轻稀土离子对钙调蛋白激活的磷酸二酯酶活力作用的影响

研究了轻稀土离子（Ｌｎ３＋）对钙调蛋白（ＣａＭ）调控的磷酸二酯酶（ＰＤＥ）活力的影响。结果表明,在无Ｃａ２＋的ＣａＭ（Ａｐｏ—ＣａＭ）体系中,由ＣａＭ调节的ＰＤＥ的活力随Ｌｎ３＋浓度的变化曲线是双相效应,即在高浓度时,Ｌｎ３＋具有抑制ＣａＭ调节ＰＤＥ活力的能力;低浓度的Ｌｎ３＋可以提高ＣａＭ调节ＰＤＥ活力的能力。在Ｃａ２＋４－ＣａＭ－ＰＤＥ体系中,高浓度的Ｌｎ３＋的加入能抑制ＣｈＭ调节ＰＤＥ活力的能力,其抑制程度因其离子不同而异。ＣａＭ的两类拮抗剂ＪｕＡ（非竞争性抑制剂）和ＴＦＰ（竞争性抑制剂）都能抑制ＣａＭ－Ｌｎ３＋－ＰＤＥ系统的活性。最后对Ｌｎ３＋和ＣａＭ相互作用的分子机制进行了初步的讨论。     

嗜热脂肪芽孢杆菌DNA解链蛋白1的纯化及性质

以嗜热脂肪芽孢杆菌为材料,通过ＰｏｌｙｍｉｎＰ沉淀,硫酸铵分级及Ｐｈｅｎｙｌ－Ｓｅｐｈａｒｏｓｅ,ＤＥＡＥ纤维素,磷酸纤维素,ＦＰＬＣ ＭｏｎｏＱ,ＦＰＬＣ Ｓｕｐｅｒｏｓｅ１２等柱层析,得到部分纯化的ＤＮＡ解链蛋白１。ＢｓｔＨ１具有依赖ＤＮＡ和Ｍｇ＾２＋的ＡＴＰ酶活力,不同类型的核酸对ＢｓｔＨ１的ＡＴＰ酶活力的促进作用不同。     

小球藻RubisCO的纯化及其在异养向自养转变过程中的含量变化

经硫酸铵分部沉淀、ＳｅｐｈａｃｒｙｌＳ－３００和ＤＥＡＥ－纤维素柱层析纯化了小球藻ＲｕｂｉｓＣＯ,得率为１５％,比活力达１．２３２μｍｏｌＣＯ２ｍｓ－１ｍｉｎ－１,分子量是５００ｋＤ,它和菠菜叶片ＲｕｂｉｓＣＯ在分子量、亚基组成和免疫特性等方面相似,反映ＲｕｂｉｓＣＯ在高等和低等植物中有较高的同源性。自养小球藻ＲｕｂｉｓＣＯ占细胞可溶性蛋白质的２４％。而异养转变后的小球藻细胞内不含ＲｕｂｉｓＣＯ。异养小球藻向自养生长转变过程中,２０ｈ后细胞内叶绿素含量逐渐增加,２４ｈ时细胞内出现ＲｕｂｉｓＣＯ,２４ｈ后大量增加,至４１ｈ时含量达最高峰;标志着小球藻细胞光合作用能力的恢复和加强。     

大肠杆菌表达的重组细胞色素P—450nor的分离纯化

用ＤＥ－５２纤维素柱色谱法和ＦＰＬＣ法分离纯化了大肠杆菌表达的重组缣孢菌色素Ｐ－４５０ｎｏｒ，经梯度洗脱ＭｏｎｏＱ纯化后的ｆＲ．Ｐ－４５０ｎｏｒ为单一色谱峰，比活达５５．２０Ｕ／ｍｇ、纯化倍数约为１１００倍，ＳＤＳ－ＰＡＧＥ检测为单一谱带。     

鼠肝DNA甲基化酶的纯化及物理化学性质的研究

以Ｗｉｓｔａｒ大鼠肝为材料,确立了一个简便的纯化鼠肝ＤＮＡ甲基化酶的程序,包括：细胞的超声破碎,去内源核酸,硫酸铵盐析,磷酸纤维素亲和层析,ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－５０柱层析及Ｓｅｐｈａｄｅｘ Ｇ－１５０凝胶过滤,用不同浓度聚丙烯酰胺凝胶电泳和孔梯度凝胶电泳检测,纯化后的酶已达电泳均一,且酶的比活力提高１１２倍,以聚丙烯酰胺孔梯度凝胶电泳测得天然酶的分子量为３６５ｋＤ,以ＳＤＳ－聚然酰胺凝     

