Schulz W, Lichtenstein I, Siebe H, Hierholzer K
Institut für Klinische Physiologie, Klinikum Steglitz, Free University of Berlin, F.R.G.
J Steroid Biochem. 1989 Apr;32(4):581-90. doi: 10.1016/0022-4731(89)90393-2.
11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) from rat renal cortex microsomes was solubilized using several detergents, the most effective being Zwittergent 3-10 and Triton X-100. The activity ratio oxidation/reduction of the reversible reaction corticosterone in equilibrium 11-dehydrocoticosterone varied depending on the detergent used. We attribute this variation to direct effects of different detergents on enzyme kinetics. In contrast, comparable results obtained with liver 11-HSD have been attributed to the possibility of spatially separated 11-oxidase and 11-reductase activities. In order to test whether renal 11-HSD represents a uniform oxido-reductase as generally assumed, or a dual enzyme system as has been recently proposed an attempt was made to characterize 11-HSD solubilized from renal microsomal fractions using isoelectric focusing (IEF). When 11-HSD was extracted with 1% Triton X-100 (= partially solubilized fraction) a heterogenous peak pattern was obtained. In contrast, IEF of 11-HSD extracted with 10% Triton X-100 (= delipidated fraction) resulted in a single peak at about pH 5.9 with both oxidative and reductive activity at practically identical positions within the gels. From this observation we conclude that the degree of detergent solubilization of a membrane bound protein affects its amphoteric properties and that removal of membranous lipids is a prerequisite for the analysis of its behaviour. Since the more delipidated fraction of 11-HSD revealed only one activity peak the data are compatible with the uniform enzyme concept since oxidative and reductive activities of renal cortical 11-HSD could not be separated.
用几种去污剂使大鼠肾皮质微粒体中的11β-羟基类固醇脱氢酶（11-HSD，EC 1.1.1.146）溶解，其中最有效的是两性离子去污剂3-10和曲拉通X-100。平衡状态下，可逆反应中皮质酮氧化/还原为11-脱氢皮质酮的活性比因所用去污剂而异。我们将这种差异归因于不同去污剂对酶动力学的直接影响。相比之下，肝脏11-HSD得到的类似结果被归因于11-氧化酶和11-还原酶活性在空间上分离的可能性。为了检验肾11-HSD是如通常所认为的那样代表一种单一的氧化还原酶，还是如最近所提出的那样是一种双酶系统，我们尝试用等电聚焦（IEF）来表征从肾微粒体组分中溶解的11-HSD。当用1%曲拉通X-100提取11-HSD时（=部分溶解组分），得到了一个异质峰型。相反，用10%曲拉通X-100提取的11-HSD（=脱脂组分）的IEF在约pH 5.9处产生一个单峰，氧化和还原活性在凝胶内的实际相同位置。从这一观察结果我们得出结论，膜结合蛋白的去污剂溶解程度会影响其两性性质，去除膜脂是分析其行为的前提条件。由于11-HSD的脱脂程度更高的组分仅显示一个活性峰，这些数据与单一酶概念相符，因为肾皮质11-HSD的氧化和还原活性无法分离。
