细胞膜蛋白质氨基酸组成对自絮凝颗粒酵母耐酒精能力的影响及作用机制

揭示细胞膜组分与酵母菌耐酒精能力的一种新颖关系及其机制。实验显示 ,培养于添加和未添加 3种氨基酸 (异亮氨酸、甲硫氨酸和苯丙氨酸 ,添加浓度分别为 1 0、0 5和 2 0g L)条件下的自絮凝颗粒酵母于 30℃经2 0 % (V V)酒精冲击 9h的存活率分别为 5 7%和 0 ,表明添加该 3种氨基酸能显著提高菌体的耐酒精能力。细胞膜蛋白质氨基酸组成分析和细胞膜流动性测定表明 ,所添加 3种氨基酸是通过组入菌体细胞膜、改变细胞膜流动性从而提高菌体的耐酒精能力的 ,即当细胞膜蛋白质氨基酸组成中异亮氨酸、甲硫氨酸和苯丙氨酸含量明显增加时 ,菌体能有效抵抗高浓度酒精冲击引发的细胞膜流动性的升高 ,从而维持细胞膜的稳定。细胞膜蛋白质氨基酸组成会影响细胞膜的流动性 (膜蛋白中异亮氨酸、甲硫氨酸和苯丙氨酸含量明显增加时膜流动性降低 )是一种新的实验现象。     

细胞膜磷脂脂肪酸组成对自絮凝酵母质膜ATP酶响应酒精刺激的影响及其与菌体耐酒精的关系

研究揭示细胞膜磷脂脂肪酸组成与质膜ATP酶在酵母菌耐酒精中的一种新颖关系。实验表明 ,细胞膜磷脂脂肪酸组成特点对生长于未添加酒精条件下的自絮凝颗粒酵母质膜ATP酶活性没有影响 ,但却明显影响生长于添加酒精 (1 %～ 10 % ,V/V)条件下的菌体质膜ATP酶对酒精激活的敏感性 :预培养于添加 0.6mmol L棕榈酸、亚油酸、或亚麻酸条件下的菌体的质膜ATP酶的最大激活水平分别为各自酶的基态水平 (未激活 )的 3.6、1.5和 1.2倍 ,而对照组 (预培养于未添加脂肪酸条件下的菌体 )的相应值为2.3倍 ,酶产生上述最大激活水平时的酒精浓度分别为 7%、6 %、6 %、和 7% (V/V)。酶激活后米氏常数K_m 、最适pH和对钒酸钠 (质膜ATP酶特异性抑制剂 )的敏感性等性质不变 ,但最大反应速度v_max明显增加。实验表明 ,细胞膜磷脂脂肪酸组成特点对提高菌体的耐酒精能力越有利 ,则其质膜ATP酶被酒精激活的幅度越大 ,说明菌体耐酒精能力的提高与其质膜ATP酶对酒精激活的敏感性的增加密切相关。细胞膜磷脂脂肪酸组成会影响酵母菌质膜ATP酶对酒精激活的敏感性是观察到的新的实验现象.     

