大熊猫乳酸脱氢酶同工酶H_4的分离纯化和某些性质的研究

采用８－（６－氨己基）－氨基－５＇－ＡＭＰＳｅｐｈａｒｏｓｅ亲和层析法和ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ离子交换层析法从大熊猫心肌中分离纯化出了乳酸脱氢酶同工酶Ｈ４．纯化的大熊猫ＬＤＨ－Ｈ４,比活为４４５Ｕ／ｍｇ蛋白,经ＳＤＳ－ＰＡＧＥ,ＰＡＧＥ,等电聚焦电泳鉴定均为一条带,其亚基分子量为３６０００,等电点为５．４５．经测定大熊猫ＬＤＨ－Ｈ亚基Ｎ端被封闭,Ｃ端氨基酸残基经测定为Ｌｅｕ．氨基酸组成分析表明每个亚基含有５个Ｃｙｓ,９个Ｍｅｔ．     

昆虫谷胱甘肽S-转移酶分离纯化的新方法

谷胱甘肽Ｓ－转移酶（ｇｌｕｔａｔｈｉｏｎｅＳ－ｔｒａｎｓｆｅｒａｓｅｓ,ＧＳＴ）是一类具有多种生理功能的同功酶．从蜡螟幼虫（Ｇａｌｅｒｉａｍｅｌｏｎｅｌａ）的提取液中分离纯化谷胱甘肽Ｓ－转移酶的基本方法如下：首先将冷冻的蜡螟幼虫在磷酸缓冲液中匀桨,经１００００ｇ和１０００００ｇ分级离心;取上清液通过ＱＡＥ－ＳｅｐｈａｄｅｘＡ－２５离子交换柱层析除去部分色素和杂蛋白;然后采用谷胱甘肽－琼脂糖凝胶亲和层析（ＧＳＨ－ＱＴ４）,四溴酚酞二磺酸盐－琼脂糖凝胶亲和层析（ＢＳＰ－ＱＴ４）,铜离子－琼脂糖凝胶螯合层析（Ｃｕ２＋－ＱＴ４）及ＰＢＥ９４－Ｓｅｐｈａｒｏｓｅ（ＰＢＥ９４）聚焦层析等层析技术进一步分离纯化．将上述方法获得的色谱峰以ＣＤＮＢ和ＤＣＮＢ为底物检测生物活性．具有生物活性部分的蛋白质,通过ＳＤＳ－ＰＡＧＥ测定其分子量．实验结果表明,采用ＧＳＨ－ＱＴ４亲和层析法获得的活性峰,在ＳＤＳ－ＰＡＧＥ图谱上呈现出两条带,分子量为２４ｋＤ,２４．５ｋＤ左右;Ｃｕ２＋－ＱＴ６螯合层析法分离的活性峰,呈现出一条带,分子量为２４ｋＤ左右;ＰＢＥ９４－聚焦层析法分离获得三个活性峰：第一色谱峰,呈现出一条带,分子量为２３ｋＤ左右     

甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶还原酶组分的纯化和性质

Ｍｅｔｙｌｏｍｏｎａｓｓｐ．ＧＹＪ３菌的甲烷单加氧酶（ＭＭＯ）粗酶提取液经ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析、ＳｅｐｈａｄｅｘＧ－１００凝胶过滤层析和ＤＥＡＥ－ＴＳＫｇｅｌＨＰＬＣ分离纯化出ＭＭＯ还原酶组分．经ＨＰＬＣ分析,纯度大于９５％,纯化倍数为４．４,加入至ＭＭＯ羟基化酶和调节蛋白Ｂ的体系中表现比活为２２８ｎｍｏｌ环氧丙烷每分钟毫克蛋白．ＳＤＳ－ＰＡＧＥ电泳表明还原酶由一种亚基组成,分子量４２ｋＤ．ＩＣＰ－ＡＥＳ测定还原酶的Ｆｅ含量为１．８３ｍｏｌＦｅ每ｍｏｌ蛋白．ＵＶ－Ｖｉｓ光谱表明还原酶除２８０ｎｍ蛋白质特征峰外在４６０ｎｍ有最大吸收峰,且Ａ２８０ｎｍ／Ａ４６０ｎｍ为２．５０,与其它黄素一铁硫蛋白相似,推测还原酶可能含一个ＦＡＤ辅基和Ｆｅ２Ｓ２中心．在厌氧条件下,还原酶能够和ＮＡＤＨ作用,ＵＶ－Ｖｉｓ光谱分析表明还原酶４６０ｎｍ处特征吸收峰消失,说明在ＭＭＯ催化过程中还原酶接受ＮＡＤＨ的电子．ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ阴离子交换层析分离出调节蛋白Ｂ,部分纯化的调节蛋白Ｂ的分子量大约在２０ｋＤ,它能够提高ＭＭＯ比活性４０倍,ＭＭＯ还原酶和调节蛋白Ｂ单独存在时不具有ＭＭＯ     

