Wahrmann J P, Gros F, Piau J P, Schapira G
Biochim Biophys Acta. 1980 Apr 11;612(2):421-32. doi: 10.1016/0005-2744(80)90125-4.
A new procedure for the purification of rat brain adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) is presented. The enzyme solubilized in Lubrol PX was purified either by molecular sieving or by hydrophobic chromatography, followed by a preparative isoelectric focusing step. For this purpose, a new isoelectric focusing technique was developed which allows a good resolution of adenylate cyclase in a short period of time. When resolved by this procedure, the enzyme migrated as a single molecular species with a pI of 6.3. When isoelectric focusing was performed in the presence of EGTA, two distinct peaks of activity could be detected at pI 6.1 and 7.3. This suggests that adenylate cyclase consists of two subunits held together by divalent ions. It is shown that the purified adenylate cyclase has a smaller sedimentation coefficient and is less hydrophobic than the native one. We conclude that the adenylate cyclase containing complex was at least partially disaggregated by this procedure.
本文介绍了一种纯化大鼠脑腺苷酸环化酶（ATP 焦磷酸裂解酶（环化），EC 4.6.1.1）的新方法。溶解在 Lubrol PX 中的该酶通过分子筛或疏水色谱法进行纯化，随后进行制备性等电聚焦步骤。为此，开发了一种新的等电聚焦技术，该技术可在短时间内实现腺苷酸环化酶的良好分离。通过该程序分离时，该酶以单一分子形式迁移，其 pI 为 6.3。当在 EGTA 存在下进行等电聚焦时，在 pI 6.1 和 7.3 处可检测到两个不同的活性峰。这表明腺苷酸环化酶由通过二价离子结合在一起的两个亚基组成。结果表明，纯化后的腺苷酸环化酶沉降系数较小，疏水性低于天然酶。我们得出结论，该程序至少部分地使含腺苷酸环化酶的复合物解聚。
