Fora M A, Valego N K, Lively M O, Castro M I, Rose J C
Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Winston-Salem, NC 27157-1083, USA.
Reprod Fertil Dev. 1996;8(1):195-201. doi: 10.1071/rd9960195.
We wished to determine if the concentration of bioactive ACTH-like activity increased during development and if there was heterogeneity in ovine fetal anterior pituitary ACTH activity as measured by bioassay and radioimmunoassay (RIA). We obtained anterior pituitaries from eight sheep fetuses (four at 0.65 and four at 0.95 gestation; term 145 +/- 5 days) and extracted and homogenized them in ice-cold 5N acetic acid, 0.3% phenylmethanesulfonyl fluoride (PMSF) and 0.2% BSA. Fractionation of each pituitary extract was performed by size-exclusion chromatography using Sepadex G-50. The ACTH-like immunoactivity (ALI) profile for each pituitary showed two well-defined peaks. One eluted with human ACTH1-39 and the other eluted with the high molecular weight fraction in the void volume. Four fractions from the first peak representing the high molecular weight forms of ACTH activity and four fractions from the second peak representing the low molecular weight forms of ACTH activity were pooled separately. These two pools were subjected to reverse-phase chromatography (RPC) on a C-8 column using a linear gradient of 70% acetonitrile in 0.8% trifluoroacetic acid over a 60 min period. Based upon the RIA, the high molecular weight forms of ACTH from the G-50 column were resolved into three main fractions, one eluting similar to the standard ACTH1-39 and the remaining two eluting after that. The low molecular weight forms of ACTH from the G-50 column were resolved into three peaks, before, with, and after the standard. We used collagenase-dispersed rat adrenal cells to test the ACTH-like bioactivity (ALB) of the crude extracts and of the different fractions obtained from the RPC of the high and low molecular weight material. The concentration of ACTH-like bioactivity in the crude extracts was similar at the two stages of gestation. However, there was a trend for the low molecular weight peak to have more peptide eluting with human ACTH1-39 and higher ratios of ALB/ALI than did the high molecular weight peak. These results suggest that multiple ACTH molecular forms with different ALB/ALI ratios are present in the ovine fetal pituitary and that there is no selective increase in ACTH1-39 concentration in the fetal pituitary in late gestation.
我们希望确定生物活性促肾上腺皮质激素（ACTH）样活性的浓度在发育过程中是否增加，以及通过生物测定和放射免疫测定（RIA）测量的绵羊胎儿垂体前叶ACTH活性是否存在异质性。我们从八只绵羊胎儿（四只妊娠0.65期，四只妊娠0.95期；足月为145±5天）获取垂体前叶，并将其在冰冷的5N乙酸、0.3%苯甲基磺酰氟（PMSF）和0.2%牛血清白蛋白（BSA）中提取并匀浆。使用葡聚糖凝胶G-50通过尺寸排阻色谱法对每个垂体提取物进行分级分离。每个垂体的ACTH样免疫活性（ALI）图谱显示出两个明确的峰。一个与人类ACTH1-39一起洗脱，另一个与空体积中的高分子量级分一起洗脱。分别合并代表ACTH活性高分子量形式的第一个峰的四个级分和代表ACTH活性低分子量形式的第二个峰的四个级分。将这两个合并物在C-8柱上进行反相色谱（RPC），使用在60分钟内70%乙腈在0.8%三氟乙酸中的线性梯度。基于RIA，来自G-50柱的ACTH高分子量形式被分离成三个主要级分，一个洗脱情况与标准ACTH1-39相似，其余两个在其之后洗脱。来自G-50柱的ACTH低分子量形式被分离成三个峰，分别在标准峰之前、与标准峰一起以及在标准峰之后。我们使用胶原酶分散的大鼠肾上腺细胞来测试粗提物以及从高分子量和低分子量物质的RPC获得的不同级分的ACTH样生物活性（ALB）。粗提物中ACTH样生物活性的浓度在两个妊娠阶段相似。然而，存在一种趋势，即低分子量峰中与人类ACTH1-39一起洗脱的肽更多，并且其ALB/ALI比值高于高分子量峰。这些结果表明，绵羊胎儿垂体中存在多种具有不同ALB/ALI比值的ACTH分子形式，并且在妊娠后期胎儿垂体中ACTH1-39浓度没有选择性增加。
