Zinc mediation of the binding of human growth hormone to the human prolactin receptor

Human growth hormone (hGH) elicits a diverse set of biological activities including lactation that derives from binding to the prolactin (PRL) receptor. The binding affinity of hGH for the extracellular binding domain of the hPRL receptor (hPRLbp) was increased about 8000-fold by addition of 50 micromolar ZnCl2. Zinc was not required for binding of hGH to the hGH binding protein (hGHbp) or for binding of hPRL to the hPRLbp. Other divalent metal ions (Ca2+, Mg2+, Cu2+, Mn2+, and Co2+) at physiological concentrations did not support such strong binding. Scatchard analysis indicated a stoichiometry of one Zn2+ per hGH.hPRLbp complex. Mutational analysis showed that a cluster of three residues (His18, His21, and Glu174) in hGH and His188 from the hPRLbp (conserved in all PRL receptors but not GH receptors) are probable Zn2+ ligands. This polypeptide hormone.receptor "zinc sandwich" provides a molecular mechanism to explain why nonprimate GHs are not lactogenic and offers a molecular link between zinc deficiency and its association with altered functions of hGH.     

Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule

Human growth hormone (hGH) forms a 1:2 complex with the extracellular domain of its receptor-binding protein (hGHbp) as studied by crystallization, size exclusion chromatography, calorimetry, and a previously undescribed fluorescence quenching assay. These and other experiments with protein engineered variants of hGH have led to the identification of the binding determinants for two distinct but adjacent sites on hGH for the hGHbp, and the data indicated that there are two overlapping binding sites on the hGHbp for hGH. Furthermore, the binding of hGH to the hGHbp occurred sequentially; a first hGHbp molecule bound to site 1 on hGH and then a second hGHbp bound to site 2. Hormone-induced receptor dimerization is proposed to be relevant to the signal transduction mechanism for the hGH receptor and other related cytokine receptors.     

Designing small-molecule switches for protein-protein interactions

Mutations introduced into human growth hormone (hGH) (Thr175 --> Gly-hGH) and the extracellular domain of the hGH receptor (Trp104 --> Gly-hGHbp) created a cavity at the protein-protein interface that resulted in binding affinity being reduced by a factor of 10(6). A small library of indole analogs was screened for small molecules that bind the cavity created by the mutations and restore binding affinity. The ligand 5-chloro-2-trichloromethylimidazole was found to increase the affinity of the mutant hormone for its receptor more than 1000-fold. Cell proliferation and JAK2 phosphorylation assays showed that the mutant hGH activates growth hormone signaling in the presence of added ligand. This approach may allow other protein-protein and protein-nucleic acid interactions to be switched on or off by the addition or depletion of exogenous small molecules.     

High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis

A strategy, called alanine-scanning mutagenesis, was used to identify specific side chains in human growth hormone (hGH) that strongly modulate binding to the hGH receptor cloned from human liver. Single alanine mutations (62 in total) were introduced at every residue contained within the three discontinuous segments of hGH (residues 2 to 19, 54 to 74, and 167 to 191) that have been implicated in receptor recognition. The alanine scan revealed a cluster of a dozen large side chains that when mutated to alanine each showed more than a four times lower binding affinity to the hGH receptor. Many of these residues that promote binding to the hGH receptor are altered in homologs of hGH (such as placental lactogens and prolactins) that do not bind tightly to the hGH receptor. The overall folding of these mutant proteins was indistinguishable from that of the wild-type hGH, as determined by strong cross-reactivities with seven different conformationally sensitive monoclonal antibodies. The alanine scan also identified at least one side chain, Glu174, that hindered binding because when it was mutated to alanine the receptor affinity increased by more than a factor of four.     

Three-dimensional structure of favin: saccharide binding-cyclic permutation in leguminous lectins

The three-dimensional structure of favin, the glucose- and mannose-binding lectin of Vicia faba (vetch, broad bean), has been determined at a resolution of 2.8 angstroms by molecular replacement. The crystals contain specifically bound glucose and provide the first high-resolution view of specific saccharide binding in a leguminous lectin. The structure is similar to those of concanavalin A (Con A) and green pea lectin; differences from Con A show that minimal changes are needed to accommodate the cyclic permutation in amino acid sequence between the two molecules. The molecule is an ellipsoidal dimer dominated by extensive beta structures. Each protomer contains binding sites for two divalent metal ions (Mn2+ and Ca2+) and a specific saccharide. Glucose is bound by favin in a cleft in the molecular surface and has noncovalent contacts primarily with two peptide loops, one of which contains several metal ion ligands. The specific carbohydrate-binding site is similar to that of Con A in location and general peptide folding, despite several differences in specific amino acid residues.     