外源脂肪酸对酒精诱导自絮凝颗粒酵母细胞膜磷脂脂肪酸组成变化的影响

实验将自絮凝颗粒酵母培养于同时添加脂肪酸 (0.6mmol/L)和酒精 (6 %～ 9% ,V/V)条件下以考察其细胞膜磷脂脂肪酸组成的变化。与单独添加棕榈酸相比 ,同时添加酒精引起细胞膜磷脂棕榈酸含量明显增加 ,伴随 9十四碳烯酸、棕榈油酸和油酸含量明显减少 ;与单独添加亚油酸相比 ,同时添加酒精未引起细胞膜磷脂亚油酸含量明显变化 ,但引起油酸含量明显增加 ,伴随 9 十四碳烯酸、棕榈油酸和棕榈酸含量减少 ;与单独添加亚麻酸相比 ,同时添加酒精引起细胞膜磷脂亚麻酸含量减少 ,伴随油酸含量显著增加 ,同时 9 十四碳烯酸、棕榈油酸和棕榈酸含量减少。存活率实验证实 ,上述变化是菌体对酒精刺激的适应性响应 ,因为 ,与培养于仅添加脂肪酸条件下的菌体相比 ,培养于同时添加酒精条件下的菌体耐酒精能力明显提高。研究表明 ,棕榈酸和油酸都可通过加强细胞膜渗透屏障而提高菌体的耐酒精能力 ,这是饱和脂肪酸 (SFA)与不饱和脂肪酸 (UFA)可提高同一菌株耐酒精能力的新的实验现象 ,揭示UFA与SFA在影响酵母菌耐酒精能力的机制上存在共同的作用方式     

钙离子通过降低细胞膜透性提高自絮凝颗粒酵母耐酒精能力

生长阶段和冲击阶段均添加 1 6 4mmol LCa2 能显著提高自絮凝颗粒酵母于 30℃在 2 0 % (V V)酒精冲击下的存活率 ,经过 9h冲击 ,对照组的存活率为 0 ,而添加Ca2 试验组的存活率为 5 0 0 % ,表明添加适当浓度的Ca2 能显著提高菌体的耐酒精能力。通过考察Ca2 对菌体于 30℃在 15 % (V V)酒精冲击下细胞膜透性的影响发现 ,生长阶段和冲击阶段均添加 1 6 4mmol LCa2 的试验组的菌体胞外核苷酸平衡浓度和细胞膜透性系数 (P′)分别仅为对照组水平的 5 0 0 %和 2 9 3% ,表明添加适当浓度的Ca2 能显著降低受冲击菌体的细胞膜透性 ;而且 ,添加Ca2 提高存活率与添加Ca2 降低胞外核苷酸浓度和P′存在直接的对应关系。因此 ,Ca2 提高自絮凝颗粒酵母耐酒精能力是与其降低受冲击菌体的细胞膜透性密切相关的。     

细胞膜磷脂棕榈酸含量对自絮凝颗粒酵母耐酒精能力的影响及作用机制

研究揭示细胞膜磷脂脂肪酸组成与酵母菌耐酒精能力的一种新颖关系及其机制。分别培养于添加 0 6mmol L棕榈酸、亚油酸或亚麻酸不同条件下的自絮凝颗粒酵母 ,其细胞膜富含各自所添加的脂肪酸。细胞膜富含棕榈酸、亚油酸或亚麻酸的三种菌体于 30℃经 2 0 %(v v)酒精冲击 6h的存活率分别为 5 2 %、1 8%和 0。通过考察三种菌体于 30℃在 1 5 %(v v)酒精冲击下的细胞膜透性发现 ,细胞膜富含棕榈酸的菌体的胞外核苷酸平衡浓度分别仅为细胞膜富含亚油酸或亚麻酸菌体的 48%和 32 %,其细胞膜透性系数 (P′)分别仅为后者的 37%和 2 0 %,且三者的胞外核苷酸浓度和P′由小到大的排列顺序均与它们的存活率由高到低的排列顺序完全一致。因此 ,细胞膜富含棕榈酸的菌体具有较强的耐酒精能力是与其在高浓度酒精冲击下可维持较低的细胞膜透性密切相关的 的。     

