Sethi R, Dhalla K S, Shah K R, Dhalla N S
Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada.
Mol Cell Biochem. 1993 Feb 17;119(1-2):185-93. doi: 10.1007/BF00926870.
Alamethicin is commonly used as an agent for unmasking the latent enzyme activities in vesicular membrane preparations; however, relatively little is known about the effect of this agent on the characteristics of adenylyl cyclase in heart sarcolemma. By employing rat heart sarcolemmal preparation, we observed 5 to 6 fold increase in adenylyl cyclase activity upon treatment with alamethicin. Kinetic experiments using various concentrations of MgATP revealed that the increase in adenylyl cyclase activity in alamethicin treated membranes was associated with an increase in Vmax as well as affinity of the substrate for the enzyme. Dose-responses of the control and alamethicin-treated preparations to various activators of adenylyl cyclase revealed that the sensitivity of the enzyme to forskolin, NaF and GppNHp, was markedly increased upon treating sarcolemma with alamethicin. The activation of adenylyl cyclase by forskolin was also enhanced by increasing the concentration of alamethicin in the incubation medium. Furthermore, there was a greater increase in adenylyl cyclase activity with different concentrations of Mn2+ in the presence of alamethicin. These results suggest that alamethicin treatment alters the characteristics of adenylyl cyclase in addition to unmasking the enzyme activity in the purified sarcolemmal vesicular preparation.
阿拉霉素通常用作一种揭示囊泡膜制剂中潜在酶活性的试剂；然而，关于该试剂对心脏肌膜中腺苷酸环化酶特性的影响，人们了解得相对较少。通过使用大鼠心脏肌膜制剂，我们观察到用阿拉霉素处理后腺苷酸环化酶活性增加了5至6倍。使用不同浓度的MgATP进行的动力学实验表明，阿拉霉素处理的膜中腺苷酸环化酶活性的增加与Vmax以及底物对该酶的亲和力增加有关。对照和阿拉霉素处理制剂对各种腺苷酸环化酶激活剂的剂量反应表明，用阿拉霉素处理肌膜后，该酶对福斯高林、NaF和GppNHp的敏感性显著增加。在孵育介质中增加阿拉霉素的浓度也增强了福斯高林对腺苷酸环化酶的激活作用。此外，在阿拉霉素存在的情况下，不同浓度的Mn2+使腺苷酸环化酶活性有更大的增加。这些结果表明，阿拉霉素处理除了能揭示纯化的肌膜囊泡制剂中的酶活性外，还改变了腺苷酸环化酶的特性。
