Lakshmi V, Monder C
Population Council, New York, New York 10021.
Endocrinology. 1988 Nov;123(5):2390-8. doi: 10.1210/endo-123-5-2390.
We have proposed that 11 beta-hydroxysteroid dehydrogenase is composed of structurally independent units with 11 beta-dehydrogenase and 11-reductase activities. We now report the purification of rat liver 11 beta-dehydrogenase to apparent homogeneity. Starting with microsomes, 800-fold purification was achieved with agarose-NADP affinity chromatography. No 11-reductase accompanied the purification. Homogeneity of 11 beta-dehydrogenase was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid end-group analysis and immunoprecipitation. The terminal amino acid was methionine. Monomer mol wt was 34,000. The enzyme was found to be a glycoprotein. A sequence of 40 amino acid units was identified from the amino end. The amino-terminal region was found to be highly nonpolar. Unlike unpurified microsomal 11 beta-dehydrogenase, which showed curvilinear Eadie plots, homogeneous enzyme gave rectilinear plots. Michaelis constants were 1.83 +/- 0.06 microM for corticosterone and 17.3 +/- 2.24 microM for cortisol. First order rate constants were 10 times greater for corticosterone than cortisol, and maximum velocities were similar.
我们曾提出，11β-羟类固醇脱氢酶由具有11β-脱氢酶和11-还原酶活性的结构独立单元组成。我们现在报告大鼠肝脏11β-脱氢酶的纯化，直至达到表观均一性。以微粒体为起始材料，通过琼脂糖-NADP亲和层析实现了800倍的纯化。纯化过程中未伴有11-还原酶。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、氨基酸末端基团分析和免疫沉淀确定了11β-脱氢酶的均一性。末端氨基酸为蛋氨酸。单体分子量为34,000。该酶被发现是一种糖蛋白。从氨基末端鉴定出了40个氨基酸单元的序列。发现氨基末端区域高度非极性。与未纯化的微粒体11β-脱氢酶不同，后者显示出曲线形的伊迪图，而均一酶给出的是直线图。皮质酮的米氏常数为1.83±0.06微摩尔，皮质醇的米氏常数为17.3±2.24微摩尔。皮质酮的一级速率常数比皮质醇大10倍，最大速度相似。
