Neutral Red as a Redox Dye Induces K+ Efflux and Current-Voltage Changes in Eremosphaera,Lemna, and Guard Cells*

Membrane effects of the redox and pH indicator neutral red were studied with the chlorococcal alga Eremosphaera viridis, with Lemna gibba, and with “isolated” guard cells in epidermal peels of Valerianella locusta. Neutral red was extracellularly reduced and caused transmembrane current-voltage changes, an increase in membrane conductance by about 14 nS, an apparent K⁺ net efflux of up to 120 μmol g^?1 FW in 5 min, and an intracellular acidification by up to 0.7 pH units. Neutral red-triggered K⁺ net efflux was most pronounced at low pH, at an E_o more positive than ?200 mV, and without extracellular Ca²⁺. From the experimental data it is concluded that, due to the redox function of the phenazine molecule, extracellular neutral red triggers a trans-plasmalemma e^? transfer, leading to strong membrane depolarization and charge compensating K⁺ net efflux, in addition to some unspecific ion release. As a consequence the intracellular concentration of strong cations relative to strong anions (SID) decreases, resulting in intracellular acidification.     

Interference of electron donors in the measurement of the proton gradient and membrane potential by flow dialysis

The three most commonly used electron donors for flow dialysis measurements of membrane potential lead to the development of an apparent but artifactual membrane potential with the interior negative in the presence or absence of membrane vesicles. The same three electron donors used in flow dialysis determinations of delta pH in the presence or absence of membrane vesicles lead to the development of an apparent but artifactual delta pH with the interior acidic. These artifacts have been evaluated using two probes for membrane potential, namely, TPP+ and rubidium in the presence of valinomycin and for two probes of delta pH, namely, acetate and DMO. Measurements were made over a range of ionic strengths.     

Use of salicylic acid to measure the apparent intracellular pH in the ehrlich ascites-tumor cell and Escherichia coli

The distribution of salicylic acid between the intracellular and extracellular phases has been used to estimate the intracellular pH in the Ehrlich cell and Escherichia coli. The validity of the method was established by: (i) comparison of the results obtained with salicylic acid with those obtained with 5,5-dimethyloxazolidine-2,4-dione; (ii) by following changes of the apparent intracellular pH under circumstances in which such changes are predictable, e.g., the addition of weak acids or proton conductors to the incubation medium during incubation at acidic pH; (iii) by comparison of the apparent intracellular pH changes with the uptake of H⁺ by the cells estimated from the changes of the medium pH. Optimal results are obtained with this indicator when the extracellular pH is below 5.5, because in this case the indicator is to a sufficient extent in its penetrating form, so that its movement can reflect intracellular pH changes occurring in less than 30 s. When the intracellular pH falls below 5.2 measurable binding of salicylic acid to the intracellular material of the Ehrlich cell takes place, but above this pH no binding has been found.The Ehrlich cell and cells of Escherichia coli behaved similarly under various experimental circumstances tested, but striking differences were found in the inherent permeability of the membrane to H⁺ and in the changes in this parameter by lowering the temperature to 2°C.     

The effect of the uncoupler carbonyl cyanide m-chlorophenylhydrazone on K+ transport,ATP level and intracellular pH of Chlorella fusca

1. Low concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced net K⁺ uptake by Chlorella fusca, optimal concentrations being 3 μM CCCP in the light and 1 μM CCCP in the dark. Higher concentrations increasingly stimulated K⁺ release. 2. Measurements of the unidirectional K⁺ fluxes showed that CCCP-induced net K⁺ uptake in the light was mainly a consequence of an inhibition of efflux. In the dark, influx was slightly stimulated in addition. 3. In conditions of CCCP-induced net K⁺ uptake, the ATP level was decreased by less than 10%. With higher CCCP concentrations it fell drastically. 4. By means of the 5,5-dimethyloxazolidine-2,4-dione distribution technique, an acidification of the cell interior on the addition of CCCP was found. 5. It is concluded that uncoupler-induced net K⁺ uptake is due to an enhanced proton leakage into the cell across the plasmalemma. Intracellular acidification by this process stimulates ATP-dependent K⁺/H⁺ exchange which, in itself, is not affected at low uncoupler concentrations.     

