二价铅离子与金属硫蛋白相互作用的研究

通过紫外吸收光谱和平衡透析法研究了二价铅离子同脱金属硫蛋白（ａｐｏ－ＭＴ）、锌－金属硫蛋白（Ｚｎ－ＭＴ）的相互作用,证实Ｐｂ（Ⅱ）是以金属巯基复合物（金属巯基比为１∶２）的形式同金属硫蛋白结合,表观离解常数（ＫＤ）为８．７１×１０－７ｍｏｌ／Ｌ．在自由铅浓度达到６．５２×１０－６ｍｏｌ／Ｌ的条件下,铅离子即可将Ｚｎ－ＭＴ上的Ｚｎ完全取代下来．通过ＥＤＴＡ、ＤＴＮＢ竞争反应、圆二色性（ＣＤ）光谱分析,认为Ｐｂ－ＭＴ的金属巯基复合物不同于Ｚｎ－ＭＴ中Ｚｎ与巯基形成的紧密的正四面体结构,而是可能形成一种三级结构相对松散、热力学上不稳定的Ｃｙｓ－Ｓ－Ｐｂ－Ｓ－Ｃｙｓ平面形结构．研究认为金属硫蛋白的两种亚型ＭＴ－Ⅰ、ＭＴ－Ⅱ与Ｐｂ（Ⅱ）的结合能力并无显著差异     

脱金属硫蛋白与二价汞离子的络合作用及构象研究

用圆二色谱研究兔肝锐金属硫蛋白的两个亚型ａｐｏ－ＭＴ１、ａｐｏ－ＭＴ２与Ｈｇ＾２＋在不同分子数比和ｐＨ值下的络合规律。发现：（１）在ｐＨ２下与Ｈｇ＾２＋重组ＭＴｓ的ＣＤ谱特征峰是３０４ｎｍ（＋）,２６０ｎｍ（－）,２５０ｎｍ（＋）。络合Ｈｇ＾２＋的数目ｎ远远超过７。任何ｐＨ值下过量Ｈｇ＾２＋的存在都将破坏ＭＴｓ的正常构象。ｐＨ值对形成和维持ＭＴｓ稳定构象的影响很大,当ｐＨ≥４．９时,ａｐｏ－ＭＴｓ     

脱金属硫蛋白与镉离子的络合作用及构象研究

用圆二色（ＣＤ）谱地研究兔肝脱金属硫属蛋白的两个亚型与Ｃｄ＾２＋的络合作用及对重组ＭＴ构象的影响。观测了ａｐｏ－ＭＴ垢巯基在空气和室温下的稳定性。在ＰＨ４．７１,镉重组ＭＴ１的ＣＤ谱特征峰在２５７ｎｍ（＋）,２３８ｎｍ（－）,２２６ｎｍ（＋）与镉诱导的天然ＭＴ１相同。在空气存在和ＰＨ７．９０的ＣＤ谱只有２４３ｎｍ（＋）一个峰。向两亚型分别加入７ｅｑＣｄ＾２＋测定ＣＤ谱随ＰＨ值的变化,发现在ＰＨ２．     

兔肝金属硫蛋白β结构域的制备和鉴定

制备一定量的Ｃｄ、Ｚｎ兔肝金属硫蛋白（ｍｅｔａｌｏｔｈｉｏｎｅｉｎ,ＭＴ）,然后脱掉其中的金属,获得不含金属的硫蛋白（ａｐｏＭＴ）．用一价金属ＣｕＣｌ或ＡｇＮＯ３以５．８１的金属蛋白摩尔比与ａｐｏＭＴ结合,将其β结构域用金属饱和,在３７℃,ｐＨ７．０下用枯草杆菌蛋白酶水解掉未结合金属的α结构域,获得Ｃｕ（Ⅰ）、Ａｇ（Ⅰ）结合的兔肝ＭＴ－Ｉ、ＭＴ－Ⅱ的β结构域．通过ＨＰＬＣ、羧甲基化后的ＳＤＳ－ＰＡＧＥ、氨基酸组成分析、金属定量、Ｎ末端分析和紫外扫描等鉴定,证明确实得到了Ｎ末端为Ｍｅｔ,分子量３０００左右,金属蛋白摩尔比为５～５．５１的ＭＴβ结构域,其氨基酸组成与理论值基本相符     

