燕麦根细胞质膜K+刺激ATP酶的纯化(英文)

Triton X-100加KI能够有效地溶解燕麦根细胞质膜上K~+刺激的ATP酶(张堃等1989)。对这种溶解的K~+刺激的ATP酶进行甘油梯度离心,得到了一些结果:1.在pH 7.5甘油梯度离心,溶解的ATP酶活性损失约85％,在pH 4.5时活性仅损失约50％,这表明低pH条件对于ATP酶活性稳定的重要性,2.使用水平转子进行甘油梯度离心效果较好;3.甘油梯度离心纯化的ATP酶经SDS-PAGE分析,85％以上的酶蛋白分子量为34kD的多肽。这一低分子量的具有K~+刺激的ATP酶活性的多肽与100kD左右的ATP酶(Serrano 1984)之间的关系有待进一步研究;4.经甘油梯度离心纯化后,K~+刺激的ATP酶活性占K~+,Mg~(2+)-ATP酶活性的52％;而溶解的ATP酶活性中,K~+刺激的部分仅为35％(表1)。此结果类似透析对溶解的ATP酶的影响(张堃等1989)。表明溶解的ATP酶制剂中K~+的存在掩盖了K~+对ATP酶的刺激作用。     

水稻幼苗根细胞质膜和液泡膜微囊Ca^2＋－ATP酶的特性

水稻幼苗根质膜和液泡膜Ca2+-ATP酶对ATP的Km值分别为7.1和4.5 μ mol·L-1;反应的最适pH分别为8.0和7.0.两者活性均受Na3VO4和曙红B(EB)抑制;CPZ抑制质膜Ca2+-ATP酶活性,但促进液泡膜Ca2+-ATP酶活性.30mmol·L-1CaCl2浸种和CaCl2浸种结合低温锻炼预处理,均可提高此酶的活性和冷稳定性.     

CaMBP—10介导的质膜H^＋—ATP酶磷酸化对该酶活性的调节

ＣａＭＢＰ－１０在活体处理条件下,抑制ＩＡＡ诱导的质膜Ｈ＾＋－ＡＴＰ酶活性及其磷酸化,抑制作用可被ＩＡＡ逆转并在外加ＣａＭ时被消除,与前期ＢＰ－１０对ＩＡＡ生理应答的调节效应相吻合。并且在各项处理中,质膜Ｈ＾＋－ＡＴＰ酶活性与其磷酸化水平呈现极显著的正相关。     

经渗透胁迫高梁根的质膜囊泡K~＋_－ATP酶及其运输特征

经ＰＥＧ－１０００处理的高粱根,用不连续蔗糖密度梯度制备获得反转密闭的纯化质膜囊泡,显示一种特殊的ＡＴＰ酶活力,它不同于Ｈ＋－ＡＴＰ酶,其最适ＰＨ为７．５,具有高ＡＴＰ亲和力和较低的Ｋ＋转运能力。环己酰亚胺和钒酸钠能抑制其活力,表明它是新合成的Ｐ－型ＡＴＰ酶。     

水稻幼苗根细胞质膜和液泡膜微囊Ca2+-ATP酶的特性

水稻幼苗根质膜和液泡膜Ca2 -ATP酶对ATP的Km值分别为7.1和4.5 μ mol·L-1;反应的最适pH分别为8.0和7.0.两者活性均受Na3VO4和曙红B(EB)抑制;CPZ抑制质膜Ca2 -ATP酶活性,但促进液泡膜Ca2 -ATP酶活性.30mmol·L-1CaCl2浸种和CaCl2浸种结合低温锻炼预处理,均可提高此酶的活性和冷稳定性.     

