The generation of metabolic energy by solute transport

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed.     

Three proton pumps,morphology and movements

The diameter of F₁ coupling factor and the distance it protrudes from the membrane of bovine heart submitochondrial particles were measured quantitatively using horse spleen ferritin as a standard. Employing the freeze-etch technique, particles of similar size were found on membranes of submitochondrial particles and on membranes of particles first depleted by F₁, then reconstituted by addition of F₁. The extramembranous size of F₁ is 9.7 nm and F₁ protrudes from the membrane surface by about 13.6 nm. Bacteriorhodopsin and cytochrome oxidase were incorporated into lipids derived from membranes of extremely thermoacidophilic microorganisms by the octylglucoside dilution method. The bacteriorhodopsin pump was fully functional provided high concentrations of valinomycin were added. With decanoyl-N-methylglucamide as detergent the pump was very active in the absence of valinomycin. Concentrations of gramicidin that collapsed the pH in bacteriorhodopsin liposomes prepared with soybean phospholipid had little or no effect on these rigid proteoliposomes. Very high concentrations (30 µg per ml) were partially effective, suggesting a mechanism other than formation of a gramicidin dimer channel. Cytochrome oxidase lost virtually all activity when incorporated into these rigid liposomes but was fully reactivated on addition of suitable detergents.Abbreviations SMP submitochondrial vesicles prepared from bovine heart mitochondria exposed to sonic oscillation in the presence of pyrophosphate - F₁ the water-soluble coupling factor of the mitochondrial ATPase complex - CF₁ the water-soluble coupling factor of the chloroplast ATPase complex - ASU vesicles submitochondrial vesicles prepared from bovine heart mitochondria disrupted by sonic oscillation in ammonia, then passed through Sephadex and treated with urea - OSCP oligomycin sensitivity-conferring protein - Mega 8, 9, and 10 for octoylnanoyl, and decanoyl-N-methylglucamide - 1799 bis-(hexafluoroacetonyl)acetone - PMS N-methylphenazonium methosulfate     

A new method in growth-electrophysiology: Pressurized intra-organ perfusion

Abstract A new experimental system was devised for the simultaneous measurement of elongation rate and the activity of the spatially separate electrogenic ion pumps of a hypocotyl segment excised from a seedling of Vigna unguiculata L. Walp. under enforced intra-organ perfusion by artificial solutions. The pathway of the perfusion medium was apoplastic space, including xylem vessels as main routes. The elongation rate of the segment was highly dependent on the perfusion pressure applied. It was possible to increase the growth rate under pressurized perfusion by 10-30 times as much as that without perfusion. Elongation rate was also dependent on respiration under perfusion, being retarded reversibly by anoxia a few minutes after the activities of the electrogenic ion pumps were stopped. Perfusion pressure had a little influence on the membrane potential (V_px) below a breakdown level (c. 130 kPa). Perfusion of mannitol or sorbitol solution of appropriate concentration reduced the elongation rate reversibly.     

Computer simulation and interpretation of45Ca efflux profile patterns

Summary Stimulations or inhibitions by various agents of⁴⁵Ca efflux from prelabeled cells or tissues display distinct and reproducible profile patterns when the results are plotted against time as fractional efflux ratios (FER). FER is the fractional efflux of⁴⁵Ca from stimulated cells divided by the fractional efflux from a control unstimulated group. These profile patterns fall into three categories: peak patterns, exponential patterns, and mixed patterns. Each category can be positive (stimulation) or negative (inhibition). The interpretation of these profiles is difficult because⁴⁵Ca efflux depends on three variables: the rate of calcium transport out of the cell, the specific activity of the cell compartment from which the calcium originates, and the concentration of free calcium in this compartment. A computer model based on data obtained by kinetic analyses of⁴⁵Ca desaturation curves and consisting of two distinct intracellular pools was designed to follow the concentration of the traced substance (⁴⁰Ca), the tracer (⁴⁵Ca), and the specific activity of each compartment before, during, and after the stimulation or the inhibition of calcium fluxes at various pool boundaries. The computer model can reproduce all the FER profiles obtained experimentally and bring information which may be helpful to the interpretation of this type of data. Some predictions of the model were tested experimentally, and the results support the views that a peak pattern may reflect a sustained change in calcium transport across the plasma membrane, that an exponential pattern arises from calcium mobilization from an internal subcellular pool, and that a mixed pattern may be caused by a simultaneous change in calcium fluxes at both compartment boundaries.     