细胞膜磷脂脂肪酸组成对自絮凝酵母质膜ATP酶响应酒精刺激的影响及其与菌体耐酒精的关系

研究揭示细胞膜磷脂脂肪酸组成与质膜ATP酶在酵母菌耐酒精中的一种新颖关系。实验表明,细胞膜磷脂脂肪酸组成特点对生长于未添加酒精条件下的自絮凝颗粒酵母质膜ATP酶活性没有影响,但却明显影响生长于添加酒精(1％～10％,V／V)条件下的菌体质膜ATP酶对酒精激活的敏感性：预培养于添加0．6mmol／L棕榈酸、亚油酸、或亚麻酸条件下的菌体的质膜ATP酶的最大激活水平分别为各自酶的基态水平(未激活)的3．6、1．5和1．2倍,而对照组(预培养于未添加脂肪酸条件下的菌体)的相应值为2．3倍,酶产生上述最大激活水平时的酒精浓度分别为7％、6％、6％、和7％(V/V)。酶激活后米氏常数Km、最适pH和对钒酸钠(质膜ATP酶特异性抑制剂)的敏感性等性质不变,但最大反应速度υmax明显增加。实验表明,细胞膜磷脂脂肪酸组成特点对提高菌体的耐酒精能力越有利,则其质膜ATP酶被酒精激活的幅度越大,说明菌体耐酒精能力的提高与其质膜ATP酶对酒精激活的敏感性的增加密切相关。细胞膜磷脂脂肪酸组成会影响酵母菌质膜ATP酶对酒精激活的敏感性是观察到的新的实验现象。     

絮凝特性对自絮凝颗粒酵母耐酒精能力的影响及作用机制

首次报道絮凝特性提高酵母菌耐酒精能力的现象及其机制。融合株SPSC与其两亲本粟酒裂殖酵母变异株和酿酒酵母变异株于 30℃经 18% (V/V)酒精冲击 7h的存活率分别为 52%、37%和 9%。细胞膜磷脂脂肪酸组成分析表明 ,两絮凝酵母 (融合株SPSC和粟酒裂殖酵母变异株 )的棕榈酸含量均约为非絮凝酵母 (酿酒酵母变异株 )的两倍 ,而棕榈油酸和油酸的含量明显低于后者。研究表明 ,当两絮凝酵母在培养中由于柠檬酸钠的作用 (抑制絮凝体的形成 )而以游离细胞生长存在时 ,其细胞膜磷脂棕榈酸含量显著下降 ,而棕榈油酸和油酸的含量明显增加 ,结果细胞膜磷脂脂肪酸组成特点与酿酒酵母变异株相似 ;而且实验表明 ,絮凝特性的消失伴随菌体耐酒精能力的急剧下降 ,变得与酿酒酵母变异株的水平相当。这些结果提示两絮凝酵母具有较强的耐酒精能力与其细胞膜磷脂脂肪酸组成中含有更高比例的棕榈酸有关。     

酵母属间原生质体融合改进菌株木糖发酵性能

通过单倍体分离和紫外诱变,获得了１４株树干毕赤酵母（Ｐｉｃｈｉａｓｔｉｐｉｔｉｓ）７１２４和酿酒酵母（Ｓａｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ）１３００的营养缺陷型突变株。用聚乙二醇（ＰＥＧ）和电诱导融合及致死融合等方法,实现了树干毕赤酵母和酿酒酵母的属间原生质体融合。融合子能发酵木糖产生酒精,其厌氧发酵木糖和木糖葡萄糖混合液的能力明显优于亲株,耐酒精的性能也比亲株树干毕赤酵母７１２４有所提高。融合子经ＤＮＡ含量、细胞体积测定和稳定性能实验证明为稳定融合子。     

酿酒酵母X330高浓度发酵时耐酒精性能的初步研究

在完全合成培养基条件下,就渗透压保护剂和营养物质对一株产高浓度酒精的酿酒酵母X330高浓度发酵时耐酒精性能的影响进行了初步研究。结果表明,与渗透压相比,营养缺乏对酿酒酵母高浓度发酵时酒精耐受性能可能起着更为关键和重要的作用。发酵培养基中各营养元素对耐酒精性能的影响不同,由高到低的顺序是酵母抽提物>蛋白胨>硫酸镁>维生素C=磷酸二氢钾>氯化钙=硫酸铵。渗透压保护剂(甘氨酸和脯氨酸)能有效提高菌体酒精耐受性能。当甘氨酸添加浓度为20mmol/L或脯氨酸添加浓度为10mmol/L时,发酵终点酒精浓度最高,菌体于30℃在18%(V/V)酒精冲击下的存活率最大,且均高于对照组(未添加甘氨酸且未添加脯氨酸)水平,但甘氨酸的促进作用强于脯氨酸。     