大熊猫乳酸脱氢酶同工酶H4的分离纯化和某些性质的研究

采用８－（６－氨己基）－氨基－５－ＡＭＰＳｅｐｈａｒｏｓｅ亲和层分析法和ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ离子交换层析法从大熊猫心肌中分离纯化出乳酸脱氢酶同工酶Ｈ４,纯化的大熊猫ＬＤＨ－Ｈ４,比活为４４５Ｕ／ｍｇ蛋白,经ＳＤＳ－ＰＡＧＥ,ＡＰＧＥ,等电聚焦电泳鉴定均为一条带,其亚基分了量为３６０００,等电点为５．４５。经测定大熊猫ＬＤＨ－Ｈ亚基Ｎ端被封闭,Ｃ端氨基酸残基经测定为Ｌｅｕ。氨基酸组成分析表明     

甲基单胞菌Methylomonassp.GYJ3中甲烷单加氧酶还原酶组分的…

Ｍｅｙｌｏｍｏｎａｓ ｓｐ．ＧＹＪ３菌的甲烷单加氧酶（ＭＭＯ）粗酶提取液经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ阴离子交换层析,Ｓｅｐｈａｄｅｘ Ｇ－１００凝胶过滤层析和ＤＥＡＥ－ＴＳＫｇｅｌ ＨＰＬＣ分离纯化出ＭＭＯ还原酶组分,经ＨＰＬＣ分析,纯度大于９５％,纯化倍数为４．４,加入至ＭＭＯ羟基化酶和调节蛋白Ｂ的体系中表现比活为２２８ｎｍｏｌ环氧丙烷每分钟毫克蛋白,ＳＤＳ－ＰＡＧＥ电泳表明的酶由     

菠菜和蚕豆叶绿体CF1结构与功能的比较

比较了菠菜和蚕豆叶绿体的光合磷酸化活力以及由不同活化方法活化的叶绿体及可溶ＣＦ１的Ｍｇ＾２＋－ＡＴＰａｓｅ和Ｃａ＾２＋－ＡＴＰａｓｅ的活力,观测到两种叶绿体ＡＴＰａｓｅ的合成和水解ＡＴＰ的功能有明显差异。从两种叶绿体ＣＦ１的ＳＤＳ－ＰＡＧＥ图谱上可见蚕豆ＣＦ１的ε亚基分子量明显上于菠菜的,蚕豆ＣＦ１的α和β亚基间分子量的差别也比菠菜的小。     

蜈蚣碱性蛋白SSmp-d的分离纯化及其部分理化性质的鉴定

少棘巨蜈蚣（ＳｃｏｌｏｐｅｎｄｒａｓｕｂｓｐｉｎｉｐｅｓｍｕｔｉｌａｎｓＬ．Ｋｏｃｈ）经９５％乙醇脱脂后,再经４℃水冷渗,水提液低温旋转浓缩,冻干,得到的冻干粉先后经过ＳｅｐｈａｄｅｘＧ－２５柱,等电聚焦制备电泳,再经ＳｅｐｈａｄｅｘＧ－１５０柱,ＳｅｐｈａｄｅｘＧ－１００柱,最后经ＨＰＬＣ制备得到一个纯的碱性蛋白,命名为ＳＳｍｐ－ｄ．该蛋白经ＨＰＬＣ、超薄等电聚焦电泳检验是均一的．采用ＨＰＬＣ和Ｐｒｏｔｅｉｎ－ＰａｋＴＭ１２５柱测定其分子量为２４．６４ｋＤ．ＩＥＦ－ＨＰＣＥ显示其等电点为９．２７．氨基酸分析表明ＳＳｍｐ－ｄ含较多的Ａｒｇ、Ｌｙｓ等碱性氨基酸,另外还含有较多的Ａｌａ、Ｌｅｕ．使用蛋白质自动序列分析仪测定了ＳＳｍｐ－ｄＮ端的１１个氨基酸,序列为ＮＨ３＋－Ａｓｐ－Ｖａｌ－Ａｓｎ－Ｐｈｅ－Ａｒｇ－Ｌｅｕ－Ｓｅｒ－Ｇｌｙ－Ａｌａ－Ａｓｐ－Ｐｒｏ．     