Electroneutral H+-K+ exchange in liver mitochondria Regulation by membrane potential

The paper analyzes the factors affecting the H⁺-K⁺ exchange catalyzed by rat liver mitochondria depleted of endogenous Mg²⁺ by treatment with the ionophore A23187. The exchange has been monitored as the rate of K⁺ efflux following addition of A23187 in low-K⁺ media. (1) The H⁺-K⁺ exchange is abolished by uncouplers and respiratory inhibitors. The inhibition is not related to the depression of ΔpH, whereas a dependence is found on the magnitude of the transmembrane electrical potential, Δψ. Maximal rate of K⁺ efflux is observed at 180–190 mV, whereas K⁺ efflux is inhibited below 140–150 mV. (2) Activation of H⁺-K⁺ exchange leads to depression of ΔpH but not of Δψ. Respiration is only slightly stimulated by the onset of H⁺-K⁺ exchange in the absence of valinomycin. These findings indicate that the exchange is electroneutral, and that the Δψ control presumably involves conformational changes of the carrier. (3) Incubation in hypotonic media at pH 7.4 or in isotonic media at alkaline pH results in a marked activation of the rate of H⁺-K⁺ exchange, while leaving unaffected the level of Mg²⁺ depletion. This type of activation results in partial ‘uncoupling’ from the Δψ control, suggesting that membrane stretching and alkaline pH induce conformational changes on the exchange carrier equivalent to those induced by high Δψ. (4) The available evidence suggests that the activity of the H⁺-K⁺ exchanger is modulated by the electrical field across the inner mitochondrial membrane.     

Dicamba Monooxygenase: Structural Insights into a Dynamic Rieske Oxygenase that Catalyzes an Exocyclic Monooxygenation

Dicamba (2-methoxy-3,6-dichlorobenzoic acid) O-demethylase (DMO) is the terminal Rieske oxygenase of a three-component system that includes a ferredoxin and a reductase. It catalyzes the NADH-dependent oxidative demethylation of the broad leaf herbicide dicamba. DMO represents the first crystal structure of a Rieske non-heme iron oxygenase that performs an exocyclic monooxygenation, incorporating O₂ into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer (α₃) in the crystallographic asymmetric unit with similar arrangement of neighboring inter-subunit Rieske domain and non-heme iron site enabling electron transport consistent with other structurally characterized Rieske oxygenases. While the Rieske domain is similar, differences are observed in the catalytic domain, which is smaller in sequence length than those described previously, yet possessing an active-site cavity of larger volume when compared to oxygenases with larger substrates. Consistent with the amphipathic substrate, the active site is designed to interact with both the carboxylate and aromatic ring with both key polar and hydrophobic interactions observed. DMO structures were solved with and without substrate (dicamba), product (3,6-dichlorosalicylic acid), and either cobalt or iron in the non-heme iron site. The substitution of cobalt for iron revealed an uncommon mode of non-heme iron binding trapped by the non-catalytic Co²⁺, which, we postulate, may be transiently present in the native enzyme during the catalytic cycle. Thus, we present four DMO structures with resolutions ranging from 1.95 to 2.2 Å, which, in sum, provide a snapshot of a dynamic enzyme where metal binding and substrate binding are coupled to observed structural changes in the non-heme iron and catalytic sites.     

奥利亚罗非鱼DMO蛋白结构和功能预测的研究

目的:对奥利亚罗非鱼DNO蛋白的结构和功能进行预测研究.方法:利用生物信息学的方法对DMO基因推导的氨基酸序列进行结构特征和功能域预测分析,探讨了DMO的信号肽、亲/疏水性、跨膜拓扑结构、卷曲螺旋结构、基序、功能域及高级结构.结果:DMO在第1～17,39～113,153～301.372～410区段为高亲水性区域,属亲水性基团,具有两个螺旋卷曲区域,97～112,155～168氨基酸区域,没有信号肽,含有两个跨膜结构域,发生跨膜运动;DMO包含两个保守的DM-domain和DNA锌指结构等功能结构域;DNO含有1个亮氨酸拉链结构(21～42),7个蛋白激酶C磷酸化位点(49～51,184～186,191～193,235～237,270～272,294～296,333～335),5个酪蛋白激酶Ⅱ磷酸化位点(189～192,225～228,227～230,231～234,233～236).1个cAMP-andcGMP-依赖蛋白激酶磷酸化位点(296～299)10个肉豆蔻酰化位点(59～64,121～126,125～130,129～134,135～140,301～306,304～309,307～312,310～315,350～355);DMO蛋白含有27.38%的α-螺旋,8.8%的β-转角和53.79%的无规卷曲;DMO的高级结构中含有两个α-螺旋区域.结论:DMO蛋白与性别调控有关,并在细胞信号传导中发挥作用,且其生物活性可能接受信号途径中多种信号的调控,这为进一步研究奥利亚罗非鱼DMO基因编码蛋白的功能,明确DMO基因与性别调控的关系奠定了基础.