兔肝金属硫蛋白β结构域的制备和鉴定

制备一定量的Ｃｄ、Ｚｎ兔肝金属硫蛋白,然后脱掉其中的金属,获得不含金属的硫蛋白,用一价金属ＣｕＣｌ或ＡｇＮＯ３以５．８：１的金属：蛋白摩尔比与ａｐｏＭＴ结合,将其β结构域用金属饱和,在３７℃,ＰＨ７．０下用枯草杆菌蛋白酶水解掉未结合金属的α结构域,获得Ｃｕ（Ⅰ）、Ａｇ（Ⅰ）结合的兔肝ＭＴ－Ⅰ、ＭＴ－Ⅱ的β结构域,通过ＨＰＬＣ、羧甲基化后的ＳＤＳ－ＰＡＧＥ、氨基酸组成分析、金属定量、Ｎ末端分析和紫外     

金属硫蛋白清除自由基及其对自由基引起的核酸损伤保护作用的研究

通过化学反应体系产生ＯＨ＾－和Ｏ＾－２自由基,采用荧光和化学发光检测体系,比较研究了不同亚型及不同结合金属的金属硫蛋白（ＭＴ）清除自由基能力的大小。结果表明,对于同一亚型,Ｚｎ结合ＭＴ清除自由基的能力大于Ｃｄ结合ＭＴ;同一结合金属的ＭＴ,ＭＴ１清除自由基的能力大于ＭＴ２。通过比较ＺｎＭＴ１与谷胱甘肽（ＧＳＨ）及超氧化物歧化酶（ＳＯＤ）清除自由基的能力大小发现,ＺｎＭＴ１清除ＯＨ的能力是ＧＳＨ的１０     

脑特异性金属硫蛋白—Ⅲ研究进展

金属硫蛋白－Ⅲ（ＭＴ－Ⅲ）是富含半胱氨酸的低分子量蛋白,主要在脑内含Ｚｎ＾２＋神经元表达,与脑的发育过程有关。ＭＴ－Ⅲ可抑制体外培养的神经细胞生长和存活。ＭＴ－Ⅲ与Ｚｎ＾２＋的结合有相对特异性,可能影响依赖于Ｚｎ＾２＋的一系列生物活动。ＡＤ患者脑内ＭＴ－Ⅲ表达减少,敲除ＭＴ－Ⅲ基因的小鼠对红藻氨酸诱发癫痫更敏感,提示ＭＴ－Ⅲ可能在某些脑变性疾病中发挥作用。     

金属硫蛋白清除自由基及其对自由基引起的核酸损伤保护作用的研究

通过化学反应体系产生ＯＨ－和Ｏ自由基,采用荧光和化学发光检测体系,比较研究了不同亚型及不同结合金属的金属硫蛋白（ＭＴ）清除自由基能力的大小。结果表明,对于同一亚型,Ｚｎ结合ＭＴ清除自由基的能力大于Ｃｄ结合ＭＴ同一结合金属的ＭＴ,ＭＴ１清除自由基的能力大于ＭＴ２。通过比较ＺｎＭＴ１与谷胱甘肽（ＧＳＨ）及超氧化物歧化酶（ＳＯＤ）清除自由基的能力大小发现,ＺｎＭＴ１清除ＯＨ的能力是ＧＳＨ的１００倍,清除Ｏ自由基的能力分别是ＧＳＨ和ＳＯＤ的２５和０．０１倍。即ＭＴ是一种很好的ＯＨ自由基清除剂。以ＯＨ对核酸（ＤＮＡ）的损伤为例,研究了ＭＴ对核酸损伤的保护作用,其变化规律与上述结果相一致。     

