Lauber M, Muller J
Department of Medicine, University Hospital, Zurich, Switzerland.
Arch Biochem Biophys. 1989 Oct;274(1):109-19. doi: 10.1016/0003-9861(89)90421-9.
It is generally accepted that the last three steps of aldosterone biosynthesis are catalyzed by a single enzyme, i.e., cytochrome P450(11) beta (P450XIB). We have previously reported that rat adrenal mitochondria may be capable of producing two forms of P450(11) beta which differ in molecular weight (49 and 51 kDa). In the present study we describe the purification, the enzymatic activities, and some structural properties of these two proteins. Using zona fasciculata mitochondria, the 51-kDa protein was purified to electrophoretic homogeneity by means of octyl-Sepharose chromatography. In a reconstituted system the protein catalyzed 18- and 11 beta-hydroxylation of deoxycorticosterone, but exhibited no 18-hydroxylation or 18-hydroxydehydrogenation of corticosterone. The 49-kDa protein was isolated from zona glomerulosa mitochondria of rats kept on a low-sodium, high-potassium regimen. Using octyl-Sepharose chromatography, it could be separated from the 51-kDa protein. A reconstituted eluate fraction, containing the 49-kDa protein, converted deoxycorticosterone not only to 18-OH-deoxycorticosterone and corticosterone, but also to 18-OH-corticosterone and aldosterone. These findings indicate that the rat adrenal cortex is capable of producing two distinct forms of active cytochrome P450(11) beta. A structural relationship of the 49- and 51-kDa proteins was indicated by experiments involving limited proteolysis. Thus, digestion with alpha-chymotrypsin and V8-protease yielded very similar peptide maps for both proteins. During potassium repletion of potassium-deficient rats, the disappearance of the active 51-kDa protein coincided with the appearance of the 49-kDa protein. These results are suggestive of a post-translational processing mechanism converting the 51-kDa protein into the smaller 49-kDa form. However, the 49-kDa protein might also be encoded by a distinct gene, regulated separately depending on the physiological conditions.
一般认为，醛固酮生物合成的最后三步由单一酶催化，即细胞色素P450(11)β（P450XIB）。我们之前报道过，大鼠肾上腺线粒体可能能够产生两种分子量不同的P450(11)β形式（49和51 kDa）。在本研究中，我们描述了这两种蛋白质的纯化、酶活性及一些结构特性。利用束状带线粒体，通过辛基琼脂糖层析将51 kDa的蛋白质纯化至电泳纯。在重组系统中，该蛋白质催化脱氧皮质酮的18-和11β-羟化反应，但对皮质酮无18-羟化或18-羟基脱氢反应。49 kDa的蛋白质是从处于低钠、高钾饮食方案的大鼠的球状带线粒体中分离出来的。利用辛基琼脂糖层析，它可以与51 kDa的蛋白质分离。含有49 kDa蛋白质的重组洗脱液部分不仅将脱氧皮质酮转化为18-羟基脱氧皮质酮和皮质酮，还转化为18-羟基皮质酮和醛固酮。这些发现表明，大鼠肾上腺皮质能够产生两种不同形式的活性细胞色素P450(11)β。有限蛋白酶解实验表明了49 kDa和51 kDa蛋白质之间的结构关系。因此，用α-胰凝乳蛋白酶和V8蛋白酶消化后，两种蛋白质产生了非常相似的肽图谱。在缺钾大鼠补钾过程中，活性51 kDa蛋白质的消失与49 kDa蛋白质的出现同时发生。这些结果提示存在一种将51 kDa蛋白质转化为较小的49 kDa形式的翻译后加工机制。然而，49 kDa蛋白质也可能由一个不同的基因编码，根据生理条件分别进行调控。