NaCl胁迫对高粱根、叶鞘和叶片液泡膜ATP酶和焦磷酸酶活性的影响

NaCl胁迫初期 ,Na 主要在根和叶鞘中积累。相应地 ,根和叶鞘液泡膜ATP酶和焦磷酸酶水解活性、依赖ATP和PPi的质子泵活性及Na /H 逆向转运活性均明显增加 ,根和叶鞘的生长没有受到抑制。NaCl胁迫后期 ,Na 开始向地上部分运输并在叶片中积累。此时 ,叶片液泡膜质子泵和Na /H 逆向转运活性开始增加 ,根和叶鞘的Na/K比增加 ,其液泡膜ATP酶和焦磷酸酶水解活性、质子泵活性和Na /H 逆向转运活性下降。相应地 ,根和叶鞘的生长也下降。当保温介质中Na/K比超过 1时 ,液泡膜微囊ATP酶和焦磷酸酶活性均随Na/K比的增加而下降。表明非盐生植物液泡膜质子泵在盐胁迫的初期对Na 在液泡内的积累及其耐盐性起重要作用     

铝和铝+钙对小麦幼苗根尖质膜、液泡膜微囊ATP酶和膜流动性的影响

研究了铝和铝 钙对小麦幼苗根尖质膜、液泡膜微囊H ATP酶、Ca2 ATP酶、Mg2 ATP酶活性及其动力学参数和膜流动性的影响。在质膜和液泡膜微囊制剂中加入 1.0mmol/L的Al3 (AlCl3)时 ,H ATP酶、Ca2 ATP酶、Mg2 ATP酶活性和酶促反应的Vmax及膜流动性下降 ,而酶促反应的最适pH和Km 均不受影响。提高酶促反应介质的Ca2 (CaCl2 )浓度可以缓解Al3 对膜ATP酶活性和膜流动性的影响。推测Al3 可能通过与膜的结合而抑制膜ATP酶的活性     

水分胁迫对小麦根细胞质膜氧化还原系统的影响

水分胁迫使小麦根质膜ＮＡＤＨ和ＮＡＤＰＨ的氧化速率及Ｆｅ（ＣＮ）６３－和ＥＤＴＡ－Ｆｅ３＋的还原速率明显降低。对照与胁迫处理的质膜氧化还原系统活性均不受鱼藤酮抗霉素Ａ和ＫＣＮ等呼吸链抑制剂的影响。在不加Ｆｅ（ＣＮ）６３－作为电子受体时,水杨基羟肘酸（ＳＨＷ）可明显刺激质膜ＮＡＤＨ的氧化和Ｏ２吸收速率。水分胁迫促使ＳＨＡＭ刺激的ＮＡＤＨ氧化明显降低,但却使Ｏ２吸收略有上升。     

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.     

Unusual enthalpy changes which accompany the titration of dimyristoylphosphatidylcholine vesicles with Triton X-100

The enthalpy changes which accompany the titration of 0.1% and 0.25% small unilamellar and multiameller vesicle samples of dimyristoylphosphatidylcholine with 2% Triton X-100 in 0.067 M phosphate buffer (pH 7.4) containing 0.15 M NaCl have been determined by titration calorimetry at 21 degrees C and 28 degrees C, the enthalpy change for both type of vesicles was zero within the limits of experimental error. At 21 degrees C, the multilamellar vesicle samples exhibited an enthalpy change of 1.35 +/- 0.48 and 2.47 +/- 0.98 kcal/mol dimyristoylphosphatidylcholine which was complete at a molar ratio of dimyristoylphosphatidylcholine to Triton of 3.21 +/- 0.84 and 5.77 +/- 1.05 for 0.1% and 0.25% dimyristoylphosphatidylcholine solutions, respectively. An exothermic transition of -2.39 +/- 0.30 and -2.05 +/- 0.69 kcal/mol phospholipid followed by an endothermic transition of 1.37 +/- 0.12 and 1.94 +/- 0.20 kcal/mol dimyristoylphosphatidylcholine was observed at 21 degrees C for 0.1% and 0.25% small unilamellar vesicle samples, respectively. In addition the nearly athermal association of the small unilemellar vesicle samples at 21 degrees C was observed, which may be an appropriate model for biological membrane fusion.     