人神经生长抑制因子β结构域的高效表达及性质研究

神经生长抑制因子（ Ｇ Ｉ Ｆ）是一种特异存在于哺乳动物脑中的金属硫蛋白（ｍ ｅｔａｌｌｏｔｈｉｏｎｅｉｎ, Ｍ Ｔ）类似物,又称 Ｍ Ｔ Ⅲ．它与 Ｍ Ｔ 有相同的结合 Ｚｎ（Ⅱ）, Ｃｄ（Ⅱ）, Ｃｕ（Ⅰ）等金属的能力,但与 Ｍ Ｔ 不同的是它能够抑制神经细胞的生长,并发现在患 Ａｌｚｈｅｉｍ ｅｒ ｄｉｓｅａｓｅ（ Ａ Ｄ 症）病人的大脑中 Ｇ Ｉ Ｆ蛋白量和ｍ Ｒ Ｎ Ａ 的量均显著下降,研究证明 Ｇ Ｉ Ｆ对神经细胞的抑制活性主要存在于其 β结构域中．为进一步研究 β结构域结构和功能的关系,将 β结构域的 ｃ Ｄ Ｎ Ａ 克隆入融合表达载体ｐ Ｇ Ｅ Ｘ ４ Ｔ １ 中, Ｉ Ｐ Ｔ Ｇ 诱导并高效表达了 β结构域蛋白,通过氨基酸组成和质谱的测定,证明得到了目的蛋白．利用金属重组的方法,分别得到了结合 Ｃｄ 和 Ｚｎ 的 Ｇ Ｉ Ｆ 的 β结构域,并测定了其巯基和金属含量对蛋白量的比值,证明所得 Ｇ Ｉ Ｆ β与 Ｍ Ｔ β在结合金属能力上十分相似．用紫外光谱学的研究表明, Ｃｄ Ｍ Ｔ 的 β结构域在２５０ ｎｍ 处比 Ｃｄ Ｇ Ｉ Ｆ 的 β结构域有一明显肩峰,从而表明二者的金属—巯基结合簇的结构有明显不同,而这种结构上的差异有可能导致二者在功能上的不同．     

介绍一种高效、简单的DNA沉淀法

毫摩尔级的Ｚｎ２＋可以在适宜条件下导致核酸沉淀物的形成。优化后的ＺｎＣｌ２沉淀法的基本步骤是：５０ｋｂ和２０ｂｐ的ＤＮＡ样品分别置于５０ｍＭＴｒｉｓ（ｐＨ７．０）稀释,ＥＤ－ＴＡ到终浓度小于０．１ｍＭ中,加入０．０１Ｍ磷酸钠缓冲液（ｐＨ７．０）到终浓...     

不同亚型ZnMT使脱金属碳酸酐酶复活的比较研究

通过荧光探针、酶的活力测定及对金属硫蛋白（ＭＴ）分子中金属与巯基配位键的特征吸收检测,比较研究了锌诱导兔肾不同亚型ＺｎＭＴ使脱金属碳酸酐酶（ＡｐｏＣＡ）复活的能力大小。结果表明,不同亚型ＺｎＭＴ对ＡｐｏＣＡ具有不同的反应活性,ＭＴ１比ＭＴ_２对于ＡｎｏＣＡ的复活具有更强的反应活性,此结论与我们在研究不同亚型ＭＴ清除自由基时的结果相一致。这两种亚型ＭＴ在反应活性上的差异,很可能与其在生物体内功能上的分化密切相关。     

