油麻藤种子中凝集素的纯化及性质的研究

中药常春油麻藤种子中含有Ａ型血专一性的凝集素（ＭＳＬ）。该凝集素可经盐析、离子交换及凝胶过滤进行纯化,当其浓度为０．４９μｇ／ｍｌ时就能凝集人Ａ型血细胞,对人类Ｂ、Ｏ型及兔红细胞无作用。Ｇａｌ,ＧａｌＮＡｃ和胃粘蛋白对ＭＳＬ的凝血活性有强抑制作用。ＭＳＬ含中性糖５．７％,凝胶过滤测得分子量为１３１８００,ＳＤＳ－ＰＡＧＥ测得分子量为６６０００和３３０００,表明ＭＳＬ可能由两个不同亚基组成。ＭＳＬ还     

油麻藤种子中凝集素的纯化及性质的研究

中药常春油麻藤种子中含有Ａ型血专一性的凝集素（ＭＳＬ）．该凝集素可经盐析、离子交换及凝胶过滤进行纯化,当其浓度为０．４９μｇ／ｍｌ时就能凝集人Ａ型血细胞,对人类Ｂ、Ｏ型及兔红细胞无作用．Ｇａｌ,ＧａｌＮＡｃ和胃粘蛋白对ＭＳＬ的凝血活性有强抑制作用．ＭＳＬ含中性糖５．７％,凝胶过滤测得分子量为１３１８００,ＳＤＳ－ＰＡＧＥ测得分子量为６６０００和３３０００,表明ＭＳＬ可能由两个不同亚基组成．ＭＳＬ还是一种促有丝分裂原,对人外周血中淋巴细胞的转化率可达７６．２％．     

甘薯叶片蔗糖酶的分离纯化及其部分性质

纯化酶经聚丙烯酰胺凝胶电泳显示单一蛋白带,ＳＤＳ－ＰＡＧＥ显示一条蛋自带,其亚基分子量为３９．８ｋＤ。用ＳｅｐｈａｃｒｙｌＳ－２００凝胶过滤测得全酶的分子量为７９．４ｋＤ,该酶由两个相同亚基组成。其表观Ｋｍ为１２ｍｍｏｌ／Ｌ,Ｖｍａｘ为９９．５ｍｇ还原糖ｍｇ－１ｐｒｏｔｅｉｎｈ－１。     

盐生杜氏藻生油—3—磷酸脱氢酶的分离化及其特性的研究

利用ＰＥＧ分级,ＤＥＡＥ离子交换层析,Ｂｈｕｅ Ｓｅｐｈａｒｏｓｅ拟亲和层析,ＭｏｎｏＱ离子交换层析等手段,分离纯化直二氏藻甘油三磷酸（Ｇ－３－Ｐ）脱氢酶（ＥＣ１．１．１．８）得到比活为１２．６ｕ／ｍｇ的电泳纯的酶,并对此酶的生化特性进行了研究。４－２０％非变性聚丙烯酰胺梯度凝胶电泳测得全酶分子量约为２７０ｋＤ,ＳＤＳ－ＰＡＧＥ表明该酶只有一种分子量约为６５ｋＤ的亚基,据此推测该酶应为同四聚体。酶     

甘薯叶片蔗糖酶的分离纯化及其部分性质

纯化酶经聚丙烯酰胺凝胶电泳显示单一蛋白带,ＳＤＳ－ＰＡＧＥ显示一条蛋白带,其亚基分子量为３９．８ｋＤ。用Ｓｅｐｈａｃｒｙｌ Ｓ－２００凝胶过滤测得全酶的分子量为７９．４ｋＤ,该酶由两个相同亚基组成。其表观Ｋｍ为１２ｍｍｏｌ／Ｌ,Ｖｍａｘ为９９．５ｍｇ还原糖ｍｇ＾－１ｐｒｏｔｅｉｎ ｈ＾－１。     

人肝谷胱甘肽过氧化物酶纯化及性质研究

经硫酸铵分级沉淀,离子交换层析和凝胶过滤等步骤,从人肝中获得了ＰＡＧＥ单一条带的谷胱甘肽过氧化物酶,比活提高１２０倍,得率为２５％。凝胶过滤法测得分子量为９０９８０,ＳＤＳ－ＰＡＧＥ测定亚基分子量为２２４２３．原子吸收法测得每分子酶含有四个硒原子。等电聚焦显示该酶等电点为５．０．酶活力的最适ｐＨ为８．５,最适温度为３７℃。动力学实验提示该酶作用机理属于乒乓机制型。     

