表面活性剂TritonX－100对菌紫质蛋白紫外荧光性质的影响

用荧光光谱方法研究了ＴｒｉｔｏｎＸ－１００（以下缩写为ＴＸ－１００）对菌紫质蛋白（Ｂａｃｔｅｒｉｏｒｈｏｄｏｐｓｉｎ,ＢＲ）及视黄醛生色团漂白后的紫膜（Ｂａｔｅｒｉｏｐｓｉｎ,ＢＯ）紫外荧光性质的影响．结果表明：表面活性剂ＴＸ－１００使ＢＲ中色氨酸在３２６ｎｍ处的荧光发射强度增加。随着ＴＸ－１００对ＢＲ的增溶,改变了生色团的构象环境,破坏了ＢＲ中色氨酸与生色团之间的能量转移。增溶后的ＢＲ中,Ｔｒｐ趋向于更疏水性的环境。     

神经节苷脂对细胞膜损伤的保护作用

观察了神经节苷脂对去垢剂Ｔｒｉｔｏｎ—Ｘ—１００损伤大鼠红细胞膜的影响。适量的红细胞与１６μｇ／ｍｌ至５×１０４μｇ／ｍｌ范围内不同浓度的神经节苷脂于３７℃孵育１小时后,加入ＴｒｉｔｏｎＸ－１００在室温下作用３分钟。离心,取上清液在５４０ｎｍ测溶液透光率,取沉淀部分的红细胞在扫描电镜下观察红细胞及其膜的形态变化。实验结果显示,在神经节苷脂浓度为１０４μ／ｍｌ时,对红细胞膜的保护作用最好,上清液透光率为正常值的８５．４％;细胞形态虽有改变,但仍的部分接近正常的细胞存在。而损伤对照组上清透光度仅为正常值的１３．４％,红细胞胞体缩小,成为棘形红细胞。结果表明,神经节苷脂对红细胞膜化学损伤有良好的保护作用。     

磷脂酰胆碱和Triton X—100对光系统Ⅱ蛋白二级结构及放氧活性？…

采用傅立叶变换红外光谱技术（ＦＴ－ＩＲ）和氧电极研究了磷脂酰胆碱和Ｔｒｉｔｏｎ Ｘ－１００对光系统Ⅱ膜复合物的蛋白二级结构及放氧活性的影响。结果表明,磷脂酰胆碱对光系统Ⅱ膜复合物的蛋白二级结构没有显著的影响,但能引起放氧活性的提高,而且脂酰侧链长度不同,对放氧活性的促进程度也不一样。相比较而言,ＴＸ－１００对膜脂的扰动却引起蛋白二级结构的明显改变,并能抑致放氧活性。结果说明,完整的膜结构对维持光合     

天然雌核发育银鲫卵子控制异源精核发育的受精学机制

作者对两性融合生殖鱼和雌核发育银鲫脱膜卵受精的精核发育进行了观察,并采用鱼类卵子无细胞系对以上两类卵质提取物体外诱导经Ｔｒｉｔｏｎ—Ｘ１００处理的精子及其发育进行了初步研究,结果表明在两性融合生殖型脱膜鱼卵中精核通过解凝最终形成原核,而在雌核发育的银鲫脱膜卵子中部分精核体积虽有一定程度的增加,但始终没有观察到原核的发育;在体外诱导实验中,经Ｔｒｉｔｏｎ—Ｘ１００处理的精子在两类卵质提取物中充分发育,都出现了类似体内原核的状态。该现象提示在银鲫卵质中存在有促使精核形成原核的因子,但在正常受精状态下,由于银鲫卵质促使精核核膜解体的功能的异常,使覆盖精子头部的核膜不能象在两性融合生殖受精卵子中进行崩解,精核进一步的原核发育受到抑制。另外,建立体外诱导系统的重要意义,在于它为研究雌核发育调控的分子学机制提供了一条有效途径。     

蜂毒素与紫膜的相互作用机理研究──与三种表面活性剂的比较

用吸收光谱和圆二色谱的方法研究了蜂毒素与嗜血菌紫膜的相互作用机理．通过与三种在结构和电荷上不同的表面活性剂与紫膜的作用相比较,可以年出蜂毒作为带正电荷的分子与同样带正电的表面活性剂ＤＴＡＢ在引起紫膜凝聚方面表现相同;但在对紫膜可见光区的吸收光谱和圆二色谱的影响上却与具有刚性结构的ＣＨＡＰＳ相似,表明蜂毒可在紫膜表面以一种刚性较大的构象（如α螺旋）存在,不能进入膜蛋白流水区的深层．另外,从紫膜－Ｔｒｉｔｏｎ－蜂毒混合作用体系的研究中得到如下推测：蜂毒与Ｔｒｉｔｏｎ竞争菌紫质分子周围的结合位点,可排斥位于菌紫质周围的Ｔｒｉｔｏｎ分子．表明蜂毒具有比Ｔｒｉｔｏｎ更强的与菌紫质的亲和力,从而提供了支持蜂毒分子存在与膜蛋白－菌紫质的直接相互作用的有力证据．     

