Aluminium Effects on ATPase Activity and Lipid Composition of Plasma Membranes in Sugar Beet Roots

The effect of aluminium (Al) in vivo and in vitro on root plasmamembranes has been studied in two sugar beet (Beta vulgarisL.) cultivars, Monohill (Al-sensitive) and Regina (relativelyAl-tolerant). Although Al in vitro inhibited the MgATPase inan uncompetitive way for both cultivars raised in the absenceof Al, the specific K⁺-activation of the MgATPase was only inhibitedby Al in cv. Monohill. Arrhenius analysis of the MgATPase activity showed that theeffect of Al in vitro depended on whether or not the plantswere exposed to Al in vivo. Al treatment in vitro of the MgATPasefrom control plants cultivated at a low pH (5·4) causedan increase in the phase transition temperature from 17 to 22°C. Only at a higher pH range (pH 6·1) could a secondtransition temperature be induced (at 9 °C). By additionof Al in vitro to plants cultivated with Al at pH 5·4,the slopes of the activity plots did not change. Aluminium changedthe K_m of the ATPase for MgATP in an opposite way by treatmentin vivo and in vitro. Lipid analyses of the plasma membranes showed that the acylcomposition differed little following Al treatment in vivo,but that the ratio of phosphatidylcholine: phosphatidylethanolamineincreased. The changes correlated with the observed change inthe K_m for the MgATPase. We conclude that the main effect ofAl on the MgATPase is not due to the formation of an Al-ATPcomplex. Instead, Al may bind to the membrane-bound enzyme(s)and/or modify the lipid environment. Key words: Aluminium, ATPase, Beta vulgaris, lipids     

Cold Acclimation in Plants: Relationship Between the Lipid Composition and the Cryostability of the Plasma Membrane

Received 23 March 1999/ Accepted in revised form 9 April 1999     

Comparison of the Lipid Composition of Oat Root and Coleoptile Plasma Membranes : Lack of Short-Term Change in Response to Auxin

The total lipid composition of plasma membranes (PM), isolated by the phase partitioning method from two different oat (Avena sativa L.) tissues, the root and coleoptile, was compared. In general, the PM lipid composition was not conserved between these two organs of the oat seedling. Oat roots contained 50 mole percent phospholipid, 25 mole percent glycolipid, and 25 mole percent free sterol, whereas comparable amounts in the coleoptile were 42, 39, and 19 mole percent, respectively. Individual lipid components within each lipid class also showed large variations between the two tissues. Maximum specific ATPase activity in the root PM was more than double the activity in the coleoptile. Treatment of coleoptile with auxin for 1 hour resulted in no detectable changes in PM lipids or extractable ATPase activity. Differences in the PM lipid composition between the two tissues that may define the limits of ATPase activity are discussed.     

Lipid Composition of Plasma Membranes and Endomembranes Prepared from Roots of Barley (Hordeum vulgare L.) : Effects of Salt

Membrane fractions enriched in endoplasmic reticulum (ER), tonoplast and Golgi membranes (TG) and plasma membranes (PM) were prepared from barley (Hordeum vulgare L. cv CM 72) roots and the lipid compositions of the three fractions were analyzed and compared. Plants were grown in an aerated nutrient solution with or without 100 millimolar NaCl. Each membrane fraction had a characteristic lipid composition. The mole per cent of the individual phospholipids, glycolipids, and sterols in each fraction was not altered when roots were grown in 100 millimolar NaCl. The ER had the highest percentages of phosphatidylinositol and phosphatidylcholine of the three fractions (7 and 45 mole per cent, respectively, of the total lipid). The TG contained the highest percentage of glycosylceramide (13 mole per cent). The PM had the highest percentage of phosphatidylserine (3 mole per cent) and nearly equal percentages of phosphatidylethanolamine (15 mole per cent and phosphatidylcholine (18 mole per cent). The most abundant sterols in membranes prepared from barley roots were stigmasterol (10 mole per cent), sitosterol (50 mole per cent), and 24ζ-methylcholesterol (40 mole per cent of the total sterol). Salt-treated plants contained a slightly higher percentage of stigmasterol than controls. The percentage of stigmasterol increased with age and a simple cause and effect relationship between salt treatment and sterol composition was not observed.     

