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     

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。     

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。     

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。     

膜蛋白氨基酸组成通过改变膜流动性影响粟酒裂殖酵母和酿酒酵母融合株耐酒精能力

实验显示,一种氨基酸混合液（含异亮氨酸、甲硫氨酸和苯丙氨酸,添加浓度分别为1.0、0.5和2.0g/L）能显著提高自絮凝酵母——粟酒裂殖酵母和酿酒酵母融合株SPSC的耐酒精能力。实验将菌体分别培养于添加（试验组）和未添加（对照组）该氨基酸混合液的条件下,然后收集菌体进行酒精（20%,V/V）冲击试验（30℃,9h）,结果,试验组的菌体尚有一半以上的存活细胞,而对照组的菌体全部死亡。通过对试验组和对照组的菌体细胞膜蛋白质氨基酸组成分析发现,试验组的菌体耐酒精能力提高与所添加氨基酸组入菌体的细胞膜密切相关。以DPH为荧光探针的细胞膜流动性测定分析进一步揭示,氨基酸组入菌体的细胞膜后,细胞膜能有效抵抗高浓度酒精冲击诱发的膜流动性的提高,从而维持膜的稳定。因此,实验首次揭示膜蛋白氨基酸组成可通过改变膜流动性而影响酵母菌的耐酒精能力。     

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。     

发酵戊糖产酒精酵母菌株的选育

介绍了一种新的发酵戊糖产酒酵母菌种筛选方法并利用该方法筛选出一株性能优良的发酵戊糖酵母蓖株Z8,该菌株性能测试结果为：产酒精能力相当于发酵戊糖理论产量的60．0％,耐酒精能力14％（V／V）,在温度高达42℃的条件下仍能正常发酵,初步鉴定该菌株为管囊酵母（Pachysolen tannophilus)。     

酵母菌耐酒精机制的研究进展

乙醇是酵母菌发酵糖的重要产物之一．但是当乙醇在培养基中用积到一定浓度时,对酵母菌细胞产生有毒效应．然而,不同的酵母菌菌株对一定浓度的乙醇有不同的抗性,而且在不同培养条件下和生长在不同的培养基中同一株酵母菌对一定浓度的乙醇也有不同的抗性．最近几年来,有的学者从自然界中分离到了或通过遗传工程手段构建了一些能在短时间内产生高浓度酒精（发酵液中的乙醇浓度达到１７．５％ｖ／ｖ以上,而普通酵母菌只能产生９％一！！％ＶＩＶ乙醇）的酵母菌Ｉ‘］．因此酵母苗耐酒精的生化机制引起了许多研究者的浓厚兴趣,因为研究酵母…     

In vivo activation by ethanol of plasma membrane ATPase of Saccharomyces cerevisiae.

Ethanol, in concentrations that affect growth and fermentation rates (3 to 10% [vol/vol]), activated in vivo the plasma membrane ATPase of Saccharomyces cerevisiae. The maximal value for this activated enzyme in cells grown with 6 to 8% (vol/vol) ethanol was three times higher than the basal level (in cells grown in the absence of ethanol). The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the activated and the basal plasma membrane ATPases were virtually identical. A near-equivalent activation was also observed when cells grown in the absence of ethanol were incubated for 15 min in the growth medium with ethanol. The activated state was preserved after the extraction from the cells of the membrane fraction, and cycloheximide appeared to prevent this in vivo activation. After ethanol removal, the rapid in vivo reversion of ATPase activation was observed. While inducing the in vivo activation of plasma membrane ATPase, concentrations of ethanol equal to and greater than 3% (vol/vol) also inhibited this enzyme in vitro. The possible role of the in vivo activation of the plasma membrane proton-pumping ATPase in the development of ethanol tolerance by this fermenting yeast was discussed.     

In vivo activation by ethanol of plasma membrane ATPase of Saccharomyces cerevisiae.

Ethanol, in concentrations that affect growth and fermentation rates (3 to 10% [vol/vol]), activated in vivo the plasma membrane ATPase of Saccharomyces cerevisiae. The maximal value for this activated enzyme in cells grown with 6 to 8% (vol/vol) ethanol was three times higher than the basal level (in cells grown in the absence of ethanol). The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the activated and the basal plasma membrane ATPases were virtually identical. A near-equivalent activation was also observed when cells grown in the absence of ethanol were incubated for 15 min in the growth medium with ethanol. The activated state was preserved after the extraction from the cells of the membrane fraction, and cycloheximide appeared to prevent this in vivo activation. After ethanol removal, the rapid in vivo reversion of ATPase activation was observed. While inducing the in vivo activation of plasma membrane ATPase, concentrations of ethanol equal to and greater than 3% (vol/vol) also inhibited this enzyme in vitro. The possible role of the in vivo activation of the plasma membrane proton-pumping ATPase in the development of ethanol tolerance by this fermenting yeast was discussed.     

