生物膜组分对膜功能和膜脂相变的调控 Ⅰ.吐温表面活性剂对玉米根端线粒体ATP酶活力的影响

膜脂流动性和膜结合酶的关系极为密切,同时膜脂组分也明显地制约着膜的流动性。本试验选用了三种不同脂肪酸碳链的吐温表面活性剂(Tween 20,Tween40,Tween 80)作为线粒体的添加物,观察对玉米根端线粒体ATP酶活力的调节作用。结果看到玉米根端线粒体ATP酶在8～32℃范围内活化能在15.5℃处出现了一个折点。添加吐温20、40、80后在8～32℃范围内都出现了二个活化能的折点,分别为26.5℃和12.3℃;25.4℃和15.3℃;22.8℃和12.8℃。清洗吐温后ATP酶活化能的转折点温度与添加吐温时没有明显差异,表明吐温的脂肪酸链可能与线粒体膜相结合,因而改变了线粒体ATP酶的活化能的折点温度。     

生物膜组分对膜功能和膜脂相变的调控——Ⅰ.吐温表面活性剂对玉米根端线粒体ATP酶活力的影响

膜脂流动性和膜结合酶的关系极为密切,同时膜脂组分也明显地制约着膜的流动性。本试验选用了三种不同脂肪酸碳链的吐温表面活性剂(Tween 20,Tween40,Tween 80)作为线粒体的添加物,观察对玉米根端线粒体ATP 酶活力的调节作用。结果看到玉米根端线粒体ATP酶在8～32℃范围内活化能在15.5℃处出现了一个折点。添加吐温20、40、80后在8～32℃范围内都出现了二个活化能的折点,分别为26.5℃和12.3℃;25.4℃和15.3℃;22.8℃和12.8℃。清洗吐温后ATP 酶活化能的转折点温度与添加吐温时没有明显差异,表明吐温的脂肪酸链可能与线粒体膜相结合,因而改变了线粒体ATP 酶的活化能的折点温度。     

外源胆固醇对水稻根端线粒体ATP酶活力的影响

外源胆固醇无论是通过根系吸收或是直接与离体线粒体一起温育的方式,在试验浓度范围内均能提高水稻根端线粒体ATP酶的活力,同时观察到外源胆固醇能明显降低ATP酶表现活化能(Apparent activation energy,AEa)在Arrhenius图上的折点温度。其中通过根系吸收进入线粒体膜内后,其线粒体ATP酶AEa的两个折点温度由对照的27.7℃和15.5℃分别降低到24.5℃和12.7℃;直接与离体线粒体一起温育的两个折点温度分别降低到18.8℃和9.6℃。试验结果证明,适量的外源胆固醇不仅对水稻根端线粒体ATP酶活力具有明显的促进作用,而且对降低线粒体膜脂的相变温度也有明显的调节作用。     

生物膜组分对膜功能和膜脂相变的调控——Ⅲ.磷脂脂质体对玉米根端线粒体ATP酶活力的影响

大豆粗磷脂经硅酸柱层析分离得到端基脂肪酸链相似的PC和PE+PG两种混合物。经超声制备成PC脂质体和PE+PG脂质体,添加于玉米根端线粒体,线粒体ATP酶活化能的折点温度由15.5℃分别降低为13℃和10.7℃,表明脂质体能与线粒体膜相结合,磷脂极性头基对ATP酶活化能的折点温度具有明显的作用。     

毛白杨幼苗低温锻炼过程中Ca2+ 的作用及细胞Ca2+-ATP酶活性的变化

观察了低温锻炼以及随后的常温生长中毛白杨幼苗质膜及线粒体Ca2 ATP酶活性、CaM含量和抗冻性的变化。结果表明 ,单纯低温锻炼在一定程度上提高了毛白杨幼苗质膜及线粒体Ca2 ATP酶活性、CaM含量和抗冻性 ,减小了低温胁迫所引起的质膜及线粒体Ca2 ATP酶活性和CaM含量的下降程度 ,促进了胁迫后恢复过程中质膜及线粒体Ca2 ATP酶活性和CaM水平的迅速回升。在低温锻炼的同时 ,用CaCl2 处理能加强低温锻炼的效果 ,但这种效应可被EGTA、LaCl3和CPZ处理所抑制     

