PURIFICATION OF ADENYLYL CYCLASE

在过去的一年里， 腺苷酸环化酶的纯化，包括配体密度和阻断 协议，已优化，允许简单和可重复的合成 的支持。 对这些载体的持续实验已经建立了 它们具有连续使用18个月以上的使用寿命。 添加一个简单的选择性增溶程序的早期， 纯化得到极高纯度的腺苷酸环化酶， 使用简单的两天程序进行特定活动。 许多抗血清对应于不同区域的肽， 腺苷酸环化酶分子的特征。 联同 光亲和标记技术和分离方法， 通过酶促或免疫检测从较大分子获得的肽 在实验室中开发的化学碎片，这些 试剂可用于鉴定酶内的结合位点。 在 此外，这种类型的特异性抗体将允许评估 这种形式的腺苷酸环化酶在不同组织中的分布， 大脑区域。 将这些结果与从 平行的光亲和标记研究可以产生关于 腺苷酸环化酶家族的其他成员。

During the past year, characteristics of an affinity support for purification of adenylyl cyclase, including ligand density and blocking protocol, have been optimized, allowing simple and reproducible synthesis of the support. Continued experiments with these supports have established that they have a useful lifetime of well over 18 months of continuous use. The addition of a simple selective solubilization procedure early in the purification has yielded adenylyl cyclase of extremely high purity and specific activity using a straightforward two day procedure. A number of antisera to peptides corresponding to different regions of the adenylyl cyclase molecule have been characterized. In conjunction with the photoaffinity labeling technology and procedures for separation and immunodetection of peptides obtained from larger molecules by enzymatic or chemical fragmentation that have been developed in the laboratory, these reagents will be useful in identifying binding sites within the enzyme. In addition, specific antibodies of this type will allow assessment of the distribution of this form of adenylyl cyclase in different tissues and brain regions. Comparison of these results with those obtained from parallel photoaffinity labeling studies may yield information regarding other members of the adenylyl cyclase family.