利用钙依赖性作为研究兔肝脏中生长激素与其结合蛋白之间相互作用的一种手段。

Use of calcium dependence as a means to study the interaction between growth hormones and their binding proteins in rabbit liver.

作者信息

Barnard R, Waters M J

机构信息

Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Australia.

出版信息

Biochem J. 1988 Mar 1;250(2):533-8. doi: 10.1042/bj2500533.

DOI:10.1042/bj2500533

PMID:3355536

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1148888/

Abstract

The affinity of 22,000-Mr human growth hormone (22 K-hGH) for GH binding proteins in rabbit liver is increased approx. 19-fold by 25 mM-Ca2+. In contrast, ovine growth hormone (oGH) binding is Ca2+-independent up to 10 mM, and decreased by greater Ca2+ concentrations. The 20,000-Mr hGH variant (20K-hGH), lacking residues 32-46, exhibits intermediate behaviour. Without Ca2+ there is a residual 40% of maximum specific binding to liver microsomes, and this increases to 65% with liver cytosolic GH binding proteins. In contrast with 22K-hGH, Scatchard analysis of 20K-hGH binding to liver microsomes produces curvilinear plots in the presence of 25 mM-Ca2+. From these results and inhibition studies with monoclonal antibodies to the GH binding proteins, it is concluded that deletion of the region 32-46 from 22K-hGH has eliminated one component of high-affinity Ca2+-potentiable binding. The Ca2+-mediated increase in Ka for the 22K-hGH-binding protein interaction is consistent with convergence of unit negative charges on the hormone and binding protein towards an intercalated Ca2+ ion. A positive charge in the critical region of nonprimate GHs would render their interactions Ca2+-independent and of lower Ka compared with 22K-hGH. A likely candidate for the negatively charged interactive residue is glutamate-33, since it is unique to human GH and is replaced by a positively charged arginine in non-primate GHs. Its absence in 20K-hGH could explain the altered calcium-dependence of 20K-hGH binding to what is probably the type 2 binding protein [Barnard & Waters (1986) Biochem. J. 237, 885-892]. The Ca2+-dependence of 20K-hGH binding to a subset of GH binding proteins provides both a verification and a mechanistic basis for the proposal [Hughes, Tokuhiro, Simpson & Friesen (1983) Endocrinology (Baltimore) 113, 1904-1906] that 20K-hGH binds with high affinity to only a subset of binding proteins in rabbit liver membranes.

摘要

22,000道尔顿的人生长激素（22K-hGH）与兔肝脏中生长激素结合蛋白的亲和力在25 mM Ca²⁺存在时增加约19倍。相比之下，绵羊生长激素（oGH）的结合在高达10 mM时不依赖Ca²⁺，而在更高的Ca²⁺浓度下会降低。缺少32 - 46位残基的20,000道尔顿hGH变体（20K-hGH）表现出中间行为。在没有Ca²⁺的情况下，与肝微粒体的最大特异性结合有40%的残留，而与肝细胞溶质生长激素结合蛋白结合时，这一比例增加到65%。与22K-hGH不同，对20K-hGH与肝微粒体结合进行Scatchard分析时，在25 mM Ca²⁺存在下会产生曲线。根据这些结果以及用针对生长激素结合蛋白的单克隆抗体进行的抑制研究，可以得出结论，从22K-hGH中缺失32 - 46区域消除了高亲和力Ca²⁺可增强结合的一个成分。22K-hGH与结合蛋白相互作用时，Ca²⁺介导的Ka增加与激素和结合蛋白上的单位负电荷向插入的Ca²⁺离子汇聚一致。非灵长类生长激素关键区域的正电荷会使其相互作用不依赖Ca²⁺，且与22K-hGH相比Ka更低。带负电荷的相互作用残基的一个可能候选者是谷氨酸-33，因为它是人生长激素特有的，在非灵长类生长激素中被带正电荷的精氨酸取代。它在20K-hGH中的缺失可以解释20K-hGH与可能是2型结合蛋白的结合的钙依赖性改变[巴纳德和沃特斯（1986年）《生物化学杂志》237卷，885 - 892页]。20K-hGH与一部分生长激素结合蛋白结合的钙依赖性为[休斯、德弘、辛普森和弗里森（1983年）《内分泌学（巴尔的摩）》113卷，1904 - 1906页]提出的20K-hGH仅与兔肝细胞膜中一部分结合蛋白高亲和力结合的观点提供了验证和机制基础。