动脉粥样硬化中单核细胞膜流动性研究

为研究动脉粥样硬化中单核细胞膜流动性的变化,本实验选用20只新西兰白兔建立动物粥样硬化模型,提取模型组与对照组兔外周血中单核细胞,通过荧光漂白恢复技术检测单核细胞膜流动性,并结合动脉粥样硬化动物模型病理切片揭示其与动脉粥样硬化相关性。结果显示动物动脉粥样硬化模型建立成功,模型组单核细胞膜的荧光恢复率和扩散系数均低于对照组。本研究揭示了单核细胞细胞膜的流动性与动脉粥样硬化的发生有关,为今后深入探究单核细胞与动脉粥样硬化关系提供实验基础。     

Ethanol-induced changes in the fatty acid composition of Lactobacillus hilgardii, its effects on plasma membrane fluidity and relationship with ethanol tolerance

The effect of environmental ethanol concentration on the fatty acid composition of strains of Lactobacillus hilgardii, differing in their tolerance to ethanol, was determined. A marked increase in the proportion of lactobacillic acid (a cyclopropane fatty acid) and a decrease in oleic and vaccenic acids with increasing ethanol concentration was observed. The amount of lactobacillic acid determined at standard conditions (25°C, 0% ethanol) was found to be proportional to the ethanol tolerance of the strains studied. The effect of this alcohol on plasma membrane fluidity was studied by differential scanning calorimetry. The adaptive response to growth in the presence of high concentrations of ethanol produced membranes which, within the limits of ethanol tolerance, maintained the fluidity and integrity in an environment which tends to increase membrane rigidity. When pre-adapted cells are analysed in the absence of environmental ethanol there is a measurabie increase in fluidity. It is proposed that this phenomenon may be correlated with the increase in the proportion of lactobacillic acid. The existence of a relationship between membrane fluidity and ethanol tolerance is discussed.     

Enhancing effect of albumin hydrolysate on ethanol production employing Saccharomyces sake

The enhancing effect of albumin hydrolysate on ethanol production was investigated in ethanol fermentations using Saccharomyces sake. In batchwise ethanol production, addition of supplemental albumin hydrolysate and phosphatidylcholine, or albumin hydrolysate alone, brought about a more than 60% increase in final ethanol concentration (148 or 144 g/L compared with 88 g/L with no supplementation [control] after 72 h). The effect of the supplements is believed to be due to an enhanced alcohol tolerance of cells grown in media containing the supplements. Cells grown in media containing albumin hydrolysate were enriched in phenyalanine, tyrosine, and methionine in their plasma membranes. All three amino acids were also present in considerable amounts in the albumin hydrolysate. This fact suggests that the three amino acids, which are present in albumin hydrolysate, are incorporated into the plasma membranes of cells. Under ethanol production conditions in which only one amino acid among the components of albumin hydrolysate was excluded, namely phenlalanine, tyrosine, or methionine, significant reductions in ethanol production resulted. (c) 1995 John Wiley & Sons, Inc.     

Relationship between ethanol tolerance, lipid composition and plasma membrane fluidity in Saccharomyces cerevisiae and Kloeckera apiculata

Abstract The lipid composition of a strain of each of two yeasts, Saccharomyces csrevisiae and Kloeckera apiculata , with different ethanol tolerances, was determined for cells grown with or without added ethanol. An increase in the proportion of ergosterol, unsaturated fatty acid levels and the maintenance of phospholipid biosynthesis seemed to be responsible for ethanol tolerance. The association of ethanol tolerance of yeast cells with plasma membrane fluidity, measured by fluorescence anisotropy, is discussed. We propose that an increase in plasma membrane fluidity may be correlated with a decrease in the sterol: phospholipid and sterol: protein ratios and an increase in unsaturation index.     

