Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH

There is now good evidence that cytoplasmic pH (pHi) may have an important role in the metabolic activation of quiescent cells. In particular, growth stimulation of mammalian fibroblasts leads to a rapid increase in pHi (refs 3-6), due to activation of a Na+/H+ exchanger in the plasma membrane, and this alkalinization is necessary for the initiation of DNA synthesis. However, the mechanism by which mitogens activate the Na+/H+ exchanger to raise pHi is not known, although an increase in cytoplasmic free Ca2+ ([Ca2+]i) has been postulated as the primary trigger. We now present data suggesting that the Na+/H+ exchanger is set in motion through protein kinase C, a phospholipid- and Ca2+-dependent enzyme normally activated by diacylglycerol produced from inositol phospholipids in response to external stimuli. Using newly developed pH microelectrodes and fluorimetric techniques, we show that a tumour promoting phorbol ester and synthetic diacylglycerol, both potent activators of kinase C (refs 12-15), mimic the action of mitogens in rapidly elevating pHi in different cell types. Furthermore, we demonstrate that, contrary to previous views, an early rise in [Ca2+]i is not essential for the activation of Na+/H+ exchange and the resultant increase in pHi. Finally, we suggest that an alkaline pHi shift, mediated by Na+/H+ exchange, may be a common signal in the action of those hormones which elicit the breakdown of inositol phospholipids.     

Pumping of protons from the mitochondrial matrix by cytochrome oxidase

The stoichiometry and mechanism of redox-linked proton translocation by the mitochondrial respiratory chain is a major issue of debate in membrane bioenergetics. The function of cytochrome oxidase is a focal point of disagreement. In 1977 it was suggested that the terminal component of the respiratory chain, cytochrome oxidase, functions as a redox-linked proton pump. That and subsequent studies were based mainly on measurements of proton ejection from mitochondria or from vesicles reconstituted with isolated cytochrome oxidase, or on measurements of translocation of electrical charge equivalents across mitochondrial and vesicle membranes. This proton-translocating function of cytochrome oxidase is confirmed here by a quantitative determination of proton uptake from the inside (matrix) of intact mitochondria.     

A mechanistic principle for proton pumping by cytochrome c oxidase

In aerobic organisms, cellular respiration involves electron transfer to oxygen through a series of membrane-bound protein complexes. The process maintains a transmembrane electrochemical proton gradient that is used, for example, in the synthesis of ATP. In mitochondria and many bacteria, the last enzyme complex in the electron transfer chain is cytochrome c oxidase (CytcO), which catalyses the four-electron reduction of O2 to H2O using electrons delivered by a water-soluble donor, cytochrome c. The electron transfer through CytcO, accompanied by proton uptake to form H2O drives the physical movement (pumping) of four protons across the membrane per reduced O2. So far, the molecular mechanism of such proton pumping driven by electron transfer has not been determined in any biological system. Here we show that proton pumping in CytcO is mechanistically coupled to proton transfer to O2 at the catalytic site, rather than to internal electron transfer. This scenario suggests a principle by which redox-driven proton pumps might operate and puts considerable constraints on possible molecular mechanisms by which CytcO translocates protons.     

Solid acids as fuel cell electrolytes

Fuel cells are attractive alternatives to combustion engines for electrical power generation because of their very high efficiencies and low pollution levels. Polymer electrolyte membrane fuel cells are generally considered to be the most viable approach for mobile applications. However, these membranes require humid operating conditions, which limit the temperature of operation to less than 100 degrees C; they are also permeable to methanol and hydrogen, which lowers fuel efficiency. Solid, inorganic, acid compounds (or simply, solid acids) such as CsHSO4 and Rb3H(SeO4)2 have been widely studied because of their high proton conductivities and phase-transition behaviour. For fuel-cell applications they offer the advantages of anhydrous proton transport and high-temperature stability (up to 250 degrees C). Until now, however, solid acids have not been considered viable fuel-cell electrolyte alternatives owing to their solubility in water and extreme ductility at raised temperatures (above approximately 125 degrees C). Here we show that a cell made of a CsHSO4 electrolyte membrane (about 1.5 mm thick) operating at 150-160 degrees C in a H2/O2 configuration exhibits promising electrochemical performances: open circuit voltages of 1.11 V and current densities of 44 mA cm-2 at short circuit. Moreover, the solid-acid properties were not affected by exposure to humid atmospheres. Although these initial results show promise for applications, the use of solid acids in fuel cells will require the development of fabrication techniques to reduce electrolyte thickness, and an assessment of possible sulphur reduction following prolonged exposure to hydrogen.     