兔阑尾中一种新的21kD的钙结合蛋白的纯化与鉴定

纯化与鉴定了Ｂ淋巴细胞中一种新的分子量为２１ｋＤ的钙结合蛋白（ＣａＢＰ２１）。兔阑尾淋巴细胞匀浆经热变性,Ｐｈｅｎｙｌ－Ｓｅｐｈａｒｏｓｅ与ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱层析,自每１ｋｇ细胞沉积物中获得ＳＤＳ－ＰＡＧＥ均一的ＣａＢＰ２１５．３ｍｇ。ＨＣｌ水解后的酸性氨基酸（Ａｓｐ＋Ｇｌｕ）含量为２６％。如同大多数钙结合蛋白一样,Ｎ末端封闭阻止其进行Ｅｄｍａｎ降解。ＣａＢＰ２１中疏水性氨基酸（计Ｇｌｙ,不计Ｔｒｐ）约占４６％,碱性氨基酸１０％,酸性氨基酸与极性氨基酸约４４％。ＣａＢＰ２１有较高的Ｓｅｒ、Ｔｙｒ含量。肽谱分析等确证ＣａＢＰ２１为２个相同或相似亚基二聚体。以ＡｒｓｅｎａｚｏⅢ作Ｃａ２＋结合分析表明每分子ＣａＢＰ２１可结合４分子Ｃａ２＋,对Ｃａ２＋的结合常数约为１０－５ｍｏｌ／Ｌ。各种性质表明ＣａＢＰ２１是一种不同于其他已知钙结合蛋白的新钙结合蛋白。     

光系统Ⅱ核心天线CP43的纯化及性质

菠菜放氧的ＰＳＩＩ核心复合物经０．８ｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．０）洗涤后,用温和的非离子去垢剂ＤＭ和高浓度的ＬｉＣｌＯ４增溶,再经ＤＥＡＥ－Ｔｏｙｏｐｅａｒｌ－６５０Ｓ离子交换柱层析分离,可得到ＰＳＩＩ核心天线４３ｋＤ叶绿素ａ结合蛋白（ＣＰ４３）。ＳＤＳ－ＰＡＧＥ显示一条４３ｋＤ蛋白质带。根据Ａｒｎｏｎ法和Ｍａｒｋｅｗｌｌ法的结果表明,每个蛋白质分子结合２０～２１个分子的叶绿素ａ。室温条     

人胎肺细胞的条件化培养液中两种类型纤溶酶原活化物的分离

用正常人胎肺细胞体外培养,从其培养液中分离纤溶酶原活化物（ＰＡ）,通过ＣＭ－ＳｅｐｈａｄｅｘＣ－５０层析,硫酸铵沉淀和Ｆｉｂｒｉｎ－Ｓｅｐｈａｒｏｓｅ,Ｌｙｓｉｎｅ－Ｓｅｐｈａｒｏｓｅ亲和层析及ＳｅｐｈａｄｅｘＧ－５０凝胶过滤等步骤,从１０．５ｌ条件培养液中分离纯化得到两种类型的纤溶酶原活化物,ｔ－ＰＡ９０μｇ,ｕ－ＰＡ８００μｇ．在还原条件下ＳＤＳ－ＰＡＧＥ均显示单带,分子量ｔ－ＰＡ为７２ｋＤ,ｕ－ＰＡ为５４ｋＤ,纤溶比活分别为１５６０００ＩＵ／ｍｇ蛋白和１０６０００ＩＵ／ｍｇ蛋白．     

家蚕滞育性卵盐酸处理的靶物质

酯酶A4（EA4）是家蚕卵的滞育生物钟蛋白质。从家蚕C108品种产下后48 h的滞育性卵和盐酸活化处理卵分离纯化出EA4酶蛋白,使用合成的EA4活性多肽抑制因子PIN（氨基酸结构：SIFMTKQHSQ DDIIQHPLDY VEQQIHQQKQ KLQKQTLN）,研究了PIN对EA4酶蛋白的作用机制。滞育性卵的EA4酶蛋白和PIN在25℃混合24h后,用矩阵辅助激光解吸离子质谱法,检测到了二者的结合体,该结合体在盐酸处理后消失;盐酸活化处理蚕卵的EA4酶蛋白和合成PIN之间没有出现这种结合体。体外25℃,滞育性蚕卵EA4的ATPase特征性活性峰在6.5 h后出现,而盐酸活化处理蚕卵的EA4在1.5 h后出现活性峰值。盐酸处理可能通过解除PIN对EA4的抑制作用,在短时间内激活EA4酶蛋白,从而活化滞育性蚕卵。     