Membrane fluidity adjustments in ethanol-stressed Oenococcus oeni cells

The effect of ethanol on the cytoplasmic membrane of Oenococcus oeni cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells. To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy. Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface. Fluidity increased with increasing concentrations of ethanol. Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity. Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol. Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage. This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved. We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested. We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface.     

Effects of ethanol on the Escherichia coli plasma membrane.

The effects of ethanol on the fluidity of Escherichia coli plasma membranes were examined by using a variety of fluorescent probes: 1,6-diphenyl-1,3,5-hexatriene, perylene, and a set of n-(9-anthroyloxy) fatty acids. The anthroyloxy fatty acid probes were used to examine the fluidity gradient across the width of the plasma membrane and artificial membranes prepared from lipid extracts of plasma membranes. Ethanol caused a small decrease in the polarization of probes primarily located near the membrane surface. In comparison, hexanol decreased the polarization of probes located more deeply in the membrane. Temperature had a large effect on probes located at all depths. The effects of ethanol on E. coli membranes from cells grown with or without ethanol were also examined. Plasma membranes isolated from cells grown in the presence of ethanol were more rigid than those from control cells. In contrast to plasma membranes, artificial membranes prepared from lipid extracts of ethanol-grown cells were more fluid than those from control cells. These differences are explained by analyses of membrane composition. Membranes from cells grown in the presence of ethanol are more rigid than those from control cells due to a decrease in the lipid-to-protein ratio. This change more than compensates for the fluidizing effect of ethanol and the ethanol-induced increase in membrane C18:1 fatty acid which occurs during growth. Our results suggest that the regulation of the lipid-to-protein ratio of the plasma membrane may be an important adaptive response of E. coli to growth in the presence of ethanol.     

Membrane Fluidity Adjustments in Ethanol-Stressed Oenococcus oeni Cells

The effect of ethanol on the cytoplasmic membrane of Oenococcus oeni cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells. To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy. Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface. Fluidity increased with increasing concentrations of ethanol. Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity. Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol. Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage. This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved. We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested. We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface.     

Chronic and acute ethanol treatment modifies fluidity and composition in plasma membranes of a human hepaic cell line (WRL-68)

The aim of this study was to compare the effects of chronic (0.1 mol/L ethanol exposure during 30 days) and acute (0.5 mol/L ethanol exposure during 24 h) ethanol treatment on the physical properties and the lipid composition of plasma membranes of the WRL-68 cells (fetal human hepatic cell line). Using fluorescence polarization we found that ethanol treatment reduced membrane anisotropy due to disorganization of acyl chains in plasma membranes and consequently increased fluidity, as measured with the diphenylhexatriene probe. Addition of ethanolin vitro reduced anisotropy in control plasma membranes, whereas chronically ethanol-treated plasma membranes were relatively tolerant to thein vitro addition of ethanol. Acutely ethanol-treated plasma membranes exhibited a smaller anisotropy parameter value than control plasma membranes. We found a decrease in total phospholipid content in acute ethanol WRL-68 plasma membranes. Cholesterol content was increased in both ethanol treatments, and we also found a significant decrease in phosphatidylinositol and phosphatidylcholine and an increase in phosphatidylethanolamine content in ethanol-treated plasma membranes. Our data showed that ethanol treatment decreased the anisotropy parameter consistently with increased fluidity, while increasing the cholesterol/phospholipid ratio of plasma membranes of WRL-68 cells, but only chronically ethanol-treated plasma membranes exhibited tolerance to thein vitro addition of ethanol. It is important to note that some changes that were interpreted as a result of chronic ethanol treatment were also present in short-period ethanol treatments.Abbreviations DPH diphenylhexatriene - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SPH sphingomyelin