生物膜组分对膜功能和膜相变的调控 Ⅱ、膜脂组分对水稻线粒体α-酮戊二酸氧化酶活力的影响

以四种抗冷性不同的水稻芽鞘为材料,分析了它们的线粒体膜脂脂肪酸成分和含量、线粒体α-酮戊二酸氧化酶活力,并在线粒体上添加含油酸酯的吐温80和清洗吐温80之后测定了α-酮戊二酸氧化酶活力的变化。 四种抗冷性不同的水稻种子,其干胚膜脂脂肪酸成分相同,但是它们的脂肪酸不饱和指数(IUFA)有明显差异,这种差异与品种抗冷性成正相关。品种间芽鞘线粒体膜脂脂肪酸成分相同,它们的脂肪酸不饱和指数也有明显差异,与品种抗冷性也成正相关。四个水稻品种的芽鞘线粒体α-酮戊二酸氧化酶活力在10～42℃间存在着两个温度折点,其中低温折点可能与品种抗冷性有关。秈稻“二九青”芽鞘线粒体添加吐温80和清洗吐温80后,线粒体α-酮戊二酸氧化酶活力的温度折点均比对照线粒体低。证明增加膜脂中不饱和脂肪酸能降低膜结合酶活力的温度折点,膜脂脂肪酸不饱和度与膜结合酶活力和水稻抗冷性密切相关。     

生物膜能量偶联ATP酶的比较研究——Ⅰ.鼠肝线粒体内膜与ATP酶(F_1)的人工重组及ANS萤光探针与ATP酶的结合

本文报导了大鼠肝线粒体内膜ATP酶的析离和重组,以及膜对ATP酶的结构和功能的影响。用(1)胰蛋白酶-尿素、(2)硅钨酸盐和(3)枯草杆菌蛋白酶三种方法分别制备的去ATP酶(F_1)的线粒体内膜与可溶性的F_1重组后,完全恢复或部分恢复到天然线粒体内膜的ATP酶活力水平。寡霉素敏感性测定、ANS结合的发射萤光光谱测定、电镜负染标本观察和低温处理试验等都一致证明,ATP酶与膜重组后表现了一系列与天然膜相似的性质。ANS萤光探针与线粒体内膜结合的萤光增强效应主要在于ANS与ATP酶(F_1)的结合并与F_1的分子构象有密切关系。经胰蛋白酶-尿素处理去掉F_1的线粒体内膜基本上丧失了ANS的萤光增强效应。可溶性F_1经0℃处理2小时后,丧失酶水解活力的84%和ANS萤光增强效应的96.4%。F_1与膜结合后,则表现了对0℃低温的稳定性。结果提示,ANS可能与ATP酶分子的疏水微区相结合;ATP酶分子疏水结构的存在对于表现酶的水解活力和ANS的萤光增强效应是必要的条件;低温处理破坏了酶分子内的疏水结构;膜与ATP酶结合则有稳定酶分子的疏水结构和分子构象的作用。     

线粒体ATP合酶抑制因子1在线粒体稳态中的调节作用

线粒体作为细胞的重要能量来源,其数量、质量及功能的稳定对维持细胞的正常活动至关重要,且其稳态的调节依赖于线粒体质量控制系统(包括线粒体自噬、线粒体融合/分裂及线粒体生物合成等)。线粒体蛋白ATP合酶抑制因子1(ATP synthase inhibitor 1, IF1)是线粒体基质中抑制F1FoATP酶/合酶活性的天然小分子蛋白质。在细胞缺氧缺血等特殊生理情况下, IF1通过改变自身的聚合状态,抑制F1FoATP酶水解ATP的活性,从而抑制细胞内的ATP被过度水解。最近的研究证实, IF1的抑制作用是双向的,其即可抑制F1FoATP酶活性,又可抑制F1FoATP合酶活性。因此, IF1可通过靶向F1FoATP酶/合酶活性及相关信号通路,参与调节线粒体质量,维持线粒体稳态。该文综述IF1在线粒体质量控制中的相关调节机制,包括IF1维持线粒体氧化还原平衡、IF1介导线粒体自噬、IF1促进线粒体融合/分裂三条通路,以及三者之间相互作用的关系,为探索IF1在相关疾病的发生、发展及治疗中的作用提供理论参考。     