洋紫荆凝集素的分离纯化和性质

利用酸化处理的Ｓｅｐｈａｒｏｓｅ ６Ｂ亲和柱从洋紫荆种子中分离纯化出了洋紫荆凝集素（ＢＶＬ）,其比活性比抽提液提高了１５９倍,活力回收率为４９．０％。ＢＶＬ分子量为８１０００,由两个相同的亚基组成。等电聚焦凝胶电注测得其等电点为４．９５。其紫外吸收高峰在２７６ｎｍ处。ＢＶＬ具一定的的热稳定性和酸碱稳定性。Ｎ－乙酰－Ｄ氨基半乳能强烈地抑制ＢＶＬ对兔红细胞的凝集作用。乳糖、半乳糖也有较强的抑制定性。Ｎ     

限制性核酸内切酶Bsp 63Ⅰ的纯化及性质研究

用Ｂａｃｉｌｌｕｓｓｐｈａｅｒｉｃｕｓ６３菌为材料,经ＤＮＡ－Ｓｅｐｈａｒｏｓｅ和ＣｉｂａｃｒｏｎＢｌｕｅＦ３ＧＡ－Ｓｅｐｈａｒｏｓｅ两步亲和层析,将Ｂｓｐ６３Ⅰ纯化到均一程度。酶比活力达６１４００Ｕ／ｍｇ蛋白。用凝胶过滤法测得该酶分子量为１１３８００。该酶样品在ＳＤＳ－ＰＡＧＥ中呈现为一条蛋白带,并测得其亚基分子量为５６８００。用ＤＮＳ－Ｃｌ法测得该酶Ｎ－末端氨基酸为丙氨酸。上述结果表明该酶分子是由两个相同亚基组成。     

百合巯基蛋白酶抑制剂的纯化及部分性质研究

百合的鳞茎中含有一种对木瓜蛋白酶有强抑制作用的巯基蛋白酶抑制剂．百合的鳞茎经浸取加热处理,木瓜蛋白酶偶联的Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和层析和ＳｅｐｈａｄｅｘＧ－１００分子筛层析,可获得在ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ均为单一蛋白带的百合巯基蛋白酶抑制剂（ＣＰＩ）．此ＣＰＩ为单链蛋白,含有０．３０７％的中性糖;Ｎ端氨基酸为Ｉｌｅ;ＳＤＳ－ＰＡＧＥ测得亚基分子量为１２０００;ＳｅｐｈａｄｅｘＧ－１００测得分子量为１２５００．百合ＣＰＩ在１００℃内和ｐＨ２～１２范围内非常稳定;对木瓜蛋白酶的抑制属竞争性抑制类型,其Ｋｉ值为１．１５×１０~（－９）ｍｏｌ／Ｌ,对木瓜蛋白酶的抑制摩尔比为８．５：１．     

对硫磷真菌降解酶的分离纯化和性质

黑曲霉Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ Ｙ－８在液体培养其中３０℃培养５ｄ,其菌体用超声波破碎后,经硫酸铵沉淀、ＤＥＡＥ－纤维素层析、Ｓｅｐｈａｄｅｘ－１００凝胶过滤,得到凝胶电泳均一的对硫磷降解酶,其比活为６．９４,提纯倍数为１３．６倍,收率为１７．４％,酶作用的最适温度是５０℃,最适ＰＨ为７．５,在４０℃以下和ＰＨ６．０－９．０之间稳定,此酶为单亚基蛋白,凝胶过滤法测得分子量为４２０００,含     

洋紫荆凝集素的分离纯化和性质

利用酸化处理的Sepharose 6B 亲和柱从洋紫荆种子中分离纯化出了洋紫荆凝集素(BVL),其比活性比抽提液提高了159倍,活力回收率为49.0%。BVL分子量为81 000,由两个相同的亚基组成。等电聚焦凝胶电泳测得其等电点为4.95。其紫外吸收高峰在276 nm处。BVL具一定的热稳定性和酸碱稳定性。N-乙酰-D-氨基半乳糖能强烈地抑制BVL对兔红细胞的凝集作用,乳糖、半乳糖也有较强的抑制作用。     

巨大芽孢杆菌青霉素酰化酶在枯草杆菌中的表达及其分离纯化

青霉素酰化酶（ＰＧＡ）在医药工业起着重要的作用,它能够水解青霉素Ｇ产生６－氨基青霉烷酸（６－ＡＰＡ）和苯乙酸,６－ＡＰＡ是半合成青霉素的关键中间体．该酶广泛存在于各种微生物中如真菌和细菌中．国际上对Ｅ．ｃｏｌｉ、Ａｒｔｈｒｏｂａｃｔｅｒｖｉｓｃｏｓｕ...     