Structural homology of Torpedo californica acetylcholine receptor subunits

The nicotinic acetylcholine receptor (AChR) from the electroplax of the ray Torpedo californica is composed of five subunits present in a molar stoichiometry of alpha 2 beta gamma delta (refs 1-3) and contains both the binding site for the neurotransmitter and the cation gating unit (reviewed in refs 4-6). We have recently elucidated the complete primary structures of the alpha-, beta- and delta-subunit precursors of the T. californica AChR by cloning and sequencing cDNAs for these polypeptides. Here, we report the whole primary structure of the gamma-subunit precursor of the AChR deduced from the nucleotide sequence of the cloned cDNA. Comparison of the amino acid sequences of the four subunits reveals marked homology among them. The close resemblance among the hydrophilicity profiles and predicted secondary structures of all the subunits suggests that these polypeptides are oriented in a pseudosymmetric fashion across the membrane. Each subunit contains four putative transmembrane segments that may be involved in the ionic channel. The transmembrane topology of the subunit molecules has also been inferred.     

Identification of the electron transfers in cytochrome oxidase that are coupled to proton-pumping

Mitochondrial cytochrome oxidase is a functionally complex, membrane-bound respiratory enzyme which catalyses both the reduction of O2 to water and proton-pumping. During respiration, an exogenous donor, cytochrome c, donates four electrons to O2 bound at the bimetallic haem alpha 3 Fe-Cu centre within the enzyme. These four electron transfers are mediated by the enzyme's haem alpha and CuA redox centres and result in the translocation of four protons across the inner mitochondrial membrane. The molecular mechanism of proton translocation has not yet been delineated, however, and in the absence of direct experimental evidence all four electron transfers have been assumed to couple equally to proton-pumping. Here, I report the effects of proton-motive force and membrane potential on two equilibria involving intermediates of the bimetallic centre at different levels of O2 reduction. The results show that only two of the electron transfers, to the 'peroxy' and 'oxyferryl' intermediates of the bimetallic centre, are linked to proton translocation, a finding which strongly constrains candidate mechanisms for proton-pumping.     

Deficiency of molecular hydrogen in the disk of beta Pictoris

Molecular hydrogen (H2) is by far the most abundant material from which stars, protoplanetary disks and giant planets form, but it is difficult to detect directly. Infrared emission lines from H2 have recently been reported towards beta Pictoris, a star harbouring a young planetary system. This star is surrounded by a dusty 'debris disk' that is continuously replenished either by collisions between asteroidal objects or by evaporation of ices on Chiron-like objects. A gaseous disk has also been inferred from absorption lines in the stellar spectrum. Here we present the far-ultraviolet spectrum of beta Pictoris, in which H2 absorption lines are not seen. This allows us to set a very low upper limit on the column density of H2: N(H2) 6 x 10-4. As CO would be destroyed under ambient conditions in about 200 years (refs 9, 11), our result demonstrates that the CO in the disk arises from evaporation of planetesimals.     

Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy

Much progress has been made in our understanding of water molecule reactions on surfaces, proton solvation in gas-phase water clusters and proton transfer through liquids. Compared with our advanced understanding of these physico-chemical systems, much less is known about individual water molecules and their cooperative behaviour in heterogeneous proteins during enzymatic reactions. Here we use time-resolved Fourier transform infrared spectroscopy (trFTIR) and in situ H2(18)O/H2(16)O exchange FTIR to determine how the membrane protein bacteriorhodopsin uses the interplay among strongly hydrogen-bonded water molecules, a water molecule with a dangling hydroxyl group and a protonated water cluster to transfer protons. The precise arrangement of water molecules in the protein matrix results in a controlled Grotthuss proton transfer, in contrast to the random proton migration that occurs in liquid water. Our findings support the emerging paradigm that intraprotein water molecules are as essential for biological functions as amino acids.     