Identification and characterization of a Triton X-100 replacement for virus inactivation

Triton X-100 detergent treatment is a robust enveloped virus inactivation unit operation included in biopharmaceutical manufacturing processes. However, the European Commission officially placed Triton X-100 on the Annex XIV authorization list in 2017 because a degradation product of Triton X-100, 4-(1,1,3,3-tetramethylbutyl) phenol (also known as 4-tert-octylphenol), is considered to have harmful endocrine disrupting activities. As a result, the use of Triton X-100 in the European Economic Area (EEA) would not be allowed unless an ECHA issued authorization was granted after the sunset date of January 4, 2021. This has prompted biopharmaceutical manufacturers to search for novel, environment-friendly alternative detergents for enveloped virus inactivation. In this study, we report the identification of such a novel detergent, Simulsol SL 11W. Simulsol SL 11W is an undecyl glycoside surfactant produced from glucose and C11 fatty alcohol. We report here that Simulsol SL 11W was able to effectively inactive enveloped viruses, such as xenotropic murine leukemia virus (XMuLV) and pseudorabies virus (PRV). By using XMuLV as a representative enveloped virus, the influence of various parameters on the effectiveness of virus inactivation was evaluated. Virus inactivation by Simulsol SL 11W was effective across different clarified bioreactor harvests at broad concentrations, pH, and temperature ranges. Simulsol SL 11W concentration, temperature of inactivation, and treatment time were identified as critical process parameters for virus inactivation. Removal of Simulsol SL 11W was readily achieved by Protein A chromatography and product quality was not affected by detergent treatment. Taken together, these results have shown the potential of Simulsol SL 11W as a desirable alternative to Triton X-100 for enveloped virus inactivation that could be readily implemented into biopharmaceutical manufacturing processes.     

Spectroscopic properties of protochlorophyll forms in Triton X-100 detergent micelles

Protochlorophyll (PChl) forms were performed in Triton X-100 detergent micelles. The concentration of Triton X-100 was 7·10^?4 M (above the critical micellar concentration); the concentration of PChl varied between 1.6·10^?5 and 1.8·10^?4 M. Absorption, fluorescence and circular dichroism (CD) spectra were registered. The absorption spectra were resolved into Gaussian components by computer analysis. PChl forms with absorption bands at 632–634, 638, 652–654, 663–664, 668 and 676 nm and with fluorescence emission bands at 634–636, 640–644, 652–655, 677–678, 686 and 694–696 nm were observed in micellar solutions of different PChl concentrations. The CD spectra showed a strong dependence on the concentration of PChl: positive CD signals or positive Cotton effects were observed in the vicinity of 650 nm. The intensity of these signals increased in parallel with increasing concentration of PChl. No CD signals were found in the region of the longer wavelength absorption bands. These data show that the PChl exists in many different forms in this system, and the spectroscopic properties of these forms are determined by different molecular interactions viz., interactions of PChl with Triton X-100 or water molecules and/or by the aggregation of PChl.     

Release of remnant plasma membrane from milk fat globules by Triton X-100

The nonionic detergent, Triton X-100, was investigated as an agent for releasing plasma membrane from milk fat globules. The sedimentable material ($\text{50 000 × g}$, 1 h) derived by treating washed goat globules with the detergent (0.2%) was compared to membrane made by the classical globule churning procedure. Characterization included lipid and protein analyses, gel electrophoresis of peptide components, determination of enzymatic activities, and examination with the electron microscope. The results established that the detergent-released material is membrane with similarities to the product by churning. Evaluation of variables revealed that a detergent concentration of 0.1 to 0.2% and reaction temperature of 20–22°C appear optimum with respect to membrane yield when a reaction time of 2 min is employed. At higher detergent concentrations or temperatures removal of phospholipid from the membrane was maximized. Triton X-100 was observed to release membrane from milk fat globules of the goat, human and cow, the latter with a minor procedural modification. The detergent based method is a convenient procedure for obtaining plasma membrane material in good yield for biochemical studies. It also should aid investigations of milk fat globule structure.     