嗜热脂肪芽孢杆菌DNA解链蛋白BstH2的纯化及性质研究

以嗜热脂肪芽孢杆菌为材料,通过ＰｏｌｙｍｉｎＰ沉淀、硫酸铵分级及ＤＥＡＥ纤维素、磷酸纤维素、Ｂｌｕｅ－Ｓｅｐｈａｒｏｓｅ、ＦＰＬＣＭｏｎｏＱ、ＦＰＬＣ Ｓｕｐｅｒｏｓｅ１２等柱层析,得到了部分纯化ＤＮＡ解链蛋白ＢｓｔＨ２。ＢｓｔＨ２具有受ＤＮＡ促进的ＡＴＰ酶活力,没类型的核酸对ＢｓｔＨ２的ＡＴＰ酶活力的促进作用没。ＢｓｔＨ２在５５℃有最高ＡＴＰ酶活力。这种活力受大肠杆菌单链ＤＮＡ结合蛋白的抑制及随     

枯草芽孢杆菌BS12乳酸脱氢酶的分离纯化与部分性质的研究

枯草芽孢杆菌ＢＳ１２的乳酸脱氢酶经硫酸铵分级沉淀、ＣＭ－纤维素、ＤＥＡＥ－纤维素离子交换柱层析、ＳｅｐｈａｄｅｘＧ—２００柱层析,得到了凝胶电脉均一的样品。用ＳＤＳ—ＰＡＧＥ测得其亚基分子量为２８０００Ｄａ。酶反应的最适ｐＨ为７．０,最适温度为３５℃。     

黑曲霉突变株分解生淀粉的葡萄糖淀粉酶的提纯及其…

黑曲霉突变株Ｔ－２１产生的葡萄糖淀粉酶具有多型性,在凝胶电泳上呈现很多蛋白质带,酶活力显色表明许多条（５条以上）均表现出淀粉酶活力,酶制剂浸出液通过硫酸铵分级沉淀,ＤＥＡＥ－纤维素柱层析,Ｓｅｐｈａｄｅｘ Ｇ－１５０柱凝胶过滤和ＤＥＡＥ－纤维素柱再层析分离纯化到了一型能被生淀粉吸附和水解生淀粉的葡萄糖淀粉酶ＧＡＩ和用凝胶制备电泳分离纯化到一型既不为生淀粉吸附又不水解生淀粉的ＧＡＩＩ。ＧＡＩ被生小麦     

嗜卷书虱抗气调性的一些生物化学特性研究(英文)

嗜卷书虱成虫于２８°Ｃ、７０％—８０％ＲＨ条件下,在３５％ＣＯ２。和１％Ｏ２。组配的气调环境中连续暴露３０代,选择压力保持在７０％左右,以选育其抗气调性。至第３０代,以ＬＴ５０。为指标衡量其抗性倍数达５．６倍。生化分析表明,抗性选育过程中离体羧酸酯酶（ＣａｒＥ）和超氧化物歧化酶（ＳＯＤ）活力均随选育代数的增多而显著增强,且酶活力与抗气调水平显著相关。第３０代,抗性品系（ＣＡ－Ｒ）的ＣａｒＥ和ＳＯＤ活力分别是敏感品系（ＣＡ－Ｓ）的４．０６和５．２２倍。两品系暴露于气调环境中,其ＣａｒＥ活力均受抑制,但对ＣＡ－Ｓ品系的抑制率显著强于ＣＡ－Ｒ品系。短时间的气调暴露对ＳＯＤ具有诱导作用,但诱导作用的时间及强度ＣＡ－Ｒ品系明显长（强）于ＣＡ－Ｓ品系。离体过氧化氢酶（ＣＡＴ）活力也随着抗气调性的增强而增强,但两着之间并无显著的相关性。抗性选育过程中离休酸性磷酸酯酶（ＡＣＰ）和碱性磷酸酯酶（ＡＬＰ）活力均未发生明显的变化。过氧化物酶（ＰＯＤ）在两品系中均未检测出。由此看出,ＣａｒＥ和ＳＯＤ活力增强是嗜卷书虱抗气调性形成的主要机制,ＣＡＴ可能对低氧气调抗性的形成具有辅助作用。     