Receptor and antibody epitopes in human growth hormone identified by homolog-scanning mutagenesis

A strategy, termed homolog-scanning mutagenesis, was used to identify the epitopes on human growth hormone (hGH) for binding to its cloned liver receptor and eight different monoclonal antibodies (Mab's). Segments of sequences (7 to 30 residues long) that were derived from homologous hormones known not to bind to the hGH receptor or Mab's, were systematically substituted throughout the hGH gene to produce a set of 17 chimeric hormones. Each Mab or receptor was categorized by a particular subset of mutant hormones was categorized by a particular subset of mutant hormones that disrupted binding. Each subset of the disruptive mutations mapped within close proximity on a three-dimensional model of hGH, even though the residues changed within each subset were usually distant in the primary sequence. The mapping analysis correctly predicted those Mab's which could or could not block binding of the receptor to hGH and further suggested (along with other data) that the folding of these chimeric hormones is like that of HGH. By this analysis, three discontinuous polypeptide determinants in hGH--the loop between residues 54 and 74, the central portion of helix 4 to the carboxyl terminus, and to a lesser extent the amino-terminal region of helix 1--modulate binding to the liver receptor. Homolog-scanning mutagenesis should be of general use in identifying sequences that cause functional variation among homologous proteins.     

Mendelevium: divalency and other chemical properties

Mendelevium (element 101) is the first actinide element found to give a divalent ion stable in solution. Reduction from the 3+ oxidation state was accomplished with Zn dust, Zn-Hg amalgam, Cr(2+), Eu(2+), and V(2+); additionally, measurements of the equilibrium with V(2+) provided an estimate of +0.2 volt for the couple, Md(2+) = Md(3+) + e(-). The chemical behavior of Md(3+) is similar to that of the other trivalent actinides and lanthanides. Oxidation from the trivalent to higher valence states with sodium bismuthate was not detected.     

铁皮石斛类原球茎增殖和分化的研究

通过单因素浓度试验和正交试验研究了5种金属离子、不同激素组合及不同天然附加物对铁皮石斛类原球茎增殖、分化的影响。试验结果表明,5种金属离子对类原球茎增殖影响的主次顺序为Mg2+(Fe2+(Ca2+(Mn2+(Zn2+,类原球茎增殖的最佳培养基为1/2MS+NAA 0.5mg/L+10%马铃薯汁+3%蔗糖(含Mg2+1.0 mmol/L、Ca2+2.0 mmol/L、Fe2+0.1mmol/L、Mn2+0.1mmol/L和Zn2+0.03 mmol/L)。培养基1/2MS+NAA 0.5mg/L+20%马铃薯汁+蔗糖20g/L+琼脂0.8g/L最适于铁皮石斛类原球茎的分化。     

无机元素对狭叶重楼花粉萌发及花粉管生长的影响

采用离体花粉培养法和方差分析方法,研究了钙(Ca2+)、锌(Zn2+)、钼(Mo6+)3种无机元素对狭叶重楼花粉萌发和花粉管生长的影响。结果表明:高浓度的Ca2+明显抑制花粉的萌发和花粉管的生长,低Ca2+对花粉的萌发和花粉管的生长也有轻度的抑制作用;高浓度的Zn2+抑制花粉的萌发和花粉管的生长,适量的Zn2+能促进花粉管的生长,低浓度的Zn2+对花粉的萌发和花粉管的生长无明显影响;高浓度的Mo6+抑制花粉的萌发和花粉管的生长,低浓度的Mo6+对花粉的萌发和花粉管的生长无明显影响。     

Tat protein from human immunodeficiency virus forms a metal-linked dimer

Tat, the transactivating protein from HIV, forms a metal-linked dimer with metal ions bridging cysteine-rich regions from each monomer. This novel arrangement is distinct from the "zinc finger" domain observed in other eukaryotic regulatory proteins. Ultraviolet absorption spectra show that Tat binds two Zn2+ or two Cd2+ ions per monomer, and electrophoresis of the Tat-metal complexes demonstrates that the protein forms metal-linked dimers. Partial proteolysis and circular dichroism spectra suggest that metal binding has its primary effects in the cysteine-rich region and relatively little effect on the folding of other regions. These results suggest new directions for biological studies and new approaches to drug design.     

