Wilson S R, Houslay M D
Biochem J. 1983 Jul 1;213(1):99-105. doi: 10.1042/bj2130099.
Dithiothreitol led to the activation and solubilization of the cyclic nucleotide phosphodiesterase activities associated with the smooth and various rough subfractions of rat liver endoplasmic reticulum. The activity in each of the subfractions exhibited somewhat different time courses, and sensitivities to dithiothreitol concentration, in respect of their solubilization and activation. Both activation and solubilization by dithiothreitol could be blocked by either thiol proteinase inhibitors or excess bovine serum albumin. Freeze-thaw solubilization was not blocked by the thiol proteinase inhibitor antipain and did not lead to the activation of the enzyme. After dithiothreitol-induced solubilization, all of the enzymes exhibited non-linear Lineweaver-Burk plots indicative of apparent negative co-operativity. In contrast, after freeze-thaw solubilization the enzyme in the smooth-endoplasmic-reticulum-plus-Golgi fraction still obeys Michaelis kinetics, as does the membrane-bound enzyme. It is possible to mimic the action of dithiothreitol in solubilizing and activating the enzyme by limited proteolysis with trypsin. Triton X-100 is highly efficient at solubilizing these enzymes, yet has little effect on their activities. Charged detergents exhibit highly selective effects on the enzymes as regards their solubilization and activity expressed.
二硫苏糖醇可激活大鼠肝脏内质网的光滑亚组分和各种粗糙亚组分相关的环核苷酸磷酸二酯酶活性，并使其溶解。各亚组分中的活性在溶解和激活方面表现出略有不同的时间进程以及对二硫苏糖醇浓度的敏感性。二硫苏糖醇的激活和溶解作用均可被巯基蛋白酶抑制剂或过量的牛血清白蛋白阻断。冻融溶解不会被巯基蛋白酶抑制剂抗蛋白酶所阻断，也不会导致酶的激活。在二硫苏糖醇诱导溶解后，所有酶均呈现非线性的Lineweaver-Burk图，表明存在明显的负协同性。相比之下，冻融溶解后，光滑内质网加高尔基体组分中的酶仍遵循米氏动力学，膜结合酶也是如此。用胰蛋白酶进行有限的蛋白水解可以模拟二硫苏糖醇溶解和激活该酶的作用。Triton X-100在溶解这些酶方面效率很高，但对其活性影响很小。就酶的溶解和表达活性而言，带电荷的去污剂对酶表现出高度选择性的影响。