A Contrast of the Plasma Membrane Lipid Composition of Oat and Rye Leaves in Relation to Freezing Tolerance

The lipid composition of the plasma membrane isolated from leaves of spring oat (Avena sativa L. cv Ogle) was vastly different from that of winter rye (Secale cereale L. cv Puma). The plasma membrane of spring oat contained large proportions of phospholipids (28.8 mol% of the total lipids), cerebrosides (27.2 mol%), and acylated sterylglucosides (27.3 mol%) with lesser proportions of free sterols (8.4 mol%) and sterylglucosides (5.6 mol%). In contrast, the plasma membrane of winter rye contained a greater proportion of phospholipids (36.6 mol%), and there was a lower proportion of cerebrosides (16.4 mol%); free sterols (38.1 mol%) were the predominant sterols, with lesser proportions of sterylglucosides (5.6 mol%) and acylated sterylglucosides (2.9 mol%). Although the relative proportions of individual phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine, and the molecular species of these two phospholipids were similar in oat and rye, the relative proportions of di-unsaturated species of these two phospholipids were substantially lower in oat than in rye. The relative proportions of sterol species in oat were different from those in rye; the molecular species of cerebrosides were similar in oat and rye, with only slight differences in the proportions of the individual species. After 4 weeks of cold acclimation, the proportion of phospholipids increased significantly in both oat (from 28.8 to 36.8 mol%) and rye (from 36.6 to 43.3 mol%) as a result of increases in the proportions of phosphatidylcholine and phosphatidylethanolamine. For both oat and rye, the relative proportions of di-unsaturated species increased after cold acclimation, but the increase was greater in rye than in oat. In both oat and rye, this increase occurred largely during the first week of cold acclimation. During the 4 weeks of cold acclimation, there was a progressive decrease in the proportion of cerebrosides in the plasma membrane of rye (from 16.4 to 10.5 mol%), but there was only a small decrease in oat (from 27.2 to 24.2 mol%). In both oat and rye, there were only small changes in the proportions of free sterols and sterol derivatives during cold acclimation. Consequently, the proportions of both acylated sterylglucosides and cerebrosides remained substantially higher in oat than in rye after cold acclimation. The relationship between these differences in the plasma membrane lipid composition of oat and rye and their freezing tolerance is presented.     

Proteins ADP-ribosylated in Nuclei and Plasma Membrane Vesicles Isolated from Sea Urchin Embryos at Various Stages of Early Development

The ADP-ribosylations of proteins in nuclei, plasma membrane vesicles, mitochondria, microsome vesicles and the soluble fraction of sea urchin embryos isolated at various stages of development were examined by measuring the radioactivities of proteins after exposure of these subcellular fractions to [adenosine-¹⁴C]NAD or [adenylate-³²P]NAD. ADP-ribosylation of proteins was detected only in the nuclear and plasma membrane fractions. In the nuclear fraction, the rate of ADP-ribosylation of the histone fraction did not change appreciably during early development. In the TCA-insoluble protein fraction of the nuclei, the rate of ADP-ribosylation increased from fertilization to the morula stage, then decreased and again increased from the mesenchyme blastula to the late gastrula stage. After exposure of the nuclear fraction to [adenylate-³²P]NAD, a protein band with a molecular weight of 90 kDa was detected by SDS-polyacrylamide gel electrophoresis and radioautography at all stages examined. Its labeling intensity indicated that its ADP-ribosylation is higher at the morula and late gastrula stages than at other stages. In the plasma membrane fraction, proteins with molecular weights of 22 and 68 kDa were ADP-ribosylated and their rates of ADP-ribosylation hardly changed during early development.     