生物膜能量偶联ATP酶的比较研究——Ⅱ.鼠肝线粒体ATP酶(F_1)与人原发性肝癌线粒体内膜的杂交重组及其对肝癌发生的遗传控制的初步分析

1974到1975年,我们用人原发性肝癌细胞的线粒体内膜进行ATP酶活力测定,结果证明人肝癌线粒体ATP酶活力极低(0.04～0.1微克分子/分/毫克蛋白)只相当于正常大鼠线粒体的1/10～1/25(0.49～1.07微克分子/分/毫克蛋白)。Walker肉瘤和人肝硬变组织的线粒体与人肝癌的酶活力相近。电镜负染标本观察证明肝癌线粒体内膜大部分失去特征性的直径为90(?)的ATP酶颗粒,表现为光滑膜。ANS萤光探针的发射萤光光谱测定和2,4-二硝基酚的激活试验均证明人肝癌细胞线粒体内膜的ATP酶大量消失是肝癌细胞的特征之一。用提取的大鼠肝线粒体ATP酶(F_1)与人肝癌线粒体内膜进行人工杂交重组,结果证明,重组后的杂交膜的ATP酶活力比人肝癌线粒体内膜高6～11倍;寡霉素敏感性也显著提高。电镜负染标本观察表明杂交膜出现了典型的直径为90(?)的ATP酶的颗粒形态;ANS萤光增强效应测定证明杂交膜的萤光强度比肝癌膜高276%(相对单位);0℃低温处理2小时,ANS萤光强度不变;酶活力在0℃2小时后,仍相当于原来活力的90%。此项试验结果证明杂交重组获得成功。鼠肝线粒体ATP酶与人肝癌线粒体内膜杂交后的特性表现了与天然线粒体内膜的ATP酶的一系列相似的特性。讨论了ATP酶复合体杂交重组试验在探索肝癌发生与细胞中两个遗传系统控制的可能关系问题。     

低温、高pH胁迫对水稻幼苗根系质膜、液泡膜ATP酶活性的影响

以耐冷性不同的两个水稻品种为材料,比较研究了幼苗根系质膜、液泡膜ATP酶对低温(8℃)及高pH(8.0)胁迫的反应。结果表明水稻根细胞质膜和液泡膜上均存在Ca3+-ATP酶,但活性远低于H+-ATP酶。耐冷品种武育粳3号经低温(8℃)处理2d,根系质膜和液泡膜H+-ATP酶、Ca2+-ATP酶活性均明显升高,至冷处理12d,H+-ATP酶、Ca2+-ATP酶活性有所下降,但仍与对照相近;而冷敏感品种汕优63经低温(8℃)处理2d,根系质膜H+-ATP酶活性略有升高,而质膜Ca2+-ATP酶以及液泡膜H+-ATP酶、Ca2+-ATP酶活性已明显下降;至冷处理12d,4种酶活性均明显低于对照。高pH胁迫使质膜和液泡膜H+-ATP酶活性下降,而使Ca2+-ATP酶活性上升。高pH胁迫会加剧低温冷害。结果表明,耐冷品种质膜、液泡膜ATP酶比冷敏感品种对低温胁迫有更强的适应能力。     

The effects of unsaturated fatty acid depletion on the proton permeability and energetic functions of yeast mitochondria