The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease

Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193-197) or insertion 4 (positions 210-221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca(2+) binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70 degrees C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca(2+). In the presence of dithiothreitol, the WF146 protease suffered excessive autolysis, indicating that the Cys52-Cys65 disulfide bond played a critical role in stabilizing the WF146 protease against autolysis. The autolytic cleavage sites of the WF146 protease were identified to locate between residues Asn63-Gly64 and Cys65-Ala66 by N-terminal amino acid analysis of the autolytic product. It was noticed that the effect of the autolytic cleavage at Asn63-Gly64 could be compensated by the disulfide bond Cys52-Cys65 under non-reducing condition, and the disulfide bond cross-linked autolytic product remained active. The apparent stabilization effect of the disulfide bond Cys52-Cys65 in the WF146 protease might provide a rational basis for improving the stability of subtilase against autolysis by protein engineering.     

家蚕吡哆醛激酶基因定点突变及突变体功能

【目的】吡哆醛激酶（pyridoxal kinase,PLK）是维生素B₆关键代谢酶。前期研究克隆出家蚕Bombyx mori吡哆醛激酶cDNA,经序列比对发现几个重要且保守的氨基酸残基在此蛋白中被替换。为明确家蚕吡哆醛激酶分子若干特定位置上氨基酸残基在酶功能上的作用进行本研究。【方法】采用重叠延伸法对家蚕吡哆醛激酶Thr⁴⁷,Asn¹²¹, Ile⁵⁴, Arg⁸⁸和Trp²³⁰氨基酸残基进行定点突变, 构建表达载体pET-22b（+）-PLK并转入大肠杆菌Escherichia coli Rosetta中进行诱导表达,经亲和层析对重组蛋白进行纯化, 通过酶活性检测进行功能鉴定。【结果】家蚕吡哆醛激酶Thr⁴⁷,Ile⁵⁴和Arg⁸⁸氨基酸突变后酶的催化活力分别下降82%, 58%和85%;Asn¹²¹突变对酶的催化活力几乎没有影响;而Trp²³⁰突变导致酶丧失催化活性。【结论】本研究明确了选定氨基酸侧链基团在家蚕吡哆醛激酶催化功能上的意义。     

Structure of pentasaccharide of glycopeptide from TIME-EA4, N-glycoprotein in silkworm diapause eggs

The TIME-EA4, from silkworm diapause eggs of pure strain C108, Bombyx mori, has glycosylated chain as tetrasaccharide (Man(2)GlcNAc(2)) attaching to the Asn(22) of T3 peptide from tryptic digests. On the other hand, from Showa silkworm strain we additionally observed a pentasaccharide (Man(3)GlcNAc(2)) on T3 at the same linkage site. The linkage pattern of the 5-sugar chain was studied through Smith degradation combined with LC-MS and MS/MS analyses. These advanced methods led us to conclude that the pentasaccharide was branching as Man 1-->3(Man 1-->6)Man 1-->4GlcNAc 1-->4GlcNAc.     

Hypothesis of a proton switch in QM/MM modelling of interaction of dUMP analogues with thymidylate synthase

Quantum mechanical, molecular mechanics and molecular dynamics (MD) methods were used to investigate initial steps of 2′-deoxyuridine-5′-monophosphate (dUMP) methylation catalysed by the thymidylate synthase (TS) enzyme. The amino acid residues surrounding the active site within a 10 Å radius sphere were modelled with the combined quantum mechanical (B3LYP/LANL2DZ) and molecular mechanics ONIOM double-layer method. The results indicated the initial nucleophilic attack of Cys146 on dUMP to be concerted with formation of a hydrogen bond to the oxygen O4 of dUMP. Moreover, the proton in the vicinity of the O4 atom appears to act as a ‘proton switch’: if a proton is present near O4, it stabilises the S(Cys146)–C6(dUMP) sulphur–carbon bond, but if it is absent, the sulphur–carbon bond does not form. If the O4 oxygen is replaced by sulphur atom, the ‘switch effect’ does not occur. The suggested correlation between the strength of hydrogen bond involving O4 oxygen and the ability of dUMP to form bonds at C6 corresponds well to the crystal structures of TS complexes available in the Protein Data Bank. In the vast majority of crystal structures, the presence of the S(Cys146)–C6(dUMP) bond was coupled with the presence of hydrogen bond between the dUMP O4 atom and the conserved Asn177. The ‘proton switch’ hypothesis is supported also by the results of MD studies of TS binary complexes, suggesting that average distance separating S(Cys146) and C6(dUMP) becomes distinctly shorter in the presence of hydrogen bonding between Asn177 and O4.     

