Chouinard M L, Fevold H R
Department of Chemistry, University of Montana, Missoula 59812.
Mol Cell Endocrinol. 1990 Jan 2;68(1):29-34. doi: 10.1016/0303-7207(90)90166-6.
Rabbit adrenal 17 alpha-hydroxylase activity has previously been shown to increase dramatically following ACTH stimulation. The present study was designed to determine whether the increase in enzyme activity could be correlated with an increase in P-450(17 alpha) protein measured by immunoblotting using an anti-porcine P-450(17 alpha) antibody. It was found that the total and specific contents of rabbit adrenal immunoreactive P-450(17 alpha) were increased 6- to 8-fold and 4-fold, respectively, after ACTH stimulation. The results were similar whether the detection system was 125I-labeled protein A or an alkaline phosphatase-conjugated second antibody. Corresponding increases in 17 alpha-hydroxylase activity were also observed but were slightly less than the increases in immunoreactive P-450(17 alpha), suggesting that not all of the protein was enzymatically active. Comparatively, immunoreactive P-450(21) was increased only 1.3-fold. Antibodies to porcine P-450(17 alpha) and bovine P-450(21) reacted monospecifically with the homologous rabbit and guinea pig proteins as judged by the detection of single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition studies showed that in an assay using 125 micrograms per ml of microsomal protein ACTH-stimulated rabbit adrenal 17 alpha-hydroxylase activity was inhibited 72% at a 100 mg per ml concentration of the anti-porcine P-450(17 alpha); however, 47% inhibition was observed at the same concentration of anti-bovine P-450(21). Pre-immune IgG had no effect. Molecular weight, Mr, determinations by SDS-PAGE showed both rabbit and guinea pig P-450(17 alpha) to be 52 kDa; rabbit P-450(21), 54 kDa; and guinea pig P-450(21), 49 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
先前的研究表明，促肾上腺皮质激素（ACTH）刺激后，兔肾上腺17α-羟化酶活性会显著增加。本研究旨在确定酶活性的增加是否与使用抗猪P-450（17α）抗体通过免疫印迹法测定的P-450（17α）蛋白增加相关。结果发现，ACTH刺激后，兔肾上腺免疫反应性P-450（17α）的总量和特异性含量分别增加了6至8倍和4倍。无论检测系统是125I标记的蛋白A还是碱性磷酸酶偶联的二抗，结果均相似。同时也观察到17α-羟化酶活性相应增加，但略低于免疫反应性P-450（17α）的增加，这表明并非所有蛋白质都具有酶活性。相比之下，免疫反应性P-450（21）仅增加了1.3倍。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测单条带判断，抗猪P-450（17α）和抗牛P-450（21）抗体分别与同源的兔和豚鼠蛋白发生单特异性反应。抑制研究表明，在使用每毫升125微克微粒体蛋白的测定中，每毫升100毫克浓度的抗猪P-450（17α）可使ACTH刺激的兔肾上腺17α-羟化酶活性抑制72%；然而，相同浓度的抗牛P-450（21）则观察到47%的抑制率。免疫前IgG无作用。通过SDS-PAGE测定分子量显示，兔和豚鼠的P-450（17α）均为52 kDa；兔P-450（21）为54 kDa；豚鼠P-450（21）为49 kDa。（摘要截短于250字）