Purification and partial sequence analysis of murine B cell growth factor II (interleukin 5)

Murine B cell growth factor II (BCGF-II/interleukin 5) was purified from the conditioned media of the helper T cell line D10 . G4 . 1. The purification scheme consisted of sequential batch adsorption onto trimethylsilyl-controlled pore glass beads, high pressure ion exchange chromatography, and reverse phase high pressure liquid chromatography. The purified BCGF-II had a relative molecular weight of 45,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Identical analysis of BCGF-II under reducing conditions yielded a m.w. of 22,500, suggesting that native BCGF-II exists as a homodimer. The NH2-terminal amino acid sequence of the purified lymphokine was determined by automated Edman degradation. A single amino acid sequence of 24 residues was obtained that, upon comparison, was contained within the cDNA pSP6K-mTRF23 recently described as encoding murine BCGF-II/T cell-replacing factor. The NH2-terminal methionine in mature BCGF-II is found at position 21 of the amino acid sequence predicted from the cDNA pSP6K-mTRF23. This finding supports the contention of Kinashi et al. (Kinashi, T., N. Harada, E. Severinson, T. Tanabe, P. Sideras, M. Konishi, C. Azuma, A. Tominaga, S. Bergstedt-Lindqvist, M. Takahashi, F. Matsuda, Y. Yaoita, K. Takatsu, and T. Honjo. 1986. Nature 324:70) that amino acids 1-20 serve as the signal sequence for the BCGF-II gene. The ability of BCGF-II to stimulate the proliferation of the B cell lymphoma BCL1 was used to assess the potency of the lymphokine. BCGF-II at 13.5 pM induced 50% of the maximal proliferative response in the BCL1 cells; concentrations as low as 2 pM were still effective in stimulating the growth of the cells. Assuming that the amount of BCGF-II necessary to mount a 50% response in the BCL1 assay is defined as one unit of activity, then the purified BCGF-II has a specific activity of 16.5 U/ng of protein.     

岩溶植物光合-光响应曲线的两种拟合模型比较

用直角双曲线和非直角双曲线两种模型,对桂林岩溶区石山植物红背山麻杆（Alchornea trewioides）、九龙藤（Bauhinia championii）、青檀（Pteroceltis tatarinowii）和圆叶乌桕（Sapium rotundifolium）光合作用的光响应曲线进行拟合。结果表明,直角双曲线拟合的表观量子效率（α）、最大净光合速率（Pmax）、暗呼吸速率（Rd）、光补偿点（Lcp）和光饱和点（Lφ）数值均高于相应的非直角双曲线拟合结果;直角双曲线模型拟合的参数值大小和曲线变化趋势不符合实际情况;非直角双曲线模型拟合结果更符合生理意义。红背山麻杆和九龙藤具有较高的表观量子效率和较低的光补偿点,对光能的利用效率和耐荫性均强于圆叶乌桕和青檀。     

The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes

The CARD domain protein BCL10 and paracaspase MALT1 are essential for the activation of IkappaB kinase (IKK) and NF-kappaB in response to T cell receptor (TCR) stimulation. Here we present evidence that TRAF6 ubiquitin ligase and TAK1 protein kinase mediate IKK activation by BCL10 and MALT1. RNAi-mediated silencing of MALT1, TAK1, TRAF6, and TRAF2 suppressed TCR-dependent IKK activation and interleukin-2 production in T cells. Furthermore, we have reconstituted the pathway from BCL10 to IKK activation in vitro with purified proteins of MALT1, TRAF6, TAK1, and ubiquitination enzymes including Ubc13/Uev1A. We find that a small fraction of BCL10 and MALT1 proteins form high molecular weight oligomers. Strikingly, only these oligomeric forms of BCL10 and MALT1 can activate IKK in vitro. The MALT1 oligomers bind to TRAF6, induce TRAF6 oligomerization, and activate the ligase activity of TRAF6 to polyubiquitinate NEMO. These results reveal an oligomerization --> ubiquitination --> phosphorylation cascade that culminates in NF-kappaB activation in T lymphocytes.     