腈纶厂失活硅藻土浸渗再生研究

采用动静态浸渗法对腈纶厂处理腈纶废水后的失活硅藻土进行解吸研究。结果表明,当pH值为2和pH值为13时,动态渗滤对失活硅藻土中的金属离子（特别是Na＋）有良好的解吸效果。各金属离子的解吸量为Zn2＋〈Fe2＋〈Al3＋〈Mg2＋〈Ca2＋〈Na＋,当溶液在pH值13时,Zn2＋、Fe2＋、Al3＋的解吸量大于pH值2时的解吸量,其中以Al3＋尤为明显。和动态渗滤相比,静态浸出对失活硅藻土中SO2-解吸效果比较明显。通过浸渗处理,硅藻土吸附活性得以较好恢复。     

盐分胁迫下番茄盐分离子和重金属的分布特征

采用盆栽试验方法研究了不同盐分含量处理下番茄不同器官盐分离子(Na+、K+、Ca2+)和重金属离子(Cd2+、Pb2+、Cr2+、Zn2+、Cu2+、Ni2+)的分布特征,探讨盐分离子对番茄不同器官吸收重金属离子的影响机制,为重金属污染盐渍土壤的农业可利用性评价提供科学依据。结果表明,番茄根、茎、叶和果实Na+含量均随盐分含量增加而增加;番茄根K+含量随盐分含量增加小幅上升,茎K+含量则显著下降,叶K+含量无显著变化;番茄各器官Ca2+含量随盐分含量增加无明显变化。番茄根Cd、Pb、Cr、Zn和Cu含量以及番茄茎、叶Cd含量均随盐分含量增加而增加;番茄根Ni含量、番茄茎叶Pb、Cr、Ni、Zn和Cu含量以及番茄果实各重金属含量受盐分含量变化影响不大。因此,土壤盐分含量的增加对番茄根部吸收重金属(Ni除外)有促进作用。     

Pb2+·金属离子与牛血清白蛋白的竞争结合平衡研究

[目的]了解Pb2+在牛血清白蛋白上的结合部位。[方法]采用平衡透析方法,研究在模拟生理(pH6.3)条件下Pb2+、金属离子与牛血清白蛋白的结合平衡,并探讨了Pb2+-BSA体系的逐级稳定常数。[结果]Pb2+在BSA上存在2类结合部位,BSA对Pb2+有1.6个强结合部位和6.5个弱结合部位。Pb2+在白蛋白分子中可能存在第1个较强的结合部位,与Zn2+相同。Pb2+在白蛋白分子中可能存在第2个较强的结合部位,与Cu2+相同。Zn2+-Pb2+-BSA三元体系中强结合部位数明显减少,说明Pb2+的优先配位基团在位点siteA。通过非线性最小二乘法拟合Bjerrum方程,确定Pb2+-BSA体系的逐级稳定常数约为104。[结论]该研究可为判断Pb2+在BSA上各结合部位之间的协调性提供参考。     

微量重金属对水螅摄食行为的毒性研究

[目的]为水螅毒理学研究提供技术支持。[方法]分别探索3种微量重金属离子(Cu2+、Cr6+、Zn2+)抑制水螅摄食和摄食反应的最低可见效应浓度(LOEC)。[结果]Cu2+、Zn2+、Cr6+抑制水螅摄食的LOEC分别为0.014、0.51和6×10-5mg/L,其抑制水螅摄食反应的LOEC分别为0.030、.55和8×10-5mg/L。Cu2+、Zn2+、Cr6+对水螅摄食反应的无可见效应浓度(NOCE)分别是0.01、0.5和6×10-5mg/L。Cu2+、Zn2+、Cr6+对水螅的摄食行为均具有明显的抑制作用,其毒性为Cr6+>Cu2+>Zn2+。[结论]利用水螅的不同摄食特性来检测水体的微毒毒性是可行的。该技术具有较高的检测效率,优于常规的动物浸泡方法。     

Systemic delivery of human growth hormone by injection of genetically engineered myoblasts

A recombinant gene encoding human growth hormone (hGH) was stably introduced into cultured myoblasts with a retroviral vector. After injection of genetically engineered myoblasts into mouse muscle, hGH could be detected in serum for 3 months. The fate of injected myoblasts was assessed by coinfecting the cells with two retroviral vectors, one encoding hGH and the other encoding beta-galactosidase from Escherichia coli. These results provide evidence that myoblasts, which can fuse into preexisting multinucleated myofibers that are vascularized and innervated, may be advantageous as vehicles for systemic delivery of recombinant proteins.     