蛇毒蛋白C激活物的初步研究

目的：研究蝮蛇毒蛋白Ｃ激活物的分离纯化与理化性质。方法：经ＤＥ５２－纤维素、ＣＭ－Ｓｐｈａｄｅｘ Ｃ－５０、Ｇ－７５柱层析,从安徽芜湖产蝮蛇（Ａｇｋｉｓｔｒｏｄｏｎ ｈａｌｙｓ）蛇毒中纯化一种均一的蛋白Ｃ激活物（ＰＣＡ）。结果：ＳＤＳ－ＰＡＧＥ测定分子量约为１５５０００Ｄａ,ＩＥＦ－ＰＡＧＥ测定等电点为４．８。它能使人血浆的ＫＰＴＴ明显延长,显示出强烈的抗凝活性。通过中和试验与显色肽定量实验表明,     

不同来源溶菌酶的性质比较

比较两种从新鲜鸡蛋清中提取溶菌酶的方法。采用较为简单的并且产率较高的结晶法分别从鸡蛋清、鹌鹑蛋清中提取了溶菌酶 ,并分别测定了各溶菌酶的酶活力、最适pH值和最适温度。同时 ,证明了该法无法从鸭蛋清中提取出纯溶菌酶 ,故仅对粗提物进行了酶活力、最适 pH值和最适温度的测定     

Purification of camel milk lysozyme and its lytic effect on Escherichia coli and Micrococcus lysodeikticus

1. Camel milk lysozyme was purified using heparin-Sepharose 4B, Sephadex G-75 and hydroxyapatite chromatography. By this procedure lysozyme was separated from lactoferrin and a low molecular weight protein. 2. The lytic effect of camel milk lysozyme was assayed using Escherichia coli and Micrococcus lysodeikticus and its activity was compared with that of lysozyme from human milk and egg white. 3. The specific activity of camel milk lysozyme was found to be lower than that of lysozyme from human milk or from egg white. 4. Camel milk lactoferrin did not show a lytic effect on bacteria, while the low molecular weight protein showed lytic activity.     

Highly active fractions of the medicinal leech recombinant destabilase-lysozyme

Fractionation of the highly purified but low active recombinant protein destabilase-lysozyme (Dest-Lys) by cation exchange chromatography on a TSK CM 3-SW chromatography the non-active fraction (IV) containing 90% of total protein has been separated. Fractions I, II, and III contained proteins with lysozyme and isopeptidase activities and their lysozyme activity correlated with the activity of native Dest-Lys. However, the ratio of lysozyme and isopeptidase activities differed in these fractions; maximal lysozyme activity was found in fraction III, while maximal isopeptidase activity was associated with fraction I. Possible regulation of different functions of Dest-Lys is discussed in the context of formation of its various complexes.     

First report of a novel plant lysozyme with both antifungal and antibacterial activities

A novel lysozyme exhibiting antifungal activity and with a molecular mass of 14.4kDa in SDS-polyacrylamide gel electrophoresis was isolated from mung bean (Phaseolus mungo) seeds using a procedure that involved aqueous extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography on POROS HS-20. Its N-terminal sequence was very different from that of hen egg white lysozyme. Its pI was estimated to be above 9.7. The specific activity of the lysozyme was 355U/mg at pH 5.5 and 30 degrees C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 55 degrees C. It is reported herein, for the first time, that a novel plant lysozyme exerted an antifungal action toward Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii, and Botrytis cinerea, in addition to an antibacterial action against Staphylococcus aureus.     

Lytic activity and biochemical properties of lysozyme in the coelomic fluid of the sea urchin strongylocentrotus intermedius

Lysozyme was identified in the coelomic fluid including coelomocytes of the sea urchin Strongylocentrotus intermedius, and its lytic activity and biochemical properties were examined in this study. The urchin lysozyme was electrophoretically fractionated to a single lytic band of about 14 kDa. No distinct difference in the lytic activity of this enzyme was found between urchins held at two temperatures, 11 degrees and 25 degrees C. The lysozyme of this species was purified through several procedures: salting out with ammonium sulfate, precipitation by ethanol saturation, gel filtration with a Biogel column, and an affinity chromatography with a heparin Sepharose column. The combination method of Biogel filtration and affinity chromatography resulted in the most purified lysozyme fraction, but we could not obtain a single protein band in SDS-PAGE. In addition, anti-hen egg white lysozyme (HEWL) antibody was produced and confirmed to react specifically with the urchin lysozyme in this study. Therefore, the HEWL antibody may be available for examining the lytic activity of lysozyme at an individual level to determine the biodefense activity of sea urchins. Copyright 1999 Academic Press.     