Time-dependent changes of calcium influx and efflux rates in rabbit skeletal muscle sarcoplasmic reticulum

Unfractionated and low buoyant density sarcoplasmic reticulum vesicles released calcium spontaneously after ATP- or acetyl phosphate-supported calcium uptake when internal Ca²⁺ was stabilized by the use of 50 mM phosphate as calcium-precipitating anion. This spontaneous calcium release could not be attributed to falling Ca²⁺ concentration outside the vesicles (Ca₀²⁺), substrate depletion, ADP accumulation, nonspecific membrane deterioration or the attainment of a high vesicular calcium content. Instead, spontaneous calcium release was directly proportional to Ca₀²⁺ at the time that calcium content was maximal. A causal relationship between high Ca₀²⁺ and spontaneous calcium release was suggested by the finding that elevation of Ca₀²⁺ from less than 1 μM to 3–5 μM increased the rate and extent of calcium release.The spontaneous calcium release was due both to acceleration of calcium efflux and slowing of calcium influx that was not accompanied by a significant change in the rate of ATP hydrolysis. Neither reversal of the transmembrane KCl gradient nor incubation with cation and proton ionophores abolished the spontaneous calcium release. The persistence of calcium release under conditions where the membrane was permeable to both anions and cations makes it unlikely that this phenomenon is due to a changing transmembrane potential.     

Response of chloride efflux from skeletal muscle ofRana pipiens to changes of temperature and membrane potential and diethylpyrocarbonate treatment

Summary Efflux of³⁶Cl^– from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl^– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases.For temperatures between 0 and 20°C, the measured activation energy is 7.5 kcal/mol for Cl^– efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl^– efflux. The pH-dependent Cl^– efflux can be described by the relationu=1/(1+10n(pK _a -pH)), whereu is the Cl^– efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150mm KCl plus 120mm NaCl (internal potential about –15 mV), the apparent pK _a is 6.5 at both 0 and 20°C, andn=2.5 for 0°C and 1.5 for 20°C. For muscles equilibrated in solutions containing 7.5mm KCl plus 120mm NaCl (internal potential about –65 mV), the apparent pK _a at 0°C is 6.9 andn is 1.5. The voltage dependence of the apparent pK _a suggests that the critical pH-sensitive moiety producing the pH-dependent Cl^– efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact thatn is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl^– efflux increment on raising external pH.The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl^– efflux according to the relation, efflux=exp(–k·[DEPC]·t), wheret is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mm). At 17°C,k ^–1=188mm·min. For temperatures between 10 and 23°C,k has an apparent Q₁₀ of 2.5. The Cl^– efflux inhibitor SCN^– at a concentration of 20mm substantially retards the reduction of the pH-dependent Cl^– efflux by DEPC. The findings that the apparent pK _a is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl^– efflux, and that this action is retarded by SCN^– supports the notion that protonation of histidine groups associated with Cl^– channels is the controlling reaction for the pH-dependent Cl^– efflux.     

Mechanism of Neurotransmitter Release Elicited by the Preferential α1-Adrenergic Receptor Agonist Phenylephrine in Superfused Superior Cervical Ganglion Cells in Culture

Phenylephrine increased [3H]norepinephrine efflux and accumulation of cyclic AMP in cultured rat superior cervical ganglion cells superfused with Tyrode's solution. The purpose of this study was to determine the mechanism and relationship between these two events. Electrical stimulation (1-2 Hz), potassium chloride (50 mM), and the preferential alpha 1-adrenergic receptor agonist phenylephrine (1-100 microM) increased fractional tritium efflux, whereas methoxamine, cirazoline, and amidephrine were relatively ineffective. Phenylephrine, but not methoxamine and cirazoline, also increased cyclic AMP accumulation. Phenylephrine-induced tritium efflux was not altered by alpha- and beta-adrenergic receptor antagonists or by removal of extracellular calcium. Phenylephrine-induced cyclic AMP accumulation was blocked by the beta-adrenergic receptor antagonists propranolol and atenolol. Forskolin (10 microM) and the nonhydrolyzable cyclic AMP analogue 8-(4-chlorophenylthio)cyclic AMP (100 microM) had minimal effect on tritium efflux. However, phenylephrine-evoked increase in tritium efflux was dose dependently attenuated by the neuronal uptake blocker cocaine, and phenylephrine dose-dependently inhibited the incorporation of [3H]norepinephrine into neuronal stores. We conclude that the increase in tritium efflux induced by phenylephrine is independent of cyclic AMP accumulation and appears to be mediated by uptake of phenylephrine via the neuronal carrier-mediated amine transport process, which in turn promotes efflux of the adrenergic transmitter from its storage sites.     