以FITC作为荧光探针研究金属硫蛋白的构象变化

本文以异硫氰基荧光素（ＦＩＴＣ）作为荧光探针标记于金属硫蛋白分子上,用荧光光谱研究了Ｃｄ＾２＋及Ａｇ＾＋离子与ＺｎＭＴ２－ＦＩＴＣ进行金属交换及与ＡｐｏＭＴ２－ＦＩＴＣ进行金属重组时的构象变化。结果表明,标记后ＭＴ与Ｃｄ＾２＋离子进行金属交换及金属重组时不具有明显的结构域特征,而Ａｇ＾＋离子进行金属交换及金属重组时,分别在Ａｇ６ＭＴ、Ａｇ１２ＭＴ及Ａｇ１８ＭＴ处具有明显的结构域形成特征。此外高温下     

Silver binding to rabbit liver metallothionein. Circular dichroism and emission study of silver-thiolate cluster formation with apometallothionein and the alpha and beta fragments

We report new spectroscopic properties for a range of silver-metallothionein species. The binding reactions that take place following addition of Ag+ to rabbit liver apoMT 2, and the apo alpha and -beta fragments have been studied using the techniques of circular dichroism (CD) and emission spectroscopy. Titrations carried out at 20 degrees C and 55 degrees C reveal for the first time the formation of a sequence of clusters (Ag6-MT, Ag12-MT and, finally, Ag18-MT) as Ag+ is added to rabbit apoMT 2. (The division of mammalian metallothioneins into two major subforms, MT 1 and MT 2, is based on differences in molecular charge, which results from differences in the sequence of amino acids that do not involve the cysteines.) It is proposed that the novel Ag18-MT complex forms with a structure that involves a well defined three-dimensional structure, in the same manner as that recently reported for the Hg18-MT complex (Cai, W. and Stillman, M. J., (1988) J. Am. Chem. Soc. 110, 7872-7873). Addition of silver in excess of 20 mol equivalents leads to the collapse of this structure. At the elevated temperatures, it is suggested that the protein can exert cooperativity so that completely filled domains are formed rather than mixtures of complexes. This contrasts with the kinetic product in which metals are bound across the peptide chain forming more random "cross-linked" regions in place of the cluster structure. CD spectra were recorded as Ag+ was added to the alpha and beta fragments formed from rabbit liver MT 1. The silver-containing fragments are less stable than the Ag-MT. The alpha and beta fragments exhibit CD spectral patterns indicative of stoichiometrically defined species. The presence of Ag3- alpha MT 1 and Ag6- alpha MT 1 is suggested by the spectral data obtained at 20 and 55 degrees C. Formation of Ag3- beta MT 1 is suggested by the spectral data recorded at 20 degrees C for the beta fragment. We also report that silver-containing metallothioneins are luminescent. Both the position of the band maximum in the 460-600 nm region and the emission intensity are strongly dependent on the stoichiometry of silver to protein. In the range of molar ratios for silver:MT of 1-12, bands at 465 and 520 nm intensify to a maximum for Ag10-MT 2. A band at 575 nm reaches a maximum for Ag16-MT 2. Analysis of the emission data suggests that Ag+ binds in a domain specific mechanism to apoMT 2.     

Cadmium-thiolate clusters in metallothionein: spectrophotometric and spectropolarimetric features

Cd-thiolate cluster formation in rabbit liver metallothionein 1 (MT) has been followed at pH 8.4 by monitoring spectroscopic features below 300 nm as a function of increasing Cd-to-apometallothionein (apoMT) ratio. The emerging absorption profiles form a family of closely similar spectra attributable to tetrahedral Cd-tetrathiolate coordination previously established for Cd7-MT [Vasák, M., K?gi, J.H.R., & Hill, H.A.O. (1981) Biochemistry, 20, 2852-2856]. However, there is a 6-nm red shift of the unresolved lowest energy absorption band when greater than 3 equiv of Cd(II) is incorporated. This shift is paralleled by a changeover in the circular dichroism (CD) features of MT from a broad monophasic positive CD profile with ellipticity bands near 240 and 220 nm to a biphasic CD spectrum characterized by positive ellipticity bands at 260 and 224 nm and an interposed negative band at 240 nm. Both features can be attributed to a changeover from separate Cd-tetrathiolate units formed at low metal-to-apoMT ratio to Cd-thiolate clusters when the supply of cysteine ligands becomes limiting. A comparable red shift signaling the transition from the mononuclear to a trinuclear tetrahedral Cd-tetrathiolate complex is also observed upon titration of the synthetic tetrathiol dodecapeptide N-Ac-Pro-Cys-Orn-Cys-Pro-Glu-Cys-Glu-Cys-Arg-Arg-Val with Cd(II). The latter studies also provide evidence for the predominantly ligand (sulfur) character of the lowest energy Cd-tetrathiolate ligand-metal charge-transfer transition. As a corollary it is inferred that the biphasic CD profile arises from excitonic coupling of these sulfur-centered transition dipole moments dissymmetrically oriented within the Cd(II)-thiolate clusters.     

