Rosenberg G B, Storm D R
J Biol Chem. 1987 Jun 5;262(16):7623-8.
Previous studies using calmodulin-Sepharose affinity chromatography have suggested that bovine brain may contain a mixture of calmodulin-sensitive and -insensitive adenylate cyclase activities (Wescott, K. R., La Porte, D. C., and Storm, D. R. (1979) Proc. Natl. Acad. Sci. U.S.A. 82, 3086-3090). In this study, mice were immunized with a purified preparation of the calmodulin-sensitive adenylate cyclase from bovine brain, and a polyclonal antiserum was obtained which was specific to the calmodulin-sensitive form of the enzyme. The antiserum was not inhibitory and precipitated enzyme activity from a homogeneous preparation of the calmodulin-sensitive adenylate cyclase catalytic subunit. Furthermore, the antiserum did not interact with calmodulin-insensitive adenylate cyclase which was resolved from the calmodulin-sensitive form of the enzyme by calmodulin-Sepharose affinity chromatography. Since the only polypeptide specifically precipitated by the antiserum had an Mr of 135,000, which was identical to the Mr of the catalytic subunit of the enzyme, it is concluded that the antiserum interacted directly and specifically with the catalytic subunit of the calmodulin-sensitive isozyme of adenylate cyclase. Detergent-solubilized membranes from several rat tissues were examined for the presence of calmodulin-sensitive adenylate cyclase using anti-calmodulin-sensitive adenylate cyclase antiserum. Approximately 40-60% of the total adenylate cyclase activity of rat brain and kidney were immunoprecipitated by the antiserum, whereas liver and testes contained no detectable calmodulin-sensitive adenylate cyclase. Approximately 15% of the total adenylate cyclase activity in rat heart and lung was the calmodulin-sensitive form. These data indicate that the calmodulin-sensitive and insensitive adenylate cyclases from bovine brain are immunologically distinct and support the proposal that there may be two or more distinct adenylate cyclase isozymes in brain.
先前利用钙调蛋白-琼脂糖亲和层析法进行的研究表明，牛脑可能含有对钙调蛋白敏感和不敏感的腺苷酸环化酶活性的混合物（韦斯科特，K.R.，拉波特，D.C.，以及斯托姆，D.R.（1979年）《美国国家科学院院刊》82，3086 - 3090）。在本研究中，用从牛脑纯化得到的对钙调蛋白敏感的腺苷酸环化酶制剂免疫小鼠，获得了对该酶的钙调蛋白敏感形式具有特异性的多克隆抗血清。该抗血清无抑制作用，且能从对钙调蛋白敏感的腺苷酸环化酶催化亚基的均一制剂中沉淀酶活性。此外，该抗血清不与通过钙调蛋白-琼脂糖亲和层析法从对钙调蛋白敏感的酶形式中分离出来的对钙调蛋白不敏感的腺苷酸环化酶相互作用。由于该抗血清特异性沉淀的唯一多肽的分子量为135,000，与该酶催化亚基的分子量相同，因此得出结论，该抗血清直接且特异性地与腺苷酸环化酶的钙调蛋白敏感同工酶的催化亚基相互作用。使用抗钙调蛋白敏感腺苷酸环化酶抗血清检测来自几种大鼠组织的去污剂溶解膜中是否存在对钙调蛋白敏感的腺苷酸环化酶。该抗血清免疫沉淀了大鼠脑和肾中约40 - 60%的总腺苷酸环化酶活性，而肝和睾丸中未检测到对钙调蛋白敏感的腺苷酸环化酶。大鼠心脏和肺中约15%的总腺苷酸环化酶活性是对钙调蛋白敏感的形式。这些数据表明，来自牛脑的对钙调蛋白敏感和不敏感的腺苷酸环化酶在免疫上是不同的，并支持了脑中可能存在两种或更多种不同的腺苷酸环化酶同工酶的提议。