Effective method for purification of lysozyme from human urine

Lysozyme in the urine of a hemodialysis patient was purified in two steps: DEAE Sephadex chromatography followed by Sephacryl chromatography. The Sephacryl S-100 column chromatographed fraction showing lytic activity was proven to give one band on SDS-PAGE and to have a molecular mass of 14 500, in agreement with that of lysozyme. The N-terminal amino acid sequence of this purified protein was identical to that of lysozyme. These results indicate that the protein purified was indeed lysozyme. The specific affinity of lysozyme for Sephacryl S-100 may explain the greater purity of the same protein isolated by this method.     

Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.     

Expression of functional recombinant human lysozyme in transgenic rice cell culture

Using particle bombardment-mediated transformation, a codon-optimized synthetic gene for human lysozyme was introduced into the calli of rice (Oryza sativa) cultivar Taipei 309. The expression levels of recombinant human lysozyme in the transformed rice suspension cell culture approached approximately 4% of total soluble protein. Recombinant human lysozyme was purified to greater than 95% homogeneity using a two-step chromatography process. Amino acid sequencing verified that the N-terminus of the mature recombinant human lysozyme was identical to native human lysozyme. This indicates that the rice RAmy3D signal peptide was correctly cleaved off from the human lysozyme preprotein by endogenous rice signal peptidase. Recombinant human lysozyme was found to have the same molecular mass, isoelectric point and specific activity as native human lysozyme. The bactericidal activity of recombinant human lysozyme was determined by turbidimetric assay using Micrococcus lysodeikticus in 96-well microtiter plates. The bactericidal activity of lysozyme on Gram-negative bacteria was examined by adding purified lysozyme to mid-log phase cultures of E. coli strain JM109. In this study, significant bactericidal activity was observed after E.coli cells were exposed to recombinant human lysozyme for 60min. Both native and recombinant human lysozyme displayed the same thermostability and resistance to degradation by low pH. The potential for using rice-derived lysozyme as an antimicrobial food supplement, particularly for infant formula and baby foods, is discussed.     

Purification of a lysozyme from skin secretions of Bufo andrewsi

A novel toad lysozyme (named BA-lysozyme) was purified from skin secretions of Bufo andrewsi by a three-step chromatography procedure. BA-lysozyme is a single chain protein and the apparent molecular weight is about 15 kDa as judged by SDS-PAGE. The specific lytic activity against Micrococcus lysodeikticus of BA-lysozyme is 2.7 x 10(5) units/mg, indicating that it is a potent lysozyme. It displayed potent bactericidal activity against Staphylococcus aureus and Escherichia coli with minimal inhibitory concentrations (MIC) of 1 and 8 microM, respectively. The deduced primary structure of BA-lysozyme from cloned cDNA was confirmed by N-terminal sequencing and peptide mass fingerprinting. Its amino acid sequence shares 56.5% identity with that of chicken egg-white lysozyme. Phylogenetic analysis indicates that B. andrewsi lysozyme is closely related to that of turtle. This is the first report on the isolation and primary structure determination of amphibian lysozyme.     

A New Method for the Analysis of Fatty Acid Mixtures by Gas Chromatography

Deamidation of lysozyme was observed during storage in a buffer solution and in egg white. The peak corresponding to native lysozyme from Bio-Rex 70 column chromatography was gradually decreased, while the peaks corresponding to deamidated lysozyme were increased during storage in 0.1 m carbonate buffer at pH 9.5. A similar change was observed during storage in egg white, but the change in egg white was larger than that in the buffer solution. A detailed analysis of the elution peaks from the Bio-Rex 70 column suggested that one to three residues of amide in lysozyme were mainly deamidated during storage in the buffer solution, and that more than three residues in lysozyme were deamidated during storage in egg white. There were significant differences in lysozyme activity between native and deamidated lysozyme, the activity being decreased in proportion to the degree of deamidation.