The Role of Phospholipids in Plasma Membrane ATPase Activity in Vigna radiata L. (Mung Bean) Roots and Hypocotyls

Root and hypocotyl plasma membrane H⁺-ATPases were partially purified from deoxycholate-solubilized fractions of microsomes in mung bean (Vigna radiata L.) plants in the presence of glycerol. Certain properties of the ATPases and the manner in which phospholipids affect their activity were compared. Root ATPase was similar to hypocotyl ATPase with respect to substrate specificity, salt stimulation, pH dependence, K_m for ATP·Mg²⁺ and inhibitor sensitivity, except for inhibition by vanadate. Both purified ATPases required phospholipids for their activation. Optimum concentrations of exogenously added phospholipid mixture (asolectin) to hypocotyl and root ATPase mixture were 0.03% and 1.0%, respectively. Root ATPase activation did not decrease if more than 1.0% asolectin was added. Qualitatively, phosphatidylserine and phosphatidylcholine brought about greater ATPase activation than other phospholipids. The hypocotyl ATPase was activated by phosphatidylinositol, phosphatidylserine and phosphatidylglycerol to a greater extent than the root ATPase. Root, but not hypocotyl ATPase, was slightly inhibited by the addition of phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid. The hypocotyl plasma membrane contained phosphatidylinositol + phosphatidylserine, phosphatidylglycerol and phosphatidic acid, and unsaturated fatty acids in greater abundance than the root plasma membrane. The differential activation of the plasma membrane ATPases may arise from these differences.     

Activation of plasma membrane ATPase of Saccharomyces cerevisiae by octanoic acid.

Plasma membrane ATPase activity of Saccharomyces cerevisiae IGC 3507III grown in the presence of the lipophilic acid octanoic acid [4-50 mg l-1 (0.03-0.35 mM), pH 4.0] was 1.5-fold higher than that in cells grown in its absence. The Km for ATP, the pH profile and the sensitivity to orthovanadate of the basal and the activated forms of the membrane ATPase were identical. This activation was closely associated with a decrease in the biomass yield and an increase in the ethanol yield, and was rapidly reversed in vivo after removal of the acid. However, the activated level was preserved when membranes were extracted and subjected to manipulations which eliminated or decreased octanoic acid incorporation in the plasma membrane. The activity of the basal plasma membrane ATPase in the total membrane fraction was slightly increased by incubation at pH 6.5 with octanoic acid at 100 mg l-1 or less (2.4 mg acid form plus 97.6 mg octanoate ion l-1). However, destruction of the permeability barrier between the enzyme and its substrate could not explain the in vivo activation. A role for plasma membrane ATPase activation in the regulation of the intracellular pH (pHi) of cells grown with octanoic acid was not proven.     

Mechanism for the Activation of Plasma Membrane H-ATPase from Rice (Oryza sativa L.) Culture Cells by Molecular Species of a Phospholipid

The activation of H⁺-ATPase solubilized from plasma membrane of rice (Oryza sativa L. var Nipponbare) culture cells was examined by the exogenous addition of various phospholipids, free fatty acids, glycerides, polar head groups of phospholipids and molecular species of phosphatidylcholine (PC). H⁺-ATPase activity appeared to be stimulated by phospholipids in the following order: asolectin > phosphatidylserine > PC > lysophosphatidylcholine > phosphatidylglycerol, and maximal ATPase activation was noted at around 0.05 to 0.03% (w/v) of asolectin or molecular species of PC. Polar head groups such as glycerol, inositol, and serine only slightly activated ATPase activity or not at all, while ethanolamine and choline had no effect. Activation was dependent on the degree of saturation or unsaturation of the fatty acyl chain and its length. The activity decreased with increase in the length of fatty acyl chain from dimyristoryl(14:0)-PC to distearoyl(18:0)-PC and the degree of unsaturation from dioleoyl(18:1)-PC to dilinolenoyl(18:3)-PC. Maximum activation was observed when PC possessing 1-myristoyl(14:0)-2-oleoyl(18:1) or 1-oleoyl-2-myristoyl was added to the reaction mixture. These data show that the activation of plasma membrane H⁺-ATPase by PC depends on a combination of saturated (myristic acid 14:0, palmitic acid 16:0, and stearic acid 18:0) and unsaturated (oleic acid 18:1, linoleic acid 18:2, and arachidonic acid 20:4) fatty acids at the sn-1 and sn-2 positions of the triglycerides.     

Stimulation of guanylate cyclase activity by several fatty acids.

Guanylate cyclase of plasma membrane of isolated rat fat cells was activated 7 to 11 fold by oleic acid, linoleic acid, linolenic acid or arachidonic acid. The activation of the enzyme by linoleic acid or oleic acid was influenced by the concentration of enzyme protein and that of the fatty acid. At 158 μg/ml of enzyme protein, 0.6 mM linoleic acid produced maximal activation of 12 fold which was partially reversed by washing. Particulate guanylate cyclase of cerebral cortex and liver was also activated by linoleic acid.     