Rapid calcium exchange for protons and potassium in cell walls of Chara

Net fluxes of Ca²⁺, H⁺ and K⁺ were measured from intact Chara australis cells and from isolated cell walls, using ion-selective microelectrodes. In both systems, a stimulation in Ca²⁺ efflux (up to 100 nmol m^?2 s^?1, from an influx of ～40 nmol m^?2 s^?1) was detected as the H⁺ or K⁺ concentration was progressively increased in the bathing solution (pH 7.0 to 4.6 or K⁺ 0.2 to 10mol m^?3, respectively). A Ca²⁺ influx of similar size occurred following the reverse changes. These fluxes decayed exponentially with a time constant of about 10 min. The threshold pH for Ca²⁺ efflux (pH 5.2) is similar to a reported pH threshold for acid-induced wall extensibility in a closely related characean species. Application of NH₄⁺ to intact cells caused prolonged H⁺ efflux and also transient Ca²⁺ efflux. We attribute all these net Ca²⁺ fluxes to exchange in the wall with H⁺ or K⁺. A theoretical treatment of the cell wall ion exchanges, using the ‘weak acid Donnan Manning’ (WADM) model, is given and it agrees well with the data. The role of Ca²⁺ in the cell wall and the effect of Ca²⁺ exchanges on the measured fluxes of other ions, including bathing medium acidification by H⁺ efflux, are discussed.     

春、秋季节树干温度和液流速度对东北3树种树干表面CO2释放通量的影响

为探明温度与液流速度对树干表面CO2释放通量的影响,采用红外气体分析法(IRGA)原位连续测定白桦、兴安落叶松和水曲柳树干表面CO2释放通量,同时测定树干液流速度及树干温度。3树种树干CO2释放通量和液流速度在昼夜变化和季节变化上有一定规律,春、秋季节树干表面CO2释放通量昼夜动态均呈明显的单峰型曲线,但是峰值出现的时间有所区别: 6月份树干表面CO2释放通量峰值出现在夜间,温度峰值则在白天;9月份树干表面CO2 释放通量和液流速度及树干温度在昼夜间的变化规律总体上呈相同趋势,均为白天升高,晚上降低,呈单峰曲线,峰值基本在中午出现。3树种树干表面CO2释放通量有明显的季节性规律,即6月份的CO2释放通量明显高于9月份。9月份和6月份白桦、兴安落叶松和水曲柳的日平均树干表面CO2释放通量分别为0.82,3.32μmol m-2 s-1;0.74,3.78μmol m-2 s-1和1.98,4.98μmol m-2 s-1;6月份和9月份日平均液流速度分别为2.48,10.02g?cm-2?h-1;4.78,10.71g?cm-2?h-1和2.69-7.93g?cm-2?h-1。树干表面CO2释放通量与树干温度和液流速度相关关系显著,6月份和9月份树干表面CO2释放通量与树干温度均呈正相关;树干表面CO2释放通量与液流速度间的相关关系6月与9月不同,6月份液流速度与树干表面CO2释放通量呈负相关,而9月份呈正相关。实验发现,除落叶松外,同1株树24小时周期内水曲柳和白桦液流上升期和下降期液流速度对树干表面CO2 释放通量的影响不一致,可能是由于上下午之间温度不同,导致树干内部CO2溶解度不同,使上下午树干内部CO2与表面通量的平衡发生改变,这种改变存在种间差异,表明温度和液流的共同作用影响树干CO2释放通量。水曲柳月份间树干表面CO2释放通量的差异受液流速度影响较大,白桦和兴安落叶松树干表面CO2释放通量的月份间变化则是液流速度和温度的共同作用的结果。我们的结论是,温度是影响树干呼吸速率的关键因子,但树干呼吸产生的CO2向大气的释放也同时受树干液流速度的影响。温度与液流对春秋季节3树种树干表面CO2释放通量的影响有所不同。     

淋病流行株外排系统与外膜通透性和多重耐药性的关系

探讨外排系统、外膜通透性与淋病流行株多重耐药性的关系。应用K—B法和琼脂稀释法从湛江地区分离出62株淋球菌多重耐药株。利用SDS—PAGE测定淋球菌外膜孔蛋白的表达;应用直接荧光法测定能量抑制剂加入前后淋球菌对抗生素的摄入和积累情况,比较耐药菌与敏感菌内膜泵蛋白表达的差异;利用煮沸法提取细菌DNA,PCR扩增mtrR基因,并对扩增产物测序,比较敏感株与多重耐药株的差异。结果5株多重耐药菌均有外膜孔蛋白表达的缺失或下降,同时伴有外排泵蛋白的表达;5株敏感淋球菌无mtrR的突变,10株多重耐药株均有mtrR基因的突变。表明外排系统、外膜通透性与淋病流行株的多重耐药性密切相关。