Amit T, Hochberg Z, Barkey R J
Department of Pharmacology, Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel.
Biochem J. 1993 Jul 15;293 ( Pt 2)(Pt 2):345-9. doi: 10.1042/bj2930345.
We recently classified the growth-hormone (GH)-binding protein (GH-BP) in a wide range of mammalian [including human (h)] sera and reported the existence of a major lactogenic component in GH-BP of type-III sera (rabbit, horse, dog, pig and cat), based on the capacity of bovine (b) and ovine prolactin (PRL) to displace 125I-labelled human growth hormone (hGH) binding and on direct 125I-bPRL binding studies. In this study, we demonstrate the high degree of Mg2+ dependence of the binding of the classically lactogenic hGH and bPRL, but not that of the somatogenic bGH to various mammalian sera (types I-IV). Serum GH-BP was assayed using a previously described and validated charcoal-separation assay. 125I-hGH binding to rat, ovine, bovine, rabbit, horse, dog and human sera was enhanced 1.5-2.5-fold in the presence of 70 mM Mg2+. The Mg2+ effect was concentration-dependent between 3.7 mM and 70 mM, causing a significant and proportional increase in 125I-hGH binding to serum. Like 125I-hGH, 125I-bPRL binding to type-III sera was also Mg(2+)-dependent. In contrast, 125I-bGH binding to all types of serum GH-BP was not affected by Mg2+ concentrations of up to 35 mM, while 70 mM Mg2+ slightly, but significantly, reduced (by approx. 15%) bGH binding to rabbit serum. In keeping with the Mg(2+)-dependent stimulation of lactogenic hormone binding to GH-BP, 70 mM Mg2+ caused a shift to the left in the displacement curves of hGH and bPRL competing with 125I-hGH binding to rabbit, dog, horse and human sera, while the effects of the somatogens bGH and rabbit GH were shifted to the right. Scatchard analysis of hGH displacement curves with sera from various species yielded linear plots and revealed that Mg2+ significantly increased (2.3-3.0-fold) the affinity constants, but not the binding capacities. These results demonstrate the ability of changes in Mg2+ concentration to determine the degree of differential recognition of somatogens versus lactogens by serum GH-BP. It remains to be determined whether such bivalent cation effects may account, at least in part, for the growth retardation seen in Zn2+ or Mg2+ ion deficiencies.
我们最近对多种哺乳动物（包括人类）血清中的生长激素（GH）结合蛋白（GH-BP）进行了分类，并报告称基于牛（b）和羊催乳素（PRL）取代125I标记的人生长激素（hGH）结合的能力以及直接的125I-bPRL结合研究，III型血清（兔、马、狗、猪和猫）的GH-BP中存在一种主要的催乳成分。在本研究中，我们证明了经典催乳性的hGH和bPRL与各种哺乳动物血清（I-IV型）结合对Mg2+的高度依赖性，但促生长性的bGH与血清结合则不然。血清GH-BP采用先前描述并验证过的活性炭分离测定法进行检测。在70 mM Mg2+存在的情况下，125I-hGH与大鼠、羊、牛、兔、马、狗和人类血清的结合增强了1.5 - 2.5倍。Mg2+的作用在3.7 mM至70 mM之间呈浓度依赖性，导致125I-hGH与血清的结合显著且成比例增加。与125I-hGH一样，125I-bPRL与III型血清的结合也依赖于Mg(2+)。相比之下，高达35 mM的Mg2+浓度对125I-bGH与所有类型血清GH-BP的结合均无影响，而70 mM Mg2+则轻微但显著地降低了（约15%）bGH与兔血清的结合。与催乳激素与GH-BP结合的Mg(2+)依赖性刺激一致，70 mM Mg2+导致hGH和bPRL与125I-hGH竞争结合兔、狗、马和人类血清的置换曲线向左移动，而促生长因子bGH和兔GH的作用则向右移动。对来自不同物种血清的hGH置换曲线进行Scatchard分析得到线性图，并表明Mg2+显著增加了（2.3 - 3.0倍）亲和常数，但未增加结合容量。这些结果证明了Mg2+浓度变化能够决定血清GH-BP对促生长因子与催乳因子的差异识别程度。Zn2+或Mg2+离子缺乏时出现的生长迟缓是否至少部分归因于这种二价阳离子效应，仍有待确定。
