Newman W, Beall L D, Levine M A, Cone J L, Randhawa Z I, Bertolini D R
Department of Immunology, Otsuka Pharmaceutical Co., Rockville, Maryland 20850.
J Biol Chem. 1989 Oct 5;264(28):16359-65.
The synthetic bovine parathyroid hormone (PTH) analog (Nle8, Nle18, Tyr34) bovine PTH(1-34)amide (bPTH(1-34)amide) was reacted with biotinyl-epsilon aminocaproic acid-N-hydroxysuccinimide under conditions which yielded five isoforms which were fractionated by a combination of reversed phase and ion-exchange chromatography. These reaction products were analyzed by automated Edman degradation in a manner which allowed us to specify the location and number of biotin residues on picomole quantities of hormone. The ability of each of these isoforms to induce a rise in intracellular cAMP in the ROS 17/2.8 cell line allowed us to evaluate the effect on function of biotinylation at different residues. Derivatized PTH molecules which contained a single biotin at either lysine 13, lysine 26, or lysine 27 possessed full biological activity. However, bioactivity was significantly reduced when position 13 plus either lysine 26 or 27 were biotinylated. Biological activity was lost when all 3 lysine residues were biotinylated. Biotinylation of the alpha-NH2 group of alanine at the NH2 terminus also resulted in a total loss of activity. Hence, unlike the effect of altering the alanine at position 1, modification of a single lysine residue at positions 13, 26, and 27 has a less critical effect on biological activity of the molecule. However, biotinylation of all three lysines results in a biologically inert PTH derivative and suggests that changes in isoelectric point, hydrophobicity, or tertiary structure may strongly influence hormone function. A fully bioactive-mixture of isoforms was used to detect receptors on ROS 17/2.8 cells by flow cytometry using fluorescein isothiocyanate-avidin as a fluorescent indicator. Binding to cell surface receptors was saturable and could be inhibited by native bPTH(1-34) but not by transforming growth factor beta, calcitonin or insulin. Moreover, PTH receptors could also be detected on primary cultures of human bone cells and human fibroblasts.
合成牛甲状旁腺激素（PTH）类似物（Nle8、Nle18、Tyr34）牛PTH(1 - 34)酰胺（bPTH(1 - 34)酰胺）与生物素化的ε-氨基己酸-N-羟基琥珀酰亚胺在一定条件下反应，生成了五种异构体，通过反相和离子交换色谱联用的方法对其进行了分离。这些反应产物通过自动Edman降解进行分析，使我们能够确定皮摩尔量激素上生物素残基的位置和数量。这些异构体中的每一种在ROS 17/2.8细胞系中诱导细胞内cAMP升高的能力，使我们能够评估不同残基上生物素化对功能的影响。在赖氨酸13、赖氨酸26或赖氨酸27处含有单个生物素的衍生化PTH分子具有完整的生物活性。然而，当赖氨酸13加上赖氨酸26或27被生物素化时，生物活性显著降低。当所有3个赖氨酸残基都被生物素化时，生物活性丧失。NH2末端丙氨酸的α-NH2基团的生物素化也导致活性完全丧失。因此，与改变第1位丙氨酸的效果不同，在第13、26和27位修饰单个赖氨酸残基对分子生物活性的影响较小。然而，所有三个赖氨酸的生物素化会导致生物惰性的PTH衍生物，这表明等电点、疏水性或三级结构的变化可能强烈影响激素功能。使用异硫氰酸荧光素-抗生物素蛋白作为荧光指示剂，通过流式细胞术，利用一种具有完全生物活性的异构体混合物来检测ROS 17/2.8细胞上的受体。与细胞表面受体的结合是可饱和的，并且可以被天然bPTH(1 - 34)抑制，但不能被转化生长因子β、降钙素或胰岛素抑制。此外，在人骨细胞和人成纤维细胞的原代培养物中也可以检测到PTH受体。