电鳐β—蝮蛇毒素结合蛋白的抽提及其与毒素结合性质的研究

以加州电鳐电器官为材料,探索了用去垢剂ＴｒｉｔｏｎＸ－１００增溶抽提β－蝮蛇毒素结合蛋白 合适条件,建立了此结合蛋白活性的检测方法,并分析了该结合蛋白与同位素＾１２５Ｉ标记β－ＡｇＴＸ的结合性质。     

鼠伤寒沙门氏菌外膜蛋白诱导BALB／C小鼠免疫保护的实验研究

本实验采用超声破碎、ＴｒｉｔｏｎＸ－１００处理和超速离心技术提取了鼠伤寒沙门氏菌（Ｓａｌｍｏｎｅｌｌａｔｙｈｉｍｕｒｉｕｍ,ＳＴＭ）的外膜蛋白（Ｏｕｔｅｒｍｅｍｂｒａｎｅｐｒｏｔｅｉｎｓ,ＯＭＰｓ）,其中脂多糖（ＬＰＳ）的含量约为５％。ＯＭＰｓ经ＳＤＳ—ＰＡＧＥ显示１０余条蛋白带。对ＯＭＰｓ诱发ＢＡＬＢ／Ｃ小鼠产生典型的迟发型变态反应（ＤＴＨ）进行了检测。经腹腔免疫的ＢＡＬＢ／Ｃ小鼠用５００ＬＤ５０鼠伤寒沙门氏菌（５０１１５）攻击,１００％可得到保护;用５００ＬＤ５０伤寒杆菌（Ｅ６８６）攻击     

泛醌结合蛋白（QPs）羧基的化学修饰与重组活性

研究了羧基修饰剂ＤＣＣＤ对泛醌结合蛋白ＱＰｓ重组活力的影响;用０．１％ＴｒｉｔｏｎＸ－１００增溶ＱＰｓ后,用２５０ｍｏｌ／ｍｏｌＱＰｓ的ＤＣＣＤ于室温处理５ｍｉｎ,处理后的ＱＰｓ丧失约５０％与琥珀酸脱氢酶的重组活力。先将ＱＰｓ与琥珀酸脱氢酶重组再用ＤＣＣＤ处理没有发现重组的琥珀酸泛醌还原酶活性的降低。此结果说明ＱＰｓ中存在重组活性必需的羧基。     

泛醌结合蛋白（QPs）羧基的化学修饰与重组活性

研究了羧基修饰剂ＤＣＣＤ对泛醌结合蛋白ＱＰｓ重组活力的影响。用０．１％Ｔｒｉｔｏｎ Ｘ－１００增溶ＱＰｓ,用２５０ｍｏｌ／ｍｏｌＱＰｓ的ＤＣＣＤ于室温处理５ｍｉｎ,处理后的ＱＰｓ丧失约５０％与琥珀酸脱氢酶的重组活力。先将ＱＰｓ与琥珀酸脱氢酸重组再用ＤＣＣＤ处理没有发现重组的琥珀酸泛醌还原酶活性的降低。此结果说明ＱＰｓ中存在重组活性必需的羧基。     

甲基毒死蜱对红细胞乙酰胆碱酯酶的抑制作用及其与膜脂相互关系

以人红细胞膜为材料,研究了甲基毒死蜱与膜上乙酰胆碱酯酶的相互作用及其与膜脂的关系。结果显示,甲基毒死埤对人红细胞ＡＣｈＥ有明显的抑制作用,与膜温育３０ｍｉｎ,其半数抑制浓度约为０．１０ｍｍｏｌ／Ｌ。动力学分析表明,其抑制作用为非竞争性。０．２％Ｔｒｉｔｏｎ Ｘ－１００并不改变ＡＣｈＥ对甲基毒死蜱的敏感性,亦即ＡＣｈＥ上甲基毒死蜱的作用部位与其所处的脂质微环境无关。     

TritonX-100对含桐酸的卵磷脂脂质体的作用

本文研究了非离子型表面活性剂TritonX-100对含桐酸的卵磷脂脂质体的作用,结果表明,在TritonX-100对含桐酸的脂质体的作用中,存在一个TritonX-100的临界浓度,低于这个临界浓度时,TritonX-100的加入对脂质体的尺寸影响很小;当TritonX-100的浓度超过临界浓度时,脂质体迅速聚集成大团粒.     

