Expression of the tetA(C) tetracycline efflux pump in Escherichia coli confers osmotic sensitivity

Expression of tetA(C) in Escherichia coli confers resistance to tetracycline as well as sensitivity to nickel and cadmium salts, lipophilic chelating agents, and aminoglycoside antibiotics. In this report we determine that high-level expression of tetA(C) also confers an osmotic sensitivity. The osmotic-sensitive phenotype is distinct from the tetracycline-resistant phenotype and can be localized to a domain contained within the first 98 amino acid residues of the TetA(C) polypeptide.     

Involvement of Nha1 antiporter in regulation of intracellular pH in Saccharomyces cerevisiae

The Nha1 antiporter is involved in regulation of intracellular pH in Saccharomyces cerevisiae. We report that deletion of the NHA1 gene resulted in an increase of cytoplasmic pH in cells suspended in water or acidic buffers. Addition of KCl or NaCl to exponentially growing cells lowered the internal pH but the difference between cells with or without NHA1 was maintained. Addition of KCl to starved cells resulted in much higher alkalinization of cytoplasmic pH in a strain lacking Nha1p compared to the wild-type or Nha1p-overexpressing strains. The H+/K+(Na+) exchange mechanism of Nha1p was confirmed in reconstituted plasma membrane vesicles.     

ATP Dependence of Na+/H+ Exchange : Nucleotide Specificity and Assessment of the Role of Phospholipids

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na⁺/H⁺ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pH_i) by microfluorimetry. Na⁺/H⁺ exchange activity was measured as the Na⁺-driven pH_i recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na⁺/H⁺ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-d-glucose and oligomycin. In cells dialyzed in the presence of ATP, no “run-down” was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at ∼5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na⁺/H⁺ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca²⁺ exchanger, this requirement has been attributed to an aminophospholipid translocase, or “flippase.” The involvement of this enzyme in Na⁺/H⁺ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na⁺/H⁺ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATPγS were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTPγS was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg²⁺ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the γ-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.     

大麦根细胞质膜Ca~(2+)转运系统对盐胁迫的反应

大麦幼苗经短时间盐处理,尚未发生伤害时,虽然质膜上需Mg~(2+)和不需Mg~(2+)的两个Ca~(2+)转运系统的转运能力均基本未变,但两者的动力学特征却有所不同。在盐处理3h内,不需Mg~(2+)的Ca~(2+)转运过程对Ca~(2+)的亲和力便明显降低,而需Mg~(2+)的Ca~(2+)转运过程对Ca~(2+)的亲和力变化不大。较长时间盐处理,两个Ca~(2+)转运系统的转运能力和ATP亲和力均有不同程度的减小。这种减小与幼苗的伤害相伴出现,随处理时间加长而加剧。不同时间的盐处理下,质膜Ca~(2+)—ATP酶活性与不需Mg~(2+)的Ca~(2+)转运过程变化规律一致。Ca~(2+)—ATP酶受钙调素激活的特性在盐处理3h内即有所减小,至处理24h基本丧失。由动力学分析结果推测,短时间盐胁迫下质膜上两个Ca~(2+)转运系统的不同变化是植物的一种调节反应,它们在钙信使系统传递胁迫信号的过程中起不同作用。Ca~(2+)—ATP酶驱动的初级Ca~(2+)转运系统可能与胁迫信号的传递有关,而次级Ca~(2+)转运系统即可能起着信息传递之后将剩余 Ca~(2+)运出胞外的功能。较长时间盐胁迫下两系统Ca~(2+)转运能力的降低则是一种伤害反应。     

Proton-coupled bioenergetic processes in extremely alkaliphilic bacteria

Oxidative phosphorylation, which involves an exclusively proton-coupled ATP synthase, and pH homeostasis, which depends upon electrogenic antiport of cytoplasmic Na⁺ in exchange for H⁺, are the two known bioenergetic processes that require inward proton translocation in extremely alkaliphilic bacteria. Energy coupling to oxidative phosphorylation is particularly difficult to fit to a strictly chemiosmotic model because of the low bulk electrochemical proton gradient that follows from the maintenance of a cytoplasmic pH just above 8 during growth at pH 10.5 and higher. A large quantitative and variable discrepancy between the putative chemiosmotic driving force and the phosphorylation potential results. This is compounded by a nonequivalence between respiration-dependent bulk gradients and artificially imposed ones in energizing ATP synthesis, and by an apparent requirement for specific respiratory chain complexes that do not relate solely to their role in generation of bulk gradients. Special features of the synthase may contribute to the mode of energization, just as novel features of the Na⁺ cycle may relate to the extraordinary capacity of the extreme alkaliphiles to achieve pH homeostasis during growth at, or sudden shifts to, an external pH of 10.5 and above.     