The C-terminal Cytosolic Region of Rim21 Senses Alterations in Plasma Membrane Lipid Composition: INSIGHTS INTO SENSING MECHANISMS FOR PLASMA MEMBRANE LIPID ASYMMETRY*

Yeast responds to alterations in plasma membrane lipid asymmetry and external alkalization via the sensor protein Rim21 in the Rim101 pathway. However, the sensing mechanism used by Rim21 remains unclear. Here, we found that the C-terminal cytosolic domain of Rim21 (Rim21C) fused with GFP was associated with the plasma membrane under normal conditions but dissociated upon alterations in lipid asymmetry or external alkalization. This indicates that Rim21C contains a sensor motif. Rim21C contains multiple clusters of charged residues. Among them, three consecutive Glu residues (EEE motif) were essential for Rim21 function and dissociation of Rim21C from the plasma membrane in response to changes in lipid asymmetry. In contrast, positively charged residues adjacent to the EEE motif were required for Rim21C to associate with the membrane. We therefore propose an “antenna hypothesis,” in which Rim21C moves to or from the plasma membrane and functions as the sensing mechanism of Rim21.     

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。     

Exploration of Lipid Metabolism in Relation with Plasma Membrane Properties of Duchenne Muscular Dystrophy Cells: Influence of L-Carnitine

Duchenne muscular dystrophy (DMD) arises as a consequence of mutations in the dystrophin gene. Dystrophin is a membrane-spanning protein that connects the cytoskeleton and the basal lamina. The most distinctive features of DMD are a progressive muscular dystrophy, a myofiber degeneration with fibrosis and metabolic alterations such as fatty infiltration, however, little is known on lipid metabolism changes arising in Duchenne patient cells. Our goal was to identify metabolic changes occurring in Duchenne patient cells especially in terms of L-carnitine homeostasis, fatty acid metabolism both at the mitochondrial and peroxisomal level and the consequences on the membrane structure and function. In this paper, we compared the structural and functional characteristics of DMD patient and control cells. Using radiolabeled L-carnitine, we found, in patient muscle cells, a marked decrease in the uptake and the intracellular level of L-carnitine. Associated with this change, a decrease in the mitochondrial metabolism can be seen from the analysis of mRNA encoding for mitochondrial proteins. Probably, associated with these changes in fatty acid metabolism, alterations in the lipid composition of the cells were identified: with an increase in poly unsaturated fatty acids and a decrease in medium chain fatty acids, mono unsaturated fatty acids and in cholesterol contents. Functionally, the membrane of cells lacking dystrophin appeared to be less fluid, as determined at 37°C by fluorescence anisotropy. These changes may, at least in part, be responsible for changes in the phospholipids and cholesterol profile in cell membranes and ultimately may reduce the fluidity of the membrane. A supplementation with L-carnitine partly restored the fatty acid profile by increasing saturated fatty acid content and decreasing the amounts of MUFA, PUFA, VLCFA. L-carnitine supplementation also restored muscle membrane fluidity. This suggests that regulating lipid metabolism in DMD cells may improve the function of cells lacking dystrophin.     

Maintenance or Collapse: Responses of Extraplastidic Membrane Lipid Composition to Desiccation in the Resurrection Plant Paraisometrum mileense

Resurrection plants usually grow in specific or extreme habitats and have the capacity to survive almost complete water loss. We characterized the physiological and biochemical responses of Paraisometrum mileense to extreme desiccation and found that it is a resurrection plant. We profiled the changes in lipid molecular species during dehydration and rehydration in P. mileense, and compared these with corresponding changes in the desiccation-sensitive plant Arabidopsis thaliana. One day of desiccation was lethal for A. thaliana but not for P. mileense. After desiccation and subsequent rewatering, A. thaliana showed dramatic lipid degradation accompanied by large increases in levels of phosphatidic acid (PA) and diacylglycerol (DAG). In contrast, desiccation and rewatering of P. mileense significantly decreased the level of monogalactosyldiacylglycerol and increased the unsaturation of membrane lipids, without changing the level of extraplastidic lipids. Lethal desiccation in P. mileense caused massive lipid degradation, whereas the PA content remained at a low level similar to that of fresh leaves. Neither damage nor repair processes, nor increases in PA, occurred during non-lethal desiccation in P. mileense. The activity of phospholipase D, the main source of PA, was much lower in P. mileense than in A. thaliana under control conditions, or after either dehydration or rehydration. It was demonstrated that low rates of phospholipase D-mediated PA formation in P. mileense might limit its ability to degrade lipids to PA, thereby maintaining membrane integrity following desiccation.     

