Sharma R K, Wang J H
J Biol Chem. 1986 Oct 25;261(30):14160-6.
A rabbit lung cyclic nucleotide phosphodiesterase (PDE) prepared by successive chromatography on DEAE-cellulose and G-200 Sephadex columns in the presence of EGTA was activated by Ca2+ and contained calmodulin (CaM), suggesting that the enzyme exists as a stable CaM X PDE complex (Sharma, R. K., and Wirch, E. (1979) Biochem. Biophys. Res. Commun. 91, 338-344). An enzyme with similar properties was demonstrated to exist in bovine lung extract. C1, a monoclonal antibody previously shown to react with the 60-kDa subunit of bovine brain PDE isozymes (Sharma, R. K., Adachi, A.-M., Adachi, K., and Wang, J. H.) (1984) J. Biol. Chem. 259, 9248-9254), cross-reacted with the lung enzyme. Purification of the lung enzyme by C1 antibody immunoaffinity chromatography rendered the enzyme dependent on exogenous CaM for Ca2+ stimulation. Further purification was achieved by CaM affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed a predominant polypeptide of Mr 58,000 and a minor band of about 50,000. The purified enzyme could be reconstituted into a PDE X CaM complex upon incubation with CaM in the presence of either Ca2+ or EGTA. The reconstituted protein complex did not dissociate in buffers containing 0.1 mM EGTA. Analysis of the purified and reconstituted lung phosphodiesterase by Sephacryl S-300 gel filtration indicated that the lung enzyme is a dimeric protein and that the reconstituted enzyme contained two molecules of calmodulin. Analysis of the reconstituted phosphodiesterase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also showed it to contain equimolar calmodulin and the enzyme subunit. The CaM antagonists, fluphenazine, compound 48/80, and calcineurin at concentrations abolishing CaM stimulation of bovine brain PDE had little effect on the activity of reconstituted bovine lung phosphodiesterase.
通过在EGTA存在下依次在DEAE-纤维素和G-200葡聚糖凝胶柱上进行层析制备的兔肺环核苷酸磷酸二酯酶（PDE）被Ca2+激活，并含有钙调蛋白（CaM），这表明该酶以稳定的CaM×PDE复合物形式存在（夏尔马，R.K.，和维尔奇，E.（1979年）《生物化学与生物物理学研究通讯》91，338 - 344）。已证明牛肺提取物中存在具有类似性质的酶。C1是一种单克隆抗体，先前已证明它能与牛脑PDE同工酶的60 kDa亚基发生反应（夏尔马，R.K.，安达知，A.-M.，安达知，K.，和王，J.H.）（1984年）《生物化学杂志》259，9248 - 9254），它与肺酶发生交叉反应。通过C1抗体免疫亲和层析纯化肺酶后，该酶对Ca2+刺激依赖于外源性CaM。通过CaM亲和层析实现了进一步纯化。对纯化酶进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析，显示主要多肽的Mr为58,000，还有一条约50,000的次要条带。在Ca2+或EGTA存在下与CaM一起孵育时，纯化酶可重构为PDE×CaM复合物。重构的蛋白质复合物在含有0.1 mM EGTA的缓冲液中不会解离。通过Sephacryl S - 300凝胶过滤对纯化和重构的肺磷酸二酯酶进行分析表明，肺酶是一种二聚体蛋白，重构酶含有两个钙调蛋白分子。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳对重构的磷酸二酯酶进行分析也表明它含有等摩尔的钙调蛋白和酶亚基。氟奋乃静、48/80化合物和钙调神经磷酸酶等CaM拮抗剂在消除对牛脑PDE的CaM刺激的浓度下，对重构的牛肺磷酸二酯酶的活性几乎没有影响。
