Aubert M L, Bewley T A, Grumbach M M, Kaplan S L, Li C H
Int J Pept Protein Res. 1986 Jul;28(1):45-57.
The relationship between the conformation of human pituitary growth hormone (hGH), biological activity, and ligand binding activity was studied by comparing conformational details previously published on in vivo and in vitro studies of identical samples of hGH and its known derivatives. In vivo assays included the rat tibia test for somatotropic activity and the pigeon crop-sac assay for lactogenic hormone activity. Relative binding affinities were compared in radioimmunoassays using 125I-hGH as tracer with 1) anti-human chorionic somatomammotropin (hCS) serum (low discriminatory hybrid assay), 2) anti-hGH sera (in conventional assays), 3) monospecific anti-hGH serum (absence of cross-reaction with hCS) and 4) human anti-hGH sera obtained from GH-deficient patients on replacement therapy. In addition, binding affinities were examined in two receptor-binding assays, one specific for somatotropic activity (rabbit liver membranes, 125I-hGH), and the other, for lactogenic hormones (rabbit mammary membranes, 125I-oPRL). The conformational properties of native hGH and various chemically and enzymatically modified derivatives of the hormone were evaluated primarily from circular dichroism spectra, while conformational stabilities were estimated from the relative rates of tryptic digestion. Unfragmented, but chemically modified derivatives, exhibited good parallelism between retention or loss of native conformation and the in vivo potencies and in vitro binding affinities. None of the fragments of hGH showed activity in any of the radioreceptor assays or radioimmunoassays. Two derivatives of hGH, which contain gaps of 6 or 12 residues in the polypeptide backbone produced by partial enzymatic digestion, had full or increased in vivo potencies, full activities in the radioimmunoassays, and were the most active derivatives in both radioreceptor assays. One of these, missing the hexapeptide corresponding to residues 135-140, was also found to retain nearly all the conformational properties of native hGH. These studies proved further evidence that 1) retention by modified forms of hGH of a high degree of in vivo biological potency or in vitro binding affinity is causally related to the retention of most of the conformation and conformational stability of the molecule, and 2) the biologically active, receptor-binding and immunoreactive sites on the hGH molecule are 3-dimensional in nature.
通过比较先前发表的关于人垂体生长激素（hGH）及其已知衍生物相同样本的体内和体外研究的构象细节，研究了hGH的构象、生物活性和配体结合活性之间的关系。体内试验包括用于生长激素活性的大鼠胫骨试验和用于催乳激素活性的鸽嗉囊试验。在放射免疫分析中，以125I-hGH作为示踪剂，比较了相对结合亲和力，所用血清分别为：1）抗人绒毛膜促生长催乳素（hCS）血清（低分辨杂交试验）；2）抗hGH血清（常规试验）；3）单特异性抗hGH血清（与hCS无交叉反应）；4）从接受替代治疗的生长激素缺乏患者获得的人抗hGH血清。此外，在两种受体结合试验中检测了结合亲和力，一种针对生长激素活性（兔肝膜，125I-hGH），另一种针对催乳激素（兔乳腺膜，125I-oPRL）。天然hGH和该激素的各种化学和酶修饰衍生物的构象性质主要通过圆二色光谱进行评估，而构象稳定性则根据胰蛋白酶消化的相对速率进行估计。未断裂但经过化学修饰的衍生物在天然构象的保留或丧失与体内效价和体外结合亲和力之间表现出良好的平行关系。hGH的任何片段在任何放射受体试验或放射免疫分析中均无活性。hGH的两种衍生物，通过部分酶消化在多肽主链中产生了6个或12个残基的缺口，具有完全或增强的体内效价，在放射免疫分析中具有完全活性，并且在两种放射受体试验中是最具活性的衍生物。其中一种缺失对应于135-140位残基的六肽，还被发现保留了天然hGH几乎所有的构象性质。这些研究进一步证明：1）hGH修饰形式保留高度的体内生物效价或体外结合亲和力与分子大部分构象和构象稳定性的保留存在因果关系；2）hGH分子上的生物活性、受体结合和免疫反应位点本质上是三维的。