Membrane Lipids of Adult Human Brain: Lipid Composition of Frontal and Temporal Lobe in Subjects of Age 20 to 100 Years

Abstract: The membrane lipid composition of human frontal and temporal cortices and white matter has been studied in 118 subjects, age 20–100 years. The brain specimens were selected from subjects who lived a normal social life and died suddenly and unexpectedly with no history of neurologic or psychiatric disease. Macroscopic and microscopic examinations ruled out any signs of organic brain disorder. The sudden death eliminated all risk of changes over a long agonal stage. The data for total solids and major lipids are summarized in graphic form. Total solids, phospholipids, and cholesterol diminished linearly from 20 years of age in frontal and temporal cortices, whereas total solids phospholipids, cholesterol, cerebroside, and sulfatide showed a curvilinear diminution in frontal and temporal white matter. Gangliosides differed from the other lipids, showing an almost constant concentration between 20 and 70 years of age with a slight peak around 50 years of age. The ganglioside pattern showed continuous change with aging, with decreasing proportions of GM1 and GD1a and increasing proportions of GD1b, GM3, and GD3. Equations are given that can be used to calculate the lipid composition of normal human frontal and temporal cortices and white matter at any age between 20 and 100 years of age. These data can be used where data by direct analysis are not available for comparison with values for various pathological states.     

Nickel and Cadmium-related Changes in Growth, Plasma Membrane Lipid Composition, ATPase Hydrolytic Activity and Proton-pumping of Rice (Oryza sativa L. cv. Bahia) Shoots

Six-day-old rice plants (Oryza sativa L. cv. Bahia) were grownin the presence of 0.5 mol m^–3 Ni or Cd for 5 or 10 d.Five days after treatment, some plants were transferred to amedium containing no heavy metal for 5 or 10 d. Plasmalemmavesicles from shoots of treated, transferred (recovery experiments)and control plants were isolated, ATPase activity and proton-pumpingwere assessed, and free sterols and phospholipids determined.The ATPase hydrolytic activity was increased by 37% and 34%in 5 and 10 d Cd-treated plants, respectively; and by 66% in5 and 10 d Ni-treated plants. However, neither the initial rateof H⁺ transport nor the proton-pumping rate at steady-statewere significantly affected by the treatments. The relativeproportion (%) of the plasmalemma sterols campesterol and ⁵-avenasteroldecreased while sitosterol increased during heavy metal treatment.The overall plasmalemma phospholipid fatty acyl chain lengthand degree of unsaturation were also reduced. In experimentswhere plants recovered from Cd and Ni treatment, differencesbetween treated and control plants were reverted, particularlyin 10 d Ni-recovered plants. The possible involvement of lipidsin the regulation of the plasmalemma ATPase as well as the relationshipbetween growth, ATPase and adaptations to stress conditionsare discussed. Key words: Cadmium, nickel, sterols, phospholipids, ATPase     

Effect of Growth Temperature on the Lipid and Fatty Acid Composition, and the Dependence on Temperature of Light-induced Redox Reactions of Cytochrome f and of Light Energy Redistribution in the Thermophilic Blue-Green Alga Synechococcus lividus

The thermophilic blue-green alga Synechococcus lividus was grown at 55 and 38 C. Arrhenius plots of the transient reduction of cytochrome during actinic illumination with light that excited both pigment systems revealed breaks near 43 and 26 C for cells grown at 55 C. In cells grown at 38 C these breaks occurred near 37 and 28 C, respectively. The shift from pigment state 1 to state 2 measured by fluorescence transients also showed characteristic breaks in the Arrhenius plots at 44 C for cells grown at 55 C and at 37 to 38 C and possibly at 25 C for cells grown at 38 C. The break points in the Arrhenius plots for the state shift as well as for the cytochrome f reduction are discussed in relation to phase transitions of thylakoid membrane lipids as studied by the temperature dependence of chlorophyll a fluorescence.     