Effect of Triton X-100 on properties of acetylcholinesterase from human erythrocytes

The effect of Triton X-100 on catalytic properties of acetylcholinesterase from human erythrocytes under acetylcholine hydrolysis, on sensitivity of acetylcholinesterase to specific phosphoorganic inhibitors and eserine, and on the mobility and isoenyme spectrum under analytical electrophoresis in polyacrylamide gel is investigated. Triton X-100, independently on its concentration within 0.05-1.0%, slightly changes V and [S]opt values and increases Km value in 2-3 times. The inhibitory effect of Triton X-100 is mainly competitive, 0.5% Triton X-100 decreases bimolecular constant (kII) of the interaction of acetylcholinesterase with phosphoorganic inhibitor and eserine in 2.5-4 times. In the presence of phosphoorganic inhibitor, kII sharply decreased when 0.02% Triton X-100 was added, and then it did not change under the increase of Triton X-100 concentration up to 1.0%. On the basis of these data, an analytical method of estimating Triton X-100 content in protein solution is proposed. The introduction of 0.1% Triton X-100 into polyacrylamide gel results in considerable quantitative redistribution of acetylcholinesterase isoenzyme fractions and in the change of the mobility of one fraction under electrophoresis.     

Use of resistant mutants to study the interaction of triton X-100 with Staphylococcus aureus.

Staphylococcus aureus mutants resistant to the nonionic detergent Triton X-100, isolated from the wild-type strain H and the autolysin-deficient strain RUS3, could grow and divide in broth containing 5% (vol/vol) Triton X-100, while growth of the parental strains was markedly inhibited above the critical micellar concentration (0.02%) of the detergent. Growth-inhibitory concentrations of Triton X-100 killed wild-type cells without demonstrable cellular lysis. Triton X-100 stimulated autolysin activity of S. aureus cells under nongrowing conditions, and this lytic response was markedly reduced in energy-poisoned cells. In contrast, the detergent had no effect on the activity of autolysins in cell-free systems, and growth in the presence of Triton X-100 did not alter either the cellular autolysin activity or the susceptibility of cell walls to exogenous lytic enzymes. Treatment with either Triton X-100 or penicillin G in the growth medium stimulated release of predominantly acylated intracellular lipoteichoic acid and sensitized staphylococci to Triton X-100-induced autolysis. There was no significant difference in the cell wall and membrane compositions or Triton X-100 binding between the parental strains and the resistant mutants. The resistant mutant TXR1, derived from S. aureus H, had a higher level of L-alpha-glycerophosphate dehydrogenase activity, and its oxygen uptake was more resistant to inhibition by a submicellar concentration (0.008%) of Triton X-100. Growth in the presence of subinhibitory concentrations of Triton X-100 rendered S. aureus H cells phenotypically resistant to the detergent and greatly stimulated the level of oxygen uptake. Membranes isolated from such cells exhibited enhanced activity of the respiratory enzymes succinic dehydrogenase and L-alpha-glycerophosphate dehydrogenase.     

A simple turbidimetric method for the determination of microgram quantities of Trition X-100

A simple and sensitive procedure for the quantitative estimation of Triton X-100 is described. The method is based on the formation of turbidity from Triton X-100 with phenol. The turbidity is proportional to Triton X-100 in a range of 20–80 μg/ml. Protein, mucopolysaccharide, and nucleic acid do not interfere in this turbidity formation. The method is especially useful for detection of the residue after the removal of Triton X-100 from solubilized samples.     

Hydrogenation of Triton X-100 eliminates its fluorescence and ultraviolet light absorption while preserving its detergent properties

The ultraviolet-light absorption and fluorescence of Triton X-100 were virtually eliminated by hydrogenation to its reduced cyclohexyl analog, RTX-100. The critical micelle concentration of RTX-100 was 12% higher than that of Triton X-100. RTX-100 and Triton X-100 were quite similar in their abilities to extract proteins from human erythrocyte membranes.     