1. The fatty acid composition of the ole-1 and ole-1 petite mutants of Saccharomyces cerevisiae was manipulated by growing the organism in the presence of defined supplements of Tween 80 or by allowing cells that had first been grown in the presence of Tween 80 to deplete their unsaturated fatty acids by sequent growth in the absence of Tween 80. 2. The transition temperature of Arrhenius plots of mitochondrial ATPase (adenosine triphosphatase) increases as the unsaturated fatty acid content is lowered. 3. Cells require larger amounts of unsaturated fatty acids to grow on ethanol at lower temperatures. 4. Cells that stop growing owing to unsaturated fatty acid depletion at low temperatures are induced to grow further by raising the temperature and this results in a further depletion of unsaturated acids. This is due to a higher rate, but not a greater efficiency, of mitochondrial ATP synthesis. 5. Arrhenius plots of the passive permeability of mitochondria to protons between 4 and 37 degrees C are linear. The rate and the Arrhenius activation energy of proton entry increase greatly as the unsaturated fatty acid content is lowered. 6. Unsaturated fatty acid depletion has the same effects on the proton permeability of ole-1 petite mitochondria, indicating that the mitochondrially synthesized subunits of the ATPase are not involved in the enhanced rates of proton entry. 7. The adenylate energy charge of depleted ole-1 cells is greatly decreased by growth on ethanol medium. 8. The adenylate energy charge of isolated mitochondria is also lowered by unsaturated fatty acid depletion. 9. The results confirm that unsaturated fatty acid depletion uncouples oxidative phosphorylation in yeast both in vivo and in vitro, and is a consequence of changes in the lipid part of the membrane.     

Temperature dependence of mitochondrial respiratory activities

Arrhenius plots of succinate oxidase activity in intact beef heart mitochondria show a clear transition from a low to a high activation energy at 27°C. This temperature is significantly higher than that observed for ATPase (17°C). Arrhenius plots of succinate-cytochromec reductase and cytochromec oxidase also show anomalous curves; while the latter has a breakpoint (at 26°C) only when assayed manometrically, the former has a break at only 20°C.The succinoxidase activity of lipid-deficient mitochondria depends upon addition of exogenous phospholipids. Unsaturated phospholipids are more active than saturated phospholipids but the latter become very effective in restoration of succinoxidase at increasing temperatures. It is suggested that a liquid-crystalline state of the phospholipids is required for correct binding to the lipid-depleted membrane and for restoration of respiratory activity. The is no clear correlation between the above mentioned effects in lipid deficient mitochondria and the transitions in the Arrhenius plots of intact mitochondria.     

USE OF ARRHENIUS PLOTS OF Na-K ATPase and ACETYLCHOLINESTERASE AS A TOOL FOR STUDYING CHANGES IN LIPID-PROTEIN INTERACTIONS IN NEURONAL MEMBRANES DURING BRAIN DEVELOPMENT

Abstract— The activities of Na-K ATPase and acetylcholinesterase in the rat brain cortex were measured at different postnatal ages as a function of temperature. It was found that compared to acetylcholinesterase, Na-K ATPase is more strongly affected by the rise in temperature and that this response is further enhanced with age. Arrhenius plots of the data were prepared and the apparent energies of activation were computed for each plot. It was observed that all plots were biphasic except that for Na-K ATPase of the immature (5-day-old) brain which showed no transition temperature, with an apparent energy of activation of 15.5 kcal/mol. The enzyme from the mature brain (25-day-old) showed an average transition temperature of 22.6°C, with average apparent energies of activation of 15.3 and 27.2 kcal/mol above and below the transition temperature respectively. The cortex of 1-day-old rat showed no Na-K ATPase activity. Arrhenius plots of acetylcholinesterase studied at ages 1, 5 and 25 days postnatally all showed transition temperatures which increased from an average of 16.1°C for 1-day-old to 17 and 21.5°C for 5- and 25-day-old animals respectively. The average apparent energies of activation for acetylcholinesterase below the transition temperature changed from 8.3 kcal/mole at day 1 to 8.7 and 7.2 kcal/mol at days 5 and 25, while above the transition temperature they were 4.3, 5.2 and 4.1 at days 1, 5 and 25 respectively. The results are discussed in terms of the differences and changes in the interactions of Na-K ATPase and acetylcholinesterase with membrane lipids during the postnatal phase of brain development.     

Calcium regulatory proteins and temperature acclimation of actomyosin ATPase from a eurythermal teleost (Carassius auratus L.)