Inhibition of Tonoplast and Plasma Membrane H$-ATPase Activity in Rice (Oryza sativa L.) Culture Cells by Local Anesthetics

Tonoplast and plasma membrane vesicles were prepared from rice(Oryza sativa L. var. Yuukara) culture cells with step sucrosegradient (30% and 42.9%, w/v) and/or step dextran T-70 gradient(1% and 8%, w/w) to determine the inhibition of tonoplast andplasma membrane AT-Pases by local anesthetics. The degree towhich the anesthetics inhibited these ATPases was of the followingorder: dibucaine>lidocainetetracaine>procaineGABA. Dibucaineranging in concentration from 0.2 nui to 2 mM inhibited tonoplastATPase activity more than plasma membrane ATPase, the half inhibitionsbeing 0.8 and 1.1 mM, respectively. The K_m values of tonoplastand plasma membrane ATPases were not affected by dibucaine,but various values were noted for V_max. Dibucaine inhibitedtonoplast and plasma membrane ATPases solubilized from 0.1%DOC pellet by n-octylglucoside and zwittergent 3–14, respectively.The addition of a phospholipid mixture (asolectin) to solubilizedboth ATPases had no effect on the inhibition by dibucaine. Thus,local anesthetics may act directly on the ATPase moiety withoutlipid mediation. (Received June 15, 1987; Accepted November 13, 1987)     

Comparison of Plasma Membrane and Tonoplast H+-Translocating ATPases in Phaseolus mungo L. Roots

Two membrane fractions were obtained from 16%/26% and 34%/40%interfaces following discontinuous sucrose density gradientcentrifugation of a 10,000–80,000xg pellet from mung bean(Phaseolus mungo L.) roots. The ATPases in the fractions differedfrom each other in their sensitivity toward various inhibitors,activation with salts, dependence of activity on pH, and K_mfor ATP.Mg²⁺. Judging from their sensitivity toward inhibitors,the ATPases in the low and high density membranes are consideredmainly of tonoplast and plasma membrane origin, respectively.Both ATPases were activated by gramicidin D and nigericin. ATP-inducedquenching of quinacrine fluorescence in both fractions requiredMg²⁺ and permeant anions such as Cl^– and quenching wascollapsed by carbonylcyanide p-trifluoromethoxyphenyl hydrazone.The sensitivities of quenching to the inhibitors were essentiallythe same as those of ATPase activity in the membranes. Thesefindings suggest the involvement of ATPases in H⁺-pumping acrossa plasma membrane and tonoplast. (Received April 12, 1985; Accepted October 11, 1985)     

Ethanol Administration in the Rat Decreases Prostacyclin Production by Isolated Brain Microvessels

The effects of short- and long-term ethanol administration to rats on basal levels and formation of prostacyclin (PGI2) measured as 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and on lipid class content and fatty acid composition of isolated brain microvessels (BMV) were studied. After acute treatment (2 h, at the peak of plasma ethanol concentration) basal 6-keto-PGF1 alpha levels in BMV and release on incubation were reduced to 50% of control values. After chronic administration (15 days), PGI2 release was reduced to about 40% of control values, without changes in basal levels. Total lipid, phospholipid, and cholesterol levels in BMV, measured after prolonged administration of alcohol, were not modified. Also, only minor changes in the fatty acid composition of individual phospholipid classes were detected. The observed reduction of PGI2 synthesis in BMV thus could not be related to changes of the fatty acid precursor pool in the preparation. Precursor release and/or the biosynthetic pathways may be affected by ethanol administration.     

质膜钙离子ATP酶

钙离子(Ca2+)是重要的第二信使,通过与效应蛋白的结合和解离,以及在不同细胞器之间的穿梭运动而精确调控细胞活动,参与多种重要生命过程。细胞内具有精确调节Ca2+时空分布的调控系统。在静息状态下,细胞内的游离Ca2+浓度约为100 nmol/L;而当细胞受到信号刺激后,胞内的Ca2+浓度可上升至1000 nmol/L甚至更高。细胞中存在多种跨膜运送Ca2+的膜蛋白,以精确调节Ca2+浓度的时空动态变化,其中,细胞质膜上的多种Ca2+通道(包括电压门控通道、受体门控通道、储存控制通道等),以及内质网/肌质网和线粒体等胞内"钙库"膜上的雷诺丁受体、三磷酸肌醇受体等膜蛋白复合物,均可提升胞内Ca2+浓度,而细胞质膜上的钠钙交换体、质膜Ca2+-ATP酶、"钙库"膜上的内质网Ca2+-ATP酶、线粒体Ca2+单向转运体等,可将Ca2+浓度降低至静息态水平。质膜钙ATP酶是向细胞外运送Ca2+的关键膜蛋白,本文将对其结构、功能及其酶活性的调控机制做一简要综述。