Brennan D P, Levine M A
Department of Medicine, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1987 Oct 25;262(30):14795-800.
A bioactive biotin-containing derivative of the synthetic bovine parathyroid hormone analog [Nle8,Nle18,Tyr34]bovine parathyroid hormone-(1-34) (bPTH-(1-34] amide was prepared by reacting the peptide with N-biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide ester. The derivative was incubated with particulate renal plasma membranes or with detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) extracts of renal cortical membranes, and two membrane components were identified. Labeling of these components was competitively inhibited by underivatized bPTH-(1-34) or bPTH-(3-34) but not by insulin, adrenocorticotropin, or oxidized rat PTH-(1-34). PTH-binding components that were immobilized on nitrocellulose could be detected by incubating the membrane with biotinyl-bPTH-(1-34). Binding components of apparent molecular mass 68, 70, and 150 kDa were specifically labeled in plasma membranes derived from canine, human, and porcine renal cortex, rat liver, and human fibroblasts. The 68-kDa binding protein was found to be consistently more acidic than the 70-kDa binding protein in human, porcine, and canine renal membranes analyzed by two-dimensional electrophoresis. The 68-70-kDa receptor doublet could be specifically isolated by streptavidin-agarose chromatography of solubilized membrane extracts that had first been incubated with biotinyl-BPTH-(1-34). Biotinyl-bPTH-(1-34) should be useful as a tool for further characterization and purification of the PTH receptor.
合成牛甲状旁腺激素类似物[Nle8,Nle18,Tyr34]牛甲状旁腺激素-(1 - 34)（bPTH-(1 - 34]酰胺的一种含生物活性生物素的衍生物，是通过使该肽与N - 生物素基 - ε - 氨基己酸N - 羟基琥珀酰亚胺酯反应制备而成。将该衍生物与肾微粒体血浆膜或肾皮质膜的去污剂[3 - [(3 - 胆酰胺丙基)二甲基铵基]-1 - 丙烷磺酸盐]提取物一起温育，鉴定出两种膜成分。未衍生化的bPTH-(1 - 34)或bPTH-(3 - 34)可竞争性抑制这些成分的标记，但胰岛素、促肾上腺皮质激素或氧化型大鼠PTH-(1 - 34)则不能。通过用生物素基 - bPTH-(1 - 34)温育膜，可检测固定在硝酸纤维素上的PTH结合成分。在源自犬、人、猪肾皮质、大鼠肝脏和人成纤维细胞的质膜中，表观分子量为68、70和150 kDa的结合成分被特异性标记。通过二维电泳分析发现，在人、猪和犬肾膜中，68 kDa的结合蛋白始终比70 kDa的结合蛋白酸性更强。通过链霉亲和素 - 琼脂糖色谱法，可从首先用生物素基 - BPTH-(1 - 34)温育过的溶解膜提取物中特异性分离出68 - 70 kDa的受体双峰。生物素基 - bPTH-(1 - 34)应可作为进一步表征和纯化PTH受体的工具。
