Purification and characterization of a calmodulin-sensitive adenylate cyclase from Bordetella pertussis.
Purification and characterization of a calmodulin-sensitive adenylate cyclase from Bordetella pertussis.
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百日咳博德特氏菌中钙调蛋白敏感的腺苷酸环化酶的纯化和表征。
DOI：
10.1021/bi00344a006
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Storm,DR
中科院分区：
文献类型：
--
作者：
Shattuck,RL;Oldenburg,DJ;Storm,DR
方法磷酸腺苷酸环化酶测定。采用Salomon et al.（1974）的一般方法在30℃下测定腺苷酸环化酶，使用[32P] ATP作为底物，[3H] cAMP监测产物回收率。测定物最终体积为250pl 20 mM Tris-HCl， pH 7.5, 1 mM [32P] ATP (10 cpm/pmol)， 5 mM MgCl2和0.1%牛血清白蛋白。ATP浓度在0.01 ~ 10 mM范围内变化，用于测定ATP Km值。如所示，CaM （2.4 pM）被包括在一些检测中。腺苷酸环化酶测定不含磷酸二酯酶抑制剂，因为在制剂中没有磷酸二酯酶活性（数据未显示）。所有结果均以三次重复测定的平均值表示，标准误差小于5%。蛋白质浓度用Peterson（1977）的方法测定。
MethodsAdenylate Cyclase Assay. Adenylate cyclase was assayed at 30 C by the general method of Salomon et al.(1974), using [32P] ATP as a substrate and [3H] cAMP to monitor product recovery. Assays contained in a final volume of 250 pL 20 mM Tris-HCl, pH 7.5, 1 mM [32P] ATP (10 cpm/pmol), 5 mM MgCl2, and 0.1% bovine serum albumin. The concen-trations of ATP were varied from 0.01 to 10 mM for the determination of ATP Km values. CaM (2.4 pM) was included in some assays as indicated. Adenylate cyclase assays did not contain phosphodiesterase inhibitors since there was no phosphodiesterase activity in the preparations (data not shown). All results are presented as the mean of triplicate assays with standard errors of less than 5%. Protein concen-trations were determined by the method of Peterson (1977).