Triton X-100 alters the resistance level of methicillin-resistant Staphylococcus aureus to oxacillin

Abstract We used population analysis to examine the effects of Triton X-100 on the level of resistance to oxacillin of 18 methicillin-resistant Staphylococcus aureus . In the presence of 0.02% Triton X-100, 17 formerly methicillin-resistiant strains exhibited enhanced sensitivity to oxacillin. One homogeneous isolate, KSAF1 was barely affected by the Triton X-100. Sensitivities of lysostaphin, 51 kDa N -acetylglucosaminidase and 62 kDa N -acetylmuramoyl-l-alanine amidase to heat-inactivated cells were not affected when the bacteria were grown in 0.02% Triton X-100. Our data, together with those of a previous study, suggested that Triton X-100 alters the resistance level of methicillin-resistant S. aureus by influencing a factor(s) other than PBPs, bacteriolytic enzymes, or femAB products.     

Solubilization of spingomyelin by triton X-100. Effect of the method of mixing and the concentrations of detergent and lipid

Mixed dispersions of sphingomyelin and Triton X-100 were prepared by two procedures. In method A, aqueous dispersions of sphingomyelin were mixed with aqueous solutions of Triton X-100. In method B, solutions of sphingomyelin and Triton X-100 in organic solvent were mixed, the solvent was evaporated and the dry residue was dispersed in buffer. Measurement of turbidities, electron microscopy and sedimentation of the mixed dispersions suggested the following: Below the critical micellar concentration of Triton X-100, the sphingomyelin is present as liposomes which sediment in the ultracentrifuge. Above the CMC, mixed micelles of sphingomyelin and Triton form. Method B resulted in aggregates of sphingomyelin which contain Triton X-100 even below its critical micellar concentration and which are smaller than those obtained by method A.     

Solubilization, purification, and characterization of succinate dehydrogenase from membranes of Mycobacterium phlei.

Succinate dehydrogenase (SDH) was solubilized from membranes of Mycobacterium phlei by Triton X-100 with a recovery of about 90%. The solubilized SDH was purified about 90-fold by Sephacryl S-300, DEAE-cellulose, hydroxylapatite, and isoelectric focusing in the presence of Triton X-100 with a 20% recovery. SDH was homogeneous, as determined by polyacrylamide gel electrophoresis in nondenaturing gels containing Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme revealed two subunits with molecular weights of 62,000 and 26,000. SDH is a flavoprotein containing 1 mol of flavin adenine dinucleotide, 7 to 8 mol of nonheme iron, and 7 to 8 mol of acid-labile sulfide per mol of protein. Using phenazine methosulfate and 2,6-dichloroindophenol as electron acceptors, the enzyme had an apparent Km of 0.12 mM succinate. SDH exhibited a sigmoidal relationship of rate to succinate concentration, indicating cooperativity. The enzyme was competitively inhibited by fumarate with a Ki of 0.15 mM. In the absence of Triton X-100, the enzyme aggregated, retained 50% of the activity, and could be resolubilized with Triton X-100 with full restoration of activity. Cardiolipin had no effect on the enzyme activity in the absence of Triton X-100, but it stimulated the activity by about 30% in the presence of 0.1% Triton X-100 in the assay mixture. Menaquinone-9(2H), isolated from M. phlei, had no effect on the enzyme activity either in the presence or absence of Triton X-100.     

Formation and characterization of mixed micelles of the nonionic surfactant Triton X-100 with egg, dipalmitoyl, and dimyristoyl phosphatidylcholines

Mixed micelles of the nonionic surfactant Triton X-100 and egg phosphatidylcholine were isolated by column chromatography on 6% agarose and by centrifugation at 35,000g. It was found that egg phosphatidylcholine bilayers are able to incorporate Triton X-100 at molar ratios of Triton to phospholipid below about 1:1, whereas above a molar ratio of about 2:1 Triton/phospholipid all of the phospholipid is converted into mixed micelles. Mixed micelles at a molar ratio of about 10:1 Triton/phospholipid were found to be in the same size range as pure micelles of Triton X-100. The formation of mixed micelles with dipalmitoyl phosphatidylcholine at room temperature, when the phospholipid is below its thermotropic phase transition, is shown to require relatively high concentrations of Triton X-100. The point at which dimyristoyl phosphatidylcholine bilayers are converted to mixed micelles was found to be less clear cut than with egg phosphatidylcholine, but above a molar ratio of about 2:1 Triton/phospholipid, all of this phospholipid is also in mixed micelles. The relevance of these results to the solubilization of membrane-bound proteins with Triton X-100 and the action of phospholipase A₂, which hydrolyzes phosphatidylcholine when it is in mixed micelles with Triton X-100, is discussed.     

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.