The effect of cholesterol on the solubilization of phosphatidylcholine bilayers by the non-ionic surfactant Triton X-100

Most of the studies on the solubilization of model membranes by Triton X-100 (TR) involve one lipid. The aim of the present study was to evaluate the effect of the addition of cholesterol on the solubilization of bilayers made of palmitoyloleoylphosphatidylcholine (POPC) or dipalmitoylphosphatidylcholine (DPPC). Detailed investigation of the kinetics of solubilization of the cholesterol-containing bilayers by TR at different temperatures reveals that: (i) At 4 degrees C, solubilization of both systems is relatively slow. Hence, in order to prevent misleading conclusions from turbidity measurements it is important to monitor the solubilization after steady-state values of optical density (OD) are reached. (ii) Studies of the temperature-induced changes of the aggregates present in mixtures of TR, POPC and cholesterol indicate that the state of aggregation at all temperatures (including 4 degrees C) represents equilibrium. By contrast, for DPPC/cholesterol/TR mixtures "kinetic traps" may occur not only at 4 degrees C but at higher temperatures as well (e.g. 37 degrees C). (iii) The presence of cholesterol in POPC bilayers makes the bilayers more resistant to solubilization at low temperatures, especially at 4 degrees C. As a consequence, the temperature dependence of the TR concentration required for complete solubilization (Dt(sol)) is no longer a monotonically increasing function (as for POPC bilayers) but a bell-shaped function, with a minimum at about 25 degrees C. Inclusion of cholesterol in DPPC bilayers makes the bilayers more resistant to solubilization at all temperatures except 4 degrees C. In this system, we observe a bell-shaped dependence of Dt(sol) on temperature, with a minimum at 37 degrees C. (iv) Both the rate of vesicle size growth and the rate of the solubilization of POPC vesicles are not affected by the inclusion of cholesterol in the bilayers. Similarly, cholesterol did not affect significantly the rate of size growth of DPPC bilayers at all temperatures, but reduced the rate of solubilization at 4 degrees C.     

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.     

Triton X-100-induced lipoteichoic acid release is correlated with the methicillin resistance in Staphylococcus aureus

We previously reported that Triton X-100 (TRX) reduced methicillin resistance in Staphylococcus aureus, although the degree of reduction varied among strains. One of the biological effects of TRX on S. aureus cells was enhancement of lipoteichoic acid (LTA) release. We investigated the correlation between the amount of LTA released and the degree of reduction in methicillin resistance induced by TRX. The strains showing the greatest reduction of methicillin resistance released the largest amount of LTA, compared to those showing slight or moderate reduction. A mutant whose resistance was not affected by TRX did not increase its release of LTA. These findings suggest that LTA release is associated with a reduction in methicillin resistance in the presence of TRX.     

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.     

Differential roles for Bak in Triton X-100- and deoxycholate-induced apoptosis

We recently reported that Bax activation occurs downstream of caspase activation in Triton X-100 (TX)-induced apoptosis. Here, Bak was found to be activated in TX-induced apoptosis. Although z-VAD-fmk completely suppressed Bax activation, it only partially attenuated TX-induced Bak activation. Moreover, activation of both Bak and Bax was detected in apoptosis induced by deoxycholate, a physiological detergent in bile. z-VAD-fmk completely suppressed deoxycholate-induced Bak as well as Bax activation. Furthermore, Bak siRNA attenuated TX- but not deoxycholate-induced caspase activation. These results suggest that Bak activation may occur upstream of caspase activation in TX- but not deoxycholate-induced apoptosis and that the mechanism of TX-induced apoptosis may differ from that of deoxycholate-induced apoptosis at least with regard to the role for Bak.