The Outer Membrane Protein VhOmp of <Emphasis Type="Italic">Vibrio harveyi</Emphasis>: Pore-Forming Properties in Black Lipid Membranes

Vibrio harveyi is known to cause fatal vibriosis in marine animals. Here, an outer membrane protein from V. harveyi, namely, VhOmp, was isolated and functionally characterized in terms of pore-forming contact with artificial lipid membranes. The native VhOmp exists as a trimer of a molecular weight similar to that of the porin OmpF from Escherichia coli. Reconstitution of VhOmp into black lipid membranes demonstrated its ability to form ion channels. The average pore conductance of VhOmp was revealed to be about 0.9 and 2 nS in 0.2 and 1 M KCl, respectively. Within transmembrane potentials of ±100 mV, VhOmp pores behaved as ohmic conduits, and their conductance scaled linearly with voltage. Nonlinear plots of the pore conductance versus symmetrical salt concentrations at either side of the protein-incorporating membrane suggested the influence of interior channel functionalities on the passage of charged species. In the presence of Omp-specific polyclonal antibodies, the pore-forming property of VhOmp was modulated so that the usual step-like current increments were replaced by random transitory current fluctuations. VhOmp exhibited a strong biological activity by causing hemolysis of human red blood cells, indicating that VhOmp may act as a crucial determinant during bacterial infection to animal host cells.     

Studies on Freezing Injury in Plant Cells : II. Protein and Lipid Changes in the Plasma Membranes of Jerusalem Artichoke Tubers during a Lethal Freezing in Vivo

Plasma membranes were isolated from both unfrozen and frozen tissues of Jerusalem artichoke tubers (Helianthus tuberosus L.) in high purity utilizing an aqueous two-polymer phase partition system. Although the recovery of the plasma membranes was decreased significantly by freezing of tissues even at the nonlethal temperature (−5°C), the isolated plasma membrane samples were considered to be representative of the plasma membranes in situ. Freezing of the tissues at sublethal temperatures resulted in marked changes in the chemical composition of the plasma membrane. Those are losses of sterols and phosphatidylethanolamine from the plasma membranes, and a change of specific proteins with relatively high molecular weights into low molecular weight peptides. These specific proteins were designated as frost susceptible proteins. The properties of the plasma membrane ATPase seem to be not affected so much by the in vivo freezing of cells. However, inhibition of the plasma membrane ATPase by N,N′-dicyclohexylcarbodiimide (DCCD) was relatively low before and after freezing in vivo at the nonlethal temperature at −5°C, but was markedly enhanced by freezing in vivo at sublethal temperatures below −10°C. From the results, it is assumed either that the enzyme molecule was partially modified, especially at the presumed DCCD binding sites or that the DCCD had become more accessible to the enzyme as a result of increased permeability of the plasma membranes. These observed changes are discussed in connection with the mechanism of cell injury.     

Early Events Induced by the Elicitor Cryptogein in Tobacco Cells: Involvement of a Plasma Membrane NADPH Oxidase and Activation of Glycolysis and the Pentose Phosphate Pathway

Application of the elicitor cryptogein to tobacco (cv Xanthi) is known to evoke external medium alkalinization, active oxygen species production, and phytoalexin synthesis. These are all dependent on an influx of calcium. We show here that cryptogein also induces calcium-dependent plasma membrane depolarization, chloride efflux, cytoplasm acidification, and NADPH oxidation without changes in NAD+ and ATP levels, indicating that the elicitor-activated redox system, responsible for active oxygen species production, uses NADPH in vivo. NADPH oxidation activates the functioning of the pentose phosphate pathway, leading to a decrease in glucose 6-phosphate and to the accumulation of glyceraldehyde 3-phosphate, 3- and 2-phosphoglyceric acid, and phosphoenolpyruvate. By inhibiting the pentose phosphate pathway, we demonstrate that the activation of the plasma membrane NADPH oxidase is responsible for active oxygen species production, external alkalinization, and acidification of the cytoplasm. A model is proposed for the organization of the cryptogein responses measured to date.