A bacterial and silkworm aminoacyl-tRNA synthetase have a common epitope which maps to the catalytic domain of each

We report here the identification of a common immunological determinant in Escherichia coli and Bombyx mori (silkworm) alanine tRNA synthetases. The E. coli protein is a tetramer of identical Mr = 95,000 chains, and the silkworm enzyme is a monomer of Mr = 115,000. Antibodies against the silkworm enzyme react with E. coli Ala-tRNA synthetase. Analysis of 10 fragments of the E. coli enzyme has mapped the cross-reacting epitope to between amino acids 350 and 385. This is within the part of the enzyme which is essential for alanyladenylate synthesis. The anti-B. mori Ala-tRNA synthetase antibodies which cross-react with the E. coli enzyme were affinity-purified. They react specifically with the catalytic domain of the silkworm enzyme and not with the remaining dispensable segment of 500 amino acids. The results support the concept that the core catalytic structural elements, and not the dispensable portions, are the most related among the synthetases.     

Express analysis of protein amino acid sequences. Primary structure of Penicillium chrysogenum 152A guanyl-specific ribonuclease

The complete amino acid sequence of Penicillium chrysogenum 152A guanyl-specific RNase has been established using automated Edman degradation of two non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the protein. The RNase contains 102 amino acid residues: His2, Arg3, Asp7, Asn8, Thr5, Ser11, Glu4, Gln2, Pro4, Gly11, Ala13, Cys4, Val8, Ile3, Leu3, Tyr9, Phe5 (Mr 10 747).     

The primary structure of cassowary (Casuarius casuarius) goose type lysozyme

The complete amino acid sequence of cassowary (Casuarius casuarius) goose type lysozyme was analyzed by direct protein sequencing of peptides obtained by cleavage with trypsin, V8 protease, chymotrypsin, lysyl endopeptidase, and cyanogen bromide. The N-terminal residue of the enzyme was deduced to be a pyroglutamate group by analysis with a LC/MS/MS system equipped with the oMALDI ionization source, and then confirmed by a glutamate aminopeptidase enzyme. The blocked N-terminal is the first reported in this enzyme group. The positions of disulfide bonds in this enzyme were chemically identified as Cys4-Cys60 and Cys18-Cys29. Cassowary lysozyme was proved to consist of 185 amino acid residues and had a molecular mass of 20408 Da calculated from the amino acid sequence. The amino acid sequence of cassowary lysozyme compared to that of reported G-type lysozymes had identities of 90%, 83%, and 81%, for ostrich, goose, and black swan lysozymes, respectively. The amino acid substitutions at PyroGlu1, Glu19, Gly40, Asp82, Thr102, Thr156, and Asn167 were newly detected in this enzyme group. The substituted amino acids that might contribute to substrate binding were found at subsite B (Asn122Ser, Phe123Met). The amino acid sequences that formed three alpha-helices and three beta-sheets were completely conserved. The disulfide bond locations and catalytic amino acid were also strictly conserved. The conservation of the three alpha-helices structures and the location of disulfide bonds were considered to be important for the formation of the hydrophobic core structure of the catalytic site and for maintaining a similar three-dimensional structure in this enzyme group.     

Structural characterization of a rhamnose-binding glycoprotein (lectin) from Spanish mackerel (Scomberomorous niphonius) eggs

A rhamnose-binding glycoprotein (lectin), named SML, was isolated from the eggs of Spanish mackerel (Scomberomorous niphonius) by affinity and ion-exchange chromatographies. SML was composed of a non-covalently linked homodimer. The SML subunit was composed of 201 amino acid residues with two tandemly repeated domains, and contained 8 half-Cys residues in each domain, which is highly homologous to the N-terminal lectin domain of calcium-independent alpha-latrotoxin receptor in mammalian brains. Each domain has the same disulfide bonding pattern; Cys10-Cys40, Cys20-Cys99, Cys54-Cys86 and Cys67-Cys73 were located in the N-terminal domain, and Cys108-Cys138, Cys117-Cys195, Cys152-Cys182 and Cys163-Cys169 were in the C-terminal domain. SML was N-glycosylated at Asn168 in the C-terminal domain. The structure of the sugar chain was determined to be NeuAc-Galbeta1-4GlcNAcbeta1-2Manalpha1-6-(NeuAc-Galbeta1-4GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc-Asn.