Alcohol Inhibits the Depolarization-Induced Stimulation of Oxidative Phosphorylation in Synaptosomes

Abstract: The effects of alcohol and Ca²⁺ transport inhibitors on depolarization-induced stimulation of oxidative phosphorylation and free-Ca²⁺ concentrations in rat synaptosomes were investigated. Glucose oxidation was stimulated by depolarization with K⁺ or veratridine and by the Ca²⁺ ionophore ionomycin. The stimulation by K⁺, veratridine, and ionomycin was correlated with elevation of synaptosomal free Ca²⁺. Depolarization-stimulated respiration was inhibited by verapamil, Cd²⁺, and ruthenium red but not by diltiazem. Synaptosomal Ca²⁺ elevation was inhibited by verapamil but not by ruthenium red. These results indicate that the stimulation depends on elevation of mitochondrial free Ca²⁺. Ethanol, at pharmacological concentrations (50–200 m M ), inhibited the Ca²⁺-dependent stimulation of oxidative phosphorylation. This inhibition resulted, in part, from the inhibition of voltage-gated Ca²⁺ channels, which inhibited the elevation of synaptosomal free Ca²⁺, and, in part, from the stimulation of the mitochondrial Ca²⁺/Na⁺ antiporter, which inhibited the elevation of the mitochondrial matrix free Ca²⁺. The inhibition by ethanol of the excitation-induced stimulation of oxidative phosphorylation in the synapse may contribute to the depressant and narcotic effects of alcohol and enhance excitotoxicity.     

跨膜逆向转运蛋白NHXFS1多克隆抗体的制备及应用

NHXFS1基因是通过DNA家族改组（DNA family shuffling）技术,以拟南芥、水稻和菊花的液泡膜Na＋/H＋逆向转运蛋白基因（NHX1）为亲本获得的活性显著增强的新基因。为制备该蛋白的多克隆抗体,对该蛋白进行跨膜结构分析,选取跨膜蛋白的C末端为靶标,并将其克隆到原核表达载体pET32a中,成功构建了原核融合蛋白pET32a-NHXFS1-抗原表达载体,转化大肠杆菌BL21（DE3）并诱导表达。通过镍柱亲和层析纯化该融合表达蛋白,获得了纯度约为80%的纯化蛋白,用于免疫新西兰大白兔制备多克隆抗体。ELISA实验表明,该抗体的效价达到1：128 000,提取表达NHXFS1蛋白的酵母液泡经该多克隆抗体Western blot检测,证明该抗体具有较好的NHXFS1蛋白特异性。NHXFS1多克隆抗体的制备为进一步认识NHXFS1新蛋白结构与功能以及植物耐盐分子生物学的研究奠定了基础。     

Over-expression of a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene improves salt tolerance in an upland rice

To develop a salt-tolerant upland rice cultivar (Oryza sativa L.), OsNHX1, a vacuolar-type Na⁺/H⁺ antiporter gene from rice was transferred into the genome of an upland rice cultivar (IRAT109), using an Agrobacterium-mediated method. Seven independent transgenic calli lines were identified by polymerase chain reaction (PCR) analysis. These 35S::OsNHX1 transgenic plants displayed a little accelerated growth during seedling stage but showed delayed flowering time and a slight growth retardation phenotype during late vegetative stage, suggesting that the OsNHX1 has a novel function in plant development. Northern and western blot analyses showed that the expression levels of OsNHX1 mRNA and protein in the leaves of three independent transgenic plant lines were significantly higher than in the leaves of wild type (WT) plants. T₂ generation plants exhibited increased salt tolerance, showing delayed appearance and development of damage or death caused by salt stress, as well as improved recovery upon removal from this condition. Several physiological traits, such as increased Na⁺ content, and decreased osmotic potential in transgenic plants grown in high saline concentrations, further indicated that the transgenic plants had enhanced salt tolerance. Our results suggest the potential use of these transgenic plants for further agricultural applications in saline soil.