanylyl cyclase in RFL-6 cells. With calmodulin and NADPH as cofactors, purified soluble GAF synthase induced an increase of 1.05 mumol of cGMP per 10(6) RFL-6 cells per 3 min per mg of protein. The coproduct of this signal-transduction pathway appeared to be L-citrulline. GAF synthase catalyzed the conversion of 107 nmol of L-arginine into L-citrulline per min per mg of protein. Based on these assays, this represents a purification of GAF synthase of approximately 10,076- and 8925-fold with recoveries of 16% and 19%, respectively. Rechromatography of the purified enzyme on Mono P (isoelectric point = 6.1 +/- 0.3), Mono Q, and Superose 12 or 6 resulted in no further purification or increase in specific activity. A Stokes radius of 7.9 +/- 0.3 nm and a sedimentation coefficient s20,w of 7.8 +/- 0.2 S were used to calculate a molecular mass of about 279 +/- 25 kDa for the native enzyme. SDS/PAGE revealed a single protein band with a molecular mass of about 155 +/- 3 kDa. These data suggest that soluble GAF synthase purified from rat cerebellum is a homodimer of 155-kDa subunits and that enzyme activity is dependent upon the presence of calmodulin.
通过先后在腺苷2',5'-二磷酸（2',5'-ADP）-琼脂糖和钙调蛋白-琼脂糖上进行亲和色谱步骤，将大鼠小脑的可溶性鸟苷酸环化酶激活因子（GAF）合酶纯化至同质。在纯化过程中，通过L-精氨酸、NADPH以及Ca2+/钙调蛋白依赖性形成一种可透过质膜的一氧化氮样因子来进行酶活性的生物测定，该因子可刺激RFL-6细胞中的可溶性鸟苷酸环化酶。以钙调蛋白和NADPH作为辅因子，纯化后的可溶性GAF合酶每毫克蛋白质每3分钟可使每10(6)个RFL-6细胞中cGMP增加1.05 μmol。该信号转导途径的副产物似乎是L-瓜氨酸。GAF合酶每毫克蛋白质每分钟可催化107 nmol的L-精氨酸转化为L-瓜氨酸。基于这些测定，这代表GAF合酶的纯化倍数约为10,076倍和8925倍，回收率分别为16%和19%。将纯化后的酶在Mono P（等电点 = 6.1 ± 0.3）、Mono Q以及Superose 12或6上再次进行色谱分析，未导致进一步的纯化或比活性增加。利用7.9 ± 0.3 nm的斯托克斯半径和7.8 ± 0.2 S的沉降系数s20,w计算出天然酶的分子量约为279 ± 25 kDa。SDS/PAGE显示出一条分子量约为155 ± 3 kDa的单一蛋白带。这些数据表明，从大鼠小脑纯化得到的可溶性GAF合酶是由155-kDa亚基组成的同型二聚体，且酶活性依赖于钙调蛋白的存在。