Summary Goldfish (Carassius auratus) were acclimated for 5 months at temperatures of either 2°C or 31°C. Natural actomyosin was prepared from white myotomal muscle and its Mg²⁺Ca²⁺ ATPase activity determined. Temperature acclimation results in adaptations in substrate turnover number and thermodynamic activation parameters of the ATPase. When assayed at 31°C the Mg²⁺Ca²⁺ ATPase of natural actomyosin was 4 times higher in 31°C than 2°C acclimated fish. Arrhenius plots of natural actomyosin ATPase from cold acclimated fish show a break in slope at 15–18°C. In contrast, the temperature dependence of warm acclimated actomyosin was linear. Activation enthalpy (H ) of the ATPase, calculated over the range 0–16°C, was approximately 8,000 cal/mole lower in 2°C than 32°C acclimated fish.In contrast, desensitised actomyosins from which the calcium regulatory proteins have been removed show a linear temperature dependence in the range 0–32°C and have similar properties in 2°C and 31°C acclimated fish. Cross-hybridisation of regulatory proteins (tropomyosin-troponins complex) from cold-acclimated fish to desensitised actomyosin from warm-acclimated fish alters the ATPase towards that of cold-acclimated natural actomyosin and vice versa. The results suggest that the regulatory proteins can influence the kinetics of the ATPase and, furthermore, that they are involved in the acclimation of the actomyosin to different cell temperatures.     

Lipid-protein interactions in mitochondria. Changes in mitochondrial adenosine triphosphatase activity induced by n-butyl alcohol.

Butanol at a concentration of 0.35 m decreases the oligomycin sensitivity of the mitochondrial ATPase; at the same concentration of butanol the activation energy of enzyme is increased threefold. Butanol does not detach the ATPase from the membrane of either mitochondria or submitochondrial particles. The same effect is exerted by butanol on the sensitivity of the ATPase to DCCD, which is covalently bound to the ATPase complex in the oligomycin inhibition site. Diethyl ether also makes the ATPase oligomycin- and DCCD-insensitive; however, its effect on the activation energy of the enzyme is different from that of butanol, since ether does not increase the activation energy but lowers the temperature where a transition occurs in an Arrhenius plot of ATPase. The effect of both organic solvents on ATPase may be closely related to changes occurring in the lipid environment which might be transferred to the enzymic activity via a conformational change of the enzymic protein.     

Wheat mitochondria: oxidative activity and membrane lipid structure as a function of temperature

Mitochondrial oxidative activity and membrane lipid structure of two wheat (Triticum aestivum L.) cultivars were measured as a function of temperature. The Arrhenius activation energy for the oxidation of both succinate and α-ketoglutarate was constant over the temperature range of 3 to 27 C. The activation energy for succinate-cytochrome c oxidoreductase activity was also constant over the same temperature range. The concentration of mitochondria in the reaction, the degree of initial inhibition of state 3 respiration, and the time after isolation of mitochondria were each shown to be capable of causing a disproportionate decrease in the rate of oxidation at low temperatures which resulted in an apparent increase in the activation energy of oxidative activity. Using three spin-labeling techniques, wheat membrane lipids were shown to undergo phase changes at about 0 C and 30 C. It is concluded that the membrane lipids of wheat, a chillingresistant plant, undergo a phase transition similar to the transition observed in the membrane lipids of chilling-sensitive plants. For wheat, however, the transition is initiated at a lower temperature and extends over a wider temperature range.     

Seasonal Changes in the Structure and Function of Mitochondrial Membranes of Artichoke Tubers: A Requisite for Surviving Low Temperatures during Dormancy

The temperature limits of the order-disorder transition, and the Arrhenius activation energy of succinate oxidase activity for mitochondria of Jerusalem artichoke (Helianthus tuberosus L.) tubers were determined from the initiation to the termination of dormancy. The temperature limits for the transition at the initiation of dormancy were 25 and 3 C. These changed to 9 and −5 C at mid-dormancy and returned to 25 and 2 C at the termination of dormancy. The Arrhenius activation energy measured in the temperature range above the transition was 35 kilojoules per mole at middormancy and decreased to 17 kilojoules per mole at the termination of dormancy when sprouting was evident. The coincidence of the changes in membrane structure and function with dormancy suggests that artichokes possess a mechanism for regulating membrane lipid structure so that cellular integrity of tuber tissue is maintained even when the tubers are exposed to low temperatures.     

