Secreted adenylate cyclase of Bordetella pertussis: calmodulin requirements and partial purification of two forms
Secreted adenylate cyclase of Bordetella pertussis: calmodulin requirements and partial purification of two forms
复制标题
百日咳博德特氏菌分泌的腺苷酸环化酶：钙调蛋白需求和两种形式的部分纯化
作者：
R. Kessin
百日咳博德特氏菌的胞外腺苷酸环化酶被部分纯化，并发现含有高分子量和低分子量的物种。高分子量形式具有可变的分子量，峰值约为700，000。通过凝胶过滤测定，较小的物质具有60至70，000的分子量。低分子量形式可源自高分子量物质。从细胞上清液中纯化的高分子量复合物被钙调素高度刺激，而低分子量酶的刺激要少得多。活性酶可以从十二烷基硫酸钠（SDS）凝胶中回收，其位置对应于约50，000和65，000的分子量。从SDS凝胶中回收的活性低分子量酶迁移的分子量约为50，000，这与考马斯亮蓝染色带一致。然而，当高分子量和低分子量的制剂进行了分析，在8 M尿素等电聚焦凝胶，酶活性回收不与染色的蛋白质条带共迁移。从变性等电聚焦或SDS凝胶中回收的酶被钙调蛋白激活，表明钙调蛋白和酶的直接相互作用。高分子量形式的酶显示出增加的活性与钙调素浓度范围从0.1到500 nM，而低分子量形式的钙调素在20 nM完全激活。活细胞表面的腺苷酸环化酶被钙调素激活的方式类似于高分子量形式。
The extracellular adenylate cyclase of Bordetella pertussis was partially purified and found to contain high- and low-molecular-weight species. The high-molecular-weight form had a variable molecular weight with a peak at about 700,000. The smaller species had a molecular weight of 60 to 70,000 as determined by gel filtration. The low-molecular-weight form could be derived from the high-molecular-weight species. The high-molecular-weight complex purified from the cellular supernatant was highly stimulated by calmodulin, while the low-molecular-weight enzyme was much less stimulated. Active enzyme could be recovered from sodium dodecyl sulfate (SDS) gels at positions corresponding to molecular weights of about 50,000 and 65,000. Active low-molecular-weight enzyme recovered from SDS gels migrated with a molecular weight of about 50,000, which coincides with a coomassie blue-stained band. However, when both high- and low-molecular weight preparations were analyzed in 8 M urea isoelectrofocusing gels, the enzyme activity recovered did not comigrate with stained protein bands. The enzyme recovered from denaturing isoelectrofocusing or SDS gels was activated by calmodulin, indicating a direct interaction of calmodulin and enzyme. The high-molecular-weight form of the enzyme showed increasing activity with calmodulin concentrations ranging from 0.1 to 500 nM, while the low-molecular-weight form was fully activated by calmodulin at 20 nM. Adenylate cyclase on the surface of living cells was activated by calmodulin in a manner which resembled that found for the high-molecular-weight form.