Equilibrium isotherm studies for the uptake of cadmium and lead ions onto sugar beet pulp

The adsorption of Cd2+ and Pb2+ on sugar beet pulp (SBP), a low-cost material, has been studied. In the present work, the abilities of native (SBP) to remove cadmium (Cd2+) and lead (Pb2+) ions from aqueous solutions were compared. The (SBP) an industrial by product and solid waste of sugar industry were used for the removal of Cd2+ and Pb2+ ions from aqueous water. Batch adsorption studies were carried out to examine the influence of various parameters such as initial pH, adsorbent dose, initial metal ion concentration, and time on uptake. The sorption process was relatively fast and equilibrium was reached after about 70 min of contact. As much as 70-75% removal of Cd2+ and Pb2+ ions for (SBP) are possible in about 70 min, respectively, under the batch test conditions. Uptake of Cd2+ and Pb2+ ions on (SBP) showed a pH-dependent profile. The overall uptake for the (SBP) is at a maximum at pH 5.3 and gives up to 46.1 mg g(-1) for Cd2+ and at pH 5.0 and gives 43.5 mg g(-1) for Pb2+ for (SBP), which seems to be removed exclusively by ion exchange, physical sorption and chelation. A dose of 8 gL(-1) was sufficient for the optimum removal of both the metal ions. The Freundlich represented the sorption data for (SBP). In the presence of 0.1M NaNO3 the level of metal ion uptake was found to reach its maximum value very rapidly with the speed increasing both with the (SPB) concentration and with increasing initial pH of the suspension. The reversibility of the process was investigated. The desorption of Cd2+ and Pb2+ ions which were previously deposited on the (SBP) back into the deionised water was observed only in acidic pH values during one day study period and was generally rather low. The extent of adsorption for both metals increased along with an increase of the (SBP) dosage. (SBP), which is cheap and highly selective, therefore seems to be a promising substrate to entrap heavy metals in aqueous solutions.     

Toxic heavy metal ions activate the heme-regulated eukaryotic initiation factor-2 alpha kinase by inhibiting the capacity of hemin-supplemented reticulocyte lysates to reduce disulfide bonds.