The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (I(e)) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that I(e) is voltage-independent from -100 mV to >0 mV, but is steeply inhibited by further depolarization, and is abolished at about +190 mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates I(e), because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.     

cGMP-dependent protein kinase enhances Ca2+ current and potentiates the serotonin-induced Ca2+ current increase in snail neurones

Protein phosphorylation catalysed by cyclic AMP-dependent, Ca2+/calmodulin-dependent and Ca2+/diacylglycerol-dependent protein kinases is important both in the modulation of synaptic transmission and in the regulation of neuronal membrane permeability (for reviews see refs 5-7). However, there has previously been no evidence for the involvement of cyclic GMP-dependent protein kinase (cGMP-PK) in the regulation of neuronal function. Serotonin induces an increase of Ca2+ current in a group of identified ventral neurones of the snail Helix aspersa. This effect is probably mediated by cGMP because it is mimicked by the intracellular injection of cGMP or the application of zaprinast, an inhibitor of cGMP-dependent phosphodiesterase. We have now found that the effect of either serotonin or zaprinast on the Ca2+ current is potentiated by the intracellular injection of cGMP-PK. Moreover, the intracellular injection of activated cGMP-PK (cGMP-PK + 1 microM cGMP) greatly enhances the Ca2+ current of the identified ventral neurones seen in the absence of serotonin. These results indicate that cGMP-PK has a physiological role in the control of the membrane permeability of these neurones.     

Superoxide involvement in the bactericidal effects of negative air ions on Staphylococcus albus.

The physical nature of small air ions is well established and it is recognized that they can produce a variety of biological effects. However, in only a few instances have any underlying biochemical changes been detected. Theoretically, one can consider the hydrated superoxide radical anion (O2) (H2O)n with n congruent to 4-8 as a likely candidate for a biologically active species of negative air ion. The chemical and biological reactivity of superoxide is high and includes a leading role in bacterial killing caused by radiation, in which superoxide dismutase (SOD), an enzyme that catalyses the reaction: O2 + O2 +2H leads to H2O2 +O2 protected markedly. Other studies have also demonstrated the bactericidal effect of O2 (refs 9-11). Inasmuch as the bactericidal action of small negative air ions has been repeatedly confirmed, we decided to test for the involvement of O2 in this phenomenon by evaluating the protective effect of SOD. Our results show strong O2 involvement in negative air ion bacterial kill.     

Reduction of cytochrome c oxidase by a second electron leads to proton translocation

Cytochrome c oxidase, the terminal enzyme of cellular respiration in mitochondria and many bacteria, reduces O(2) to water. This four-electron reduction process is coupled to translocation (pumping) of four protons across the mitochondrial or bacterial membrane; however, proton pumping is poorly understood. Proton pumping was thought to be linked exclusively to the oxidative phase, that is, to the transfer of the third and fourth electron. Upon re-evaluation of these data, however, this proposal has been questioned, and a transport mechanism including proton pumping in the reductive phase--that is, during the transfer of the first two electrons--was suggested. Subsequently, additional studies reported that proton pumping during the reductive phase can occur, but only when it is immediately preceded by an oxidative phase. To help clarify the issue we have measured the generation of the electric potential across the membrane, starting from a defined one-electron reduced state. Here we show that a second electron transfer into the enzyme leads to charge translocation corresponding to pumping of one proton without necessity for a preceding turnover.     

基于金纳米棒/多壁碳纳米管-壳聚糖复合膜电极的肌红蛋白直接电化学和电催化研究

采用金纳米棒(AuNRs)/多壁碳纳米管-壳聚糖(MWCNTs-Chit)复合膜促进肌红蛋白在电极上的直接电子转移,并用于构建H2O2生物传感器.首先将金纳米棒固定到玻碳电极表面,然后把MWCNTs-Chit分散溶液和肌红蛋白(Mb)固载到玻碳电极上,得到MWCNTs-Chit/Mb/AuNRs复合膜电极.通过循环伏安法对膜电极进行表征,在pH=7.0磷酸缓冲溶液中,Mb表现出一对峰形良好且可逆的氧化还原峰,其中氧化峰和还原峰电位分别为-0.291 V、-0.235 V,式电位(Eθ’)为-0.263 V.与此同时还探讨了修饰电极的电催化活性,结果表明其对H2O2具有良好的电催化还原作用,可作为检测H2O2的生物传感器.传感器对H2O2的米氏常数为0.0494 mM,线性范围为5.0×10-5～5.0×10-3M(R=0.986 7,n=10),检测限为3.2×10-6M(信噪比为3).     