Structure of cruzipain/cruzain inhibitors isolated from Bauhinia bauhinioides seeds

The saline extract of Bauhinia bauhinioides dry seeds was shown to inhibit cruzipain, a cysteine proteinase from Trypanosoma cruzi. The inhibitory activity was assigned to a protein with 164 amino acid residues and molecular mass of 18 034 Da that was purified by chromatography on DEAE-Sephadex, trypsin-Sepharose (removal of trypsin inhibitors), Mono Q and a reversed-phase C4 column. The primary structure is homologous to other plant Kunitz-type inhibitors, but it lacks cysteine residues and therefore the disulfide bridges. No methionine residue was identified by amino acid sequencing. The inhibition of cruzipain fits into a slow-tight binding mechanism with a low dissociation constant (Ki 1.2 nM). The studied Bauhinia protein also inhibits cruzain (Ki 0.3 nM), a C-terminally truncated recombinant species of cruzipain. Cathepsin L, a cysteine proteinase with high homology to cruzipain, is also inhibited (Ki 0.22 nM), but not cathepsin B, papain, bromelain or ficin.     

Purification,primary structure and potential functions of a novel lectin from Bauhinia forficata seeds

A new lectin, BfL, was purified from Bauhinia forficata seeds by ammonium sulfate fractionation, DEAE-Sephadex ion exchange chromatography, Sepharose-4B and chitin affinity chromatographies and Superdex 75 size exclusion chromatography. The molecular homogeneity and purity of BfL were assessed by reversed-phase HPLC. BfL appeared as a single band of approximately 27.0 kDa on SDS-PAGE under non-reducing and reducing conditions, and its molecular weight was determined to be 27,850 Da by LC/ESI-MS. BfL is a glycoprotein with a carbohydrate content of 6.24% determined by the phenol–sulfuric acid method. Fetuin, asialofetuin, thyroglobulin and azocasein inhibited the hemagglutinating activity of BfL, whereas saccharides did not. BfL hemagglutinating activity was stable at 100 °C for 30 min, pH-dependent, with the highest activity at pH 6.0, and metal-independent. The primary structure of BfL shows similarity with other lectins from the genus Bauhinia. Deconvolution of the BfL circular dichroism (CD) spectrum indicated the presence of α-helix and β structures. BfL increases coagulation time, but this effect is not related to human plasma kallikrein or human factor Xa inhibition. BfL also inhibits ADP- and epinephrine-induced platelet aggregation in a dose-dependent manner and is the only currently described lectin from Bauhinia that exhibits anticoagulant and antiplatelet aggregating properties.     

Heparin-inhibitable lectins: Marked similarities in chicken and rat

Extracts of young rat lung contain a heparin-inhibitable lectin that closely resembles one recently purified from chicken liver. Both lectins interact with heparin and N-acetyl-D-galactosamine, and were purified by gel filtration on Sepharose CL-2B followed by affinity chromatography on heparin-Sepharose. They both behave as high molecular weight aggregates that can be dissociated into two peptides with apparent molecular weights of 13,000 and 16,000 by gel electrophoresis in SDS. Samples of purified lectin contained up to 20% DNA by weight, and the degree of lectin aggregation and hemagglutination activity was greatly reduced by treatment with micrococcal nuclease without inhibiting heparin-binding activity. Association of lectin with DNA is an artifact of homogenization in high salt, since only 2% of the lectin is found associated with a purified nuclear fraction.     

cDNA cloning and expression of Bauhinia purpurea lectin.

Bauhinia purpurea lectin (BPA) was purified from seeds of B. purpurea alba. The purified lectin was digested with an endoproteinase, Asp-N, or trypsin and then the amino acid sequences of the resultant fragments were analyzed. Furthermore, a cDNA library for BPA was constructed using RNA isolated from germinated Bauhinia purpurea seeds. By gene cloning, the nucleotide sequence of BPA cDNA and its deduced amino acid sequence were analyzed. The cloned BPA cDNA comprised 1,152 nucleotides and the open reading frame of the cDNA encodes a polypeptide of 290 amino acids including a signal peptide composed of 28 amino acids. BPA expressed in Escherichia coli showed a relative molecular mass of 29 kDa on sodium dodecyl sulfate-polyacrylamide gel. On comparison of its sequence with those of other leguminous seed lectins, BPA showed high homology to the others.