Addition of toxic heavy metal ions (Cd2+, Hg2+, and Pb2+) to hemin-supplemented rabbit reticulocyte lysate brings about the activation of the heme-regulated eukaryotic initiation factor 2 alpha kinase (HRI) and the inhibition of protein chain initiation. In this report we examined the effects of monothiol and dithiol compounds, metal ion-chelating agents, and metallothioneins (MT) on metal ion-induced inhibition of protein synthesis. The dithiol compounds dithiothreitol and 2,3-dimercaptopropane sulfonic acid prevented and relieved the inhibition of protein synthesis caused by Cd2+ and Hg2+ in hemin-supplemented lysates, but the monothiol compounds 2-mercaptoethanol, cysteamine, D-(-)penicillamine, and glutathione had no effect. The inhibition of protein synthesis caused by Cd2+ was reversed by the addition of excess EDTA but not by the addition of excess nitrilotriacetic acid. Toxic heavy metal ions inhibited the capacity of hemin-supplemented lysate to reduce disulfide bonds. Addition of excess EDTA to Cd(2+)-inhibited lysates restored the capacity of the lysate to reduce disulfide bonds and inhibited the phosphorylation of eukaryotic initiation factor eIF-2. MTs and their apoproteins (apoMTs) inhibited the activation of HRI and protected protein synthesis from inhibition by Cd2+, Hg2+, and Pb2+. Addition of apoMTs to heavy metal ion-inhibited lysates restored the capacity of lysates to reduce disulfide bonds. The restoration of the lysate's thioredoxin/thioredoxin reductase activity was accompanied by the inactivation of HRI and the resumption of protein synthesis, indicating that apoMTs can "detoxify" metal ions already bound to proteins. Several observations presented in this report suggest that the binding of metal ions to the alpha-domain of MT is responsible for the ability of MT to sequester bound metal in a non-toxic form. Addition of glucose 6-phosphate or NADPH had no effect on protein synthesis in metal ion-inhibited lysates, and NADPH concentrations in Cd(2+)-inhibited and hemin-supplemented control lysates were equivalent. The data suggest that the metal ions cause the inhibition of protein synthesis by binding to vicinal sulfhydryl groups present in some critical protein(s), possibly the dithiols present in the active site of thioredoxin and (or) thioredoxin reductase, which leads to the activation of HRI.     

An Apomictic Autotriploid Line TAR Identified in Oryza sativa

Since apomixis has a close correlation with polyploidy and sterility, a number of autotriploids with no sexual reproductivity were induced and apomictic germplasm were screened in Oryza sativa L. As a result, an autotriploid line, named TAR, was cytoembryologically identified which possessed apomictic property, with an average seed-set rate of 10% per panicle. Karyotype analysis proved that all the progeny seeds of TAR carried 36 chromosomes in the generations tested. Priliminary cytological observations revealed that all the ovaries of TAR had embryo sac differentiation, 33% of which developed into normal megagametophyte, 9% with previous embryogenesis prior to anthesis, and about 58% differentiated abnormally, i.e. disordered polarization, absent female generative unit and more than 2 polar nuclei. In TAR, the frequencies of chromosome configuration of 12 Ⅲ, 11 Ⅱ + 1 Ⅱ +1 Ⅰ. L0Ⅲ +2Ⅱ +2 Ⅰ, 9Ⅲ+3Ⅱ +3 Ⅰ, 8Ⅲ+4Ⅱ +4 Ⅰ and 7Ⅲ+5 Ⅱ +5 Ⅰ were ll%, 17%, 15%, 26%, 20% and 11% respectively at metaphase Ⅰ . While in the check line T-15 of autotriploid only 7 % of the ovaries observed had embryo sac development, and the progenies of this triploid line were aneuploids with chromosome number of 25～27. In T-15, the frequencies of chromosome configuration of 12 Ⅲ, 11Ⅲ +1 Ⅱ +1 Ⅰ, 10 Ⅲ +2 Ⅱ +2 Ⅰ , 9Ⅲ+3 Ⅱ +3 Ⅰ and 8 Ⅲ+4 Ⅱ +4 Ⅰ were 24%, 16%, 36%, 17% and 7% respectively at metaphse Ⅰ . The above observations indicated that some megaspore mother cell in TAR might undergo apomeiosis and where it gave rise to unreduced embryo sac, the unreduced eggs or synergids developed into embryos without fertilization and polar nuclei produced endosperm by pseudogamy.     

Gram scale purification and preparation of rabbit liver zinc metallothionein.

A simplified procedure for purifying gram quantities of rabbit liver metallothionein (MT) using gel filtration and anion exchange chromatography is presented. The MT purification made use of anion exchange batch elution chromatography which greatly shortened the procedure. Quantitation techniques for use with crude and purified MT are discussed. This paper also describes the preparation of large amounts of ZnMT from Cd,ZnMT.