Effect of cold acclimation on bulk tissue electrical impedance: I. Measurements with birdsfoot trefoil at subfreezing temperatures

The resistive and reactive components of electrical impedance were measured for birdsfoot trefoil (Lotus corniculatus L.) stems at freezing temperatures to −8°C. As temperature decreased the specific resistance at frequencies between 49 hertz and 1.11 megahertz of stems from cold acclimated plants increased more rapidly than from nonacclimated plants. This temperature dependence of specific resistance could be characterized by an Arrhenius activation energy; cold acclimated stems had a larger Arrhenius activation energy than nonacclimated stems. The low frequency resistance is believed to characterize the extracellular region of the stems and the high frequency resistance is believed to characterize the intracellular region of the stems. Cold acclimation increased the intracellular but not the extracellular resistance at nonfreezing temperatures. Cold acclimated stems were not injured by freezing to −8°C and thawing, but nonacclimated stems were injured by freezing to temperatures between −2.2 and −5.6°C and thawing. Injury to nonacclimated stems at freezing temperatures below −2.2°C was indicated by a decrease in the ratio of resistance at 49 Hz to that at 1.11 megahertz.     

Amylolytic activity in fish intestinal mucosa: temperature effects

The activity, temperature characteristics and energy of activation of amylolytic enzymes in the intestinal mucosa were studied in six species of fish living in a boreal zone [burbot (Lota lota L.), northern pike (Exos lucius L.), perch (Perca fluviatilis L.), bream (Abramis brama L.), roach (Rutilis rutilis L.), and carp (Cyprinus carpio L.)] and in three species from tropical and subtropical areas [pilchard (Sardina pilchardus W.), jack mackerel (Trachurus trecae C.) and round sardinella (Sardinella aurita V.)]. The amylolytic activity correlated with the feeding habits: it was essentially lower in predators. The enzyme activity at low temperature, relative to the maximal activity, was correlated with the natural environmental temperature where the species lived. At low temperature the relative activity was higher in boreal fish than in tropical and subtropical fish. We found a breakpoint in the Arrhenius plots in all fish species, except for jack mackerel. The energy of activation in predators decreased below the breakpoint in the low-temperature region. The energy of activation in benthophages of the Aral-Ponto-Caspian area was lower at higher temperatures above the breakpoint. A reduction in activation energy in the range of physiological temperatures might indicate adaptation to the environmental temperature.     

Mechanism of chilling injury in sweet potato Part XII. Temperature dependency of succinoxidase activity and lipid-protein interaction in mitochondria from healthy or chilling-stored tissue

The Arrhenius plot of succinoxidase activity of intact mitochondriafrom healthy sweet potato (Ipomoea batatas Lam.) roots showedtwo transition temperatures of the activation energy at 8–10?Cand 16–18?C. Two transition temperatures were also observedin the case of sweet potatoes stored for 21 days at chillingtemperature, but the activation energy was lower than that ofhealthy tissue. The results on lipiddepleted mitochondria andlipid-rebound mitochondria indicated that the membrane lipidplayed an important role in the transition temperature of succinoxidaseactivity. On the other hand, in the binding of phospholipidto lipid-depleted mitochondria from healthy tissue, the numberof binding sites (n) and the dissociation constant (K) conspicuouslychanged at a temperature between 10 and 15?C. That is, n andK values above the temperature were considerably higher thanthose below the temperature, in spite of addition of phospholipidmicelles whose phase transition temperatures were differentfrom each other. In similar binding experiments with 14-daychilling-stored tissue, n values did not remarkably change,between higher and lower ranges of the above-mentioned temperatureand were lower at higher than at lower temperature. The resultssuggest that the conformation of the protein moiety in the mitochondrialmembrane undergoes reversible change in the early stage of chillingtreatment, which becomes irreversible with prolonged chillingtreatment. ¹This paper constitutes Part 113 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. ²Present address: Division of Breeding, Fruit Tree ResearchStation (Minist. Agric. Forest)., Hiratsuka, Kanagawa 254, Japan. (Received March 11, 1974; )