Yeager R E, Heideman W, Rosenberg G B, Storm D R
Biochemistry. 1985 Jul 2;24(14):3776-83. doi: 10.1021/bi00335a054.
A calmodulin-sensitive adenylate cyclase was purified 3000-fold from bovine cerebral cortex using DEAE-Sephacel, calmodulin-Sepharose, and two heptanediamine-Sepharose column steps. The purified enzyme activity was stimulated by calmodulin, forskolin, 5'-guanylyl imidodiphosphate, and NaF. The molecular weight of the protein component was estimated as 328 000 with a smaller form of Mr 153 000 obtained in the presence of Mn2+. The most highly purified preparations contained major polypeptides of 150 000, 47 000, and 35 000 daltons on sodium dodecyl sulfate (SDS) gels. Photoaffinity labeling of the preparation with azido[125I]iodocalmodulin gave one product of 170 000 daltons on SDS gels. It is proposed that the catalytic subunit of the calmodulin-sensitive enzyme is 150 000 +/- 10 000 daltons and that the enzyme exists as a complex of one catalytic subunit and the stimulatory guanyl nucleotide regulatory complex. These data are consistent with the previous report that the catalytic subunit of this enzyme has a molecular weight of 150 000 +/- 10 000 [Andreasen, T.J., Heideman, W., Rosenberg, G.B., & Storm, D.R. (1983) Biochemistry 22,2757].
利用二乙氨基乙基葡聚糖凝胶（DEAE - Sephacel）、钙调蛋白琼脂糖凝胶（calmodulin - Sepharose）以及两步庚二胺琼脂糖凝胶（heptanediamine - Sepharose）柱层析法，从牛大脑皮层中纯化出了一种钙调蛋白敏感的腺苷酸环化酶，纯化倍数达3000倍。纯化后的酶活性受到钙调蛋白、福斯高林、5'-鸟苷酰亚胺二磷酸以及氟化钠的刺激。该蛋白质组分的分子量估计为328000，在锰离子存在的情况下可得到分子量为153000的较小形式。在十二烷基硫酸钠（SDS）凝胶上，纯度最高的制剂含有分子量为150000、47000和35000道尔顿的主要多肽。用叠氮[125I]碘钙调蛋白对该制剂进行光亲和标记，在SDS凝胶上得到一种分子量为17万道尔顿的产物。有人提出，钙调蛋白敏感酶的催化亚基分子量为150000±10000道尔顿，并且该酶以一个催化亚基与刺激性鸟苷酸调节复合物的复合物形式存在。这些数据与之前的报道一致，即该酶的催化亚基分子量为150000±10000[安德烈亚森，T.J.，海德曼，W.，罗森伯格，G.B.，& 斯托姆 D.R.（1983年）《生物化学》22,2757]。
