Monnier N, Defaye G, Chambaz E M
INSERM U 244, LBIO, Département de Recherches Fondamentales CEN BP 85X, Grenoble, France.
Eur J Biochem. 1987 Nov 16;169(1):147-53. doi: 10.1111/j.1432-1033.1987.tb13592.x.
Adrenodoxin is an iron-sulfur protein which functions as a carrier of reducing equivalents in steroid hydroxylation reactions catalyzed by specific cytochromes P-450 in steroidogenic tissues such as adrenal cortex. Purified bovine adrenocortical adrenodoxin was shown to be selectively phosphorylated upon incubation with purified cAMP-dependent protein kinase, whereas other protein kinases were ineffective. The phosphorylation reaction was completed within 45 min at 30 degrees C and resulted in the optimal incorporation of 1 mol phosphate/mol adrenodoxin. Apoadrenodoxin, lacking the iron-sulfur cluster, was also phosphorylated under similar conditions. An apparent Km of 55 microM with a Vmax of 0.3 pmol 32P incorporated min-1 mg adrenodoxin-1 was calculated. Phosphorylation resulted in a striking change in several molecular properties of adrenodoxin, such as electrophoretic behavior and hydroxyapatite affinity, thus providing the possibility of clearly separating phosphorylated from unphosphorylated adrenodoxin. In addition, phosphoadrenodoxin became refractory to mild trypsin degradation, whereas this was not the case with apoadrenodoxin. The phosphorylated site of adrenodoxin was identified as a serine residue; study of peptide products resulting from CNBr and proteolytic cleavages of phosphoadrenodoxin suggested that Ser-88 was the target of the phosphorylation reaction. The influence of phosphorylation upon adrenodoxin activity was examined using cholesterol side-chain cleavage and 11 beta-hydroxylase (11 beta) systems, reconstituted from purified components. Phosphorylation of adrenodoxin resulted in an average twofold decrease in its Km values for the two specific cytochromes P-450 involved. This effect was paralleled by a positive relationship between the degree of adrenodoxin phosphorylation and its ability to support the overall activity of reconstituted side-chain cleavage and 11 beta-hydroxylase systems. Although it remains to be examined whether adrenodoxin is phosphorylated in the intact cell, the present observations suggest that it represents a potential target in the hormonal regulation of the adrenocortical differentiated functions, especially by stimulatory agents acting through a cyclic-AMP-dependent mechanism, such as adrenocorticotropin.
肾上腺皮质铁氧还蛋白是一种铁硫蛋白，在诸如肾上腺皮质等类固醇生成组织中，由特定的细胞色素P - 450催化的类固醇羟基化反应中，它作为还原当量的载体发挥作用。经证明，纯化的牛肾上腺皮质肾上腺皮质铁氧还蛋白与纯化的环磷酸腺苷（cAMP）依赖性蛋白激酶一起温育时会被选择性磷酸化，而其他蛋白激酶则无效。磷酸化反应在30℃下45分钟内完成，导致每摩尔肾上腺皮质铁氧还蛋白最佳掺入1摩尔磷酸盐。缺乏铁硫簇的脱辅基肾上腺皮质铁氧还蛋白在类似条件下也会被磷酸化。计算得出其表观米氏常数（Km）为55微摩尔，最大反应速度（Vmax）为每分钟每毫克肾上腺皮质铁氧还蛋白掺入0.3皮摩尔32P。磷酸化导致肾上腺皮质铁氧还蛋白的几个分子特性发生显著变化，如电泳行为和羟基磷灰石亲和力，从而提供了清晰分离磷酸化和未磷酸化肾上腺皮质铁氧还蛋白的可能性。此外，磷酸化的肾上腺皮质铁氧还蛋白对温和的胰蛋白酶降解具有抗性，而脱辅基肾上腺皮质铁氧还蛋白则不然。肾上腺皮质铁氧还蛋白的磷酸化位点被确定为一个丝氨酸残基；对由溴化氰（CNBr）和磷酸化肾上腺皮质铁氧还蛋白的蛋白水解裂解产生的肽产物的研究表明，丝氨酸 - 88是磷酸化反应的靶点。使用从纯化成分重构的胆固醇侧链裂解和11β - 羟化酶（11β）系统研究了磷酸化对肾上腺皮质铁氧还蛋白活性的影响。肾上腺皮质铁氧还蛋白的磷酸化导致其对所涉及的两种特定细胞色素P - 450的米氏常数平均降低两倍。这种效应与肾上腺皮质铁氧还蛋白的磷酸化程度与其支持重构的侧链裂解和11β - 羟化酶系统整体活性的能力之间的正相关关系平行。尽管肾上腺皮质铁氧还蛋白在完整细胞中是否被磷酸化仍有待研究，但目前的观察结果表明，它是肾上腺皮质分化功能激素调节中的一个潜在靶点，特别是通过诸如促肾上腺皮质激素等通过环磷酸腺苷依赖性机制起作用的刺激剂。