紫色土中Cu~(2+)、Zn~(2+)吸附特征研究

[目的]了解紫色土对铜、锌的吸附特点。[方法]采用振荡平衡的方法研究了铜、锌在酸性、中性和石灰性3种紫色土中的吸附特征,并同时比较了不同环境条件对2种离子在3种紫色土中的吸附影响。[结果]研究得出,一定pH值条件下,在同种土壤上的吸附能力大小依次是Cu2+>Zn2+;3种紫色土在一定pH值条件下对同种重金属离子的吸附强度依次是石灰性紫色土>中性紫色土>酸性紫色土,与其表面电荷密度的大小相一致;Cu2+在3种紫色土表面以电性吸附和专性吸附的方式共存,其专性吸附的比例分别约为石灰性紫色土40%、中性紫色土25%、酸性紫色土20%;而Zn2+在土壤表面上绝大多数发生电性吸附。[结论]这2种重金属在紫色土中的吸附特征对库区土壤重金属污染评价有重要意义。     

锌对小麦抗氧化酶活性的影响

[目的]探讨内源抗氧化酶系统在作物抵抗外源重金属胁迫中的作用。[方法]用不同浓度锌溶液处理盆栽小麦幼苗,至幼苗生长缓慢时取材测定过氧化物酶（POD）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、丙二醛（MDA）等指标。[结果]20、50、100、200、400和800mg/L锌溶液处理的小麦POD活性分别比对照组提高了54.8%、58.2%、115.2%、157.8%、408.5%和564.0%。200、400和800mg/L锌溶液处理的小麦SOD活性分别是对照组的77.1%、68.4%和61.2%。20、50、100、200和400mg/L锌溶液处理的小麦CAT活性分别比对照组提高了1.7%、4.0%、13.2%、16.3%和22.8%。20和50mg/L锌溶液处理的小麦的丙二醛含量分别为对照的96.4%和97.5%。100、200、400和800mg/L锌溶液处理的小麦的丙二醛含量分别比对照组提高了57.7%、92.7%、235.4%和464.4%。[结论]在锌胁迫下的小麦活性氧清除系统中,POD是关键的抗氧化酶。     

Systemic delivery of recombinant proteins by genetically modified myoblasts

The ability to stably deliver recombinant proteins to the systemic circulation would facilitate the treatment of a variety of acquired and inherited diseases. To explore the feasibility of the use of genetically engineered myoblasts as a recombinant protein delivery system, stable transfectants of the murine C2C12 myoblast cell line were produced that synthesize and secrete high levels of human growth hormone (hGH) in vitro. Mice injected with hGH-transfected myoblasts had significant levels of hGH in both muscle and serum that were stable for at least 3 weeks after injection. Histological examination of muscles injected with beta-galactosidase-expressing C2C12 myoblasts demonstrated that many of the injected cells had fused to form multinucleated myotubes. Thus, genetically engineered myoblasts can be used for the stable delivery of recombinant proteins into the circulation.     

钝化剂对重金属的吸附及其吸附机理的研究

试验探讨了静态条件下沸石和草炭对重金属离子的吸附特性和吸附热力学性质。结果表明,重金属钝化剂沸石和草炭中加入重金属溶液(Cu2+、Mn2+、Zn2+、Pb2+、Cd2+)的浓度越大,钝化剂对重金属离子的吸附量越多;不同钝化剂对重金属离子的吸附能力略有不同,草炭>沸石;草炭对重金属的吸附亲和力顺序为Zn2+>Mn2+>Cu2+>Cd2+>Pb2+;沸石的为Zn2+>Cu2+>Mn2+>Cd2+>Pb2+;两种钝化剂对重金属的吸附等温线与Freundlich和Temkin方程均有较好的拟合性,Langmuir方程不适宜描述两种钝化剂对重金属的等温吸附过程,其等温线符合Freundlich吸附等温模型。