An H+-ATPase in opposite plasma membrane domains in kidney epithelial cell subpopulations

Vectorial solute transport by epithelia requires the polarized insertion of transport proteins into apical or basolateral plasmalemmal domains. In the specialized intercalated cells of the kidney collecting duct, the selective placement of an apical plasma membrane proton-pumping ATPase (H+-ATPase) and of a basolateral membrane anion-exchange protein results in transepithelial proton secretion. It is currently believed that amino-acid sequences of membrane proteins contain critical signalling regions involved in sorting these proteins to specific membrane domains. Recently, it was proposed that intercalated cells can reverse their direction of proton secretion under different acid-base conditions by redirecting proton pumps from apical to basolateral membranes, and anion exchangers from basolateral to apical membranes. But others have found that antibodies raised against the red cell anion-exchange protein (Band 3) only labelled intercalated cells at the basolateral plasma membrane, providing evidence against the model of polarity reversal. In this report, we have examined directly the distribution of proton pumps in kidney intercalated cells using specific polyclonal antibodies against subunits of a bovine kidney medullary H+-ATPase. We find that some cortical collecting duct intercalated cells have apical plasma membrane proton pumps, whereas others have basolateral pumps. This is the first direct demonstration of neighbouring epithelial cells maintaining opposite polarities of a transport protein. Thus, either subtle structural differences exist between proton pumps located at opposite poles of the cell, or factors other than protein sequence determine the polarity of H+-ATPase insertion.     

Proton-coupled electron transfer drives the proton pump of cytochrome c oxidase

Electron transfer in cell respiration is coupled to proton translocation across mitochondrial and bacterial membranes, which is a primary event of biological energy transduction. The resulting electrochemical proton gradient is used to power energy-requiring reactions, such as ATP synthesis. Cytochrome c oxidase is a key component of the respiratory chain, which harnesses dioxygen as a sink for electrons and links O2 reduction to proton pumping. Electrons from cytochrome c are transferred sequentially to the O2 reduction site of cytochrome c oxidase via two other metal centres, Cu(A) and haem a, and this is coupled to vectorial proton transfer across the membrane by a hitherto unknown mechanism. On the basis of the kinetics of proton uptake and release on the two aqueous sides of the membrane, it was recently suggested that proton pumping by cytochrome c oxidase is not mechanistically coupled to internal electron transfer. Here we have monitored translocation of electrical charge equivalents as well as electron transfer within cytochrome c oxidase in real time. The results show that electron transfer from haem a to the O2 reduction site initiates the proton pump mechanism by being kinetically linked to an internal vectorial proton transfer. This reaction drives the proton pump and occurs before relaxation steps in which protons are taken up from the aqueous space on one side of the membrane and released on the other.     

Oxygen activation and the conservation of energy in cell respiration.

Many of the membrane-associated oxidases that catalyse respiratory reduction of O2 to water simultaneously couple this exergonic reaction to the translocation of protons across the inner mitochondrial membrane, or the cell membrane in prokaryotes, a process by which metabolic energy is conserved for subsequent synthesis of ATP. The molecular mechanism of O2 reduction and its linkage to H+ translocation are now emerging. The bimetallic haem iron-copper reaction centre in this family of enzymes is the critical structure for catalysis of both these processes.     

Proton translocation by cytochrome c oxidase.

Cell respiration in mitochondria and some bacteria is catalysed by cytochrome c oxidase, which reduces O2 to water, coupled with translocation of four protons across the mitochondrial or bacterial membrane. The enzyme's catalytic cycle consists of a reductive phase, in which the oxidized enzyme receives electrons from cytochrome c, and an oxidative phase, in which the reduced enzyme is oxidized by O2. Previous studies indicated that proton translocation is coupled energetically only to the oxidative phase, but this has been challenged. Here, with the purified enzyme inlaid in liposomes, we report time-resolved measurements of membrane potential, which show that half of the electrical charges due to proton-pumping actually cross the membrane during reduction after a preceding oxidative phase. pH measurements confirm that proton translocation also occurs during reduction, but only when immediately preceded by an oxidative phase. We conclude that all the energy for proton translocation is conserved in the enzyme during its oxidation by O2. One half of it is utilized for proton-pumping during oxidation, but the other half is unlatched for this purpose only during re-reduction of the enzyme.     

四氮杂大环与Co(Ⅱ)、Ni(Ⅱ)配合物的合成及电子光谱研究

报道在水溶液中合成新型四氮杂四乙酸基取代大环H4L( 5,7,1 2 ,1 4-四甲基 -1 ,4,8,1 1 -四氮杂环十四烷 -N′,N″,N′″,N″″-四乙酸 )与Co(Ⅱ )、Ni(Ⅱ )的单核固体配合物。经元素分析、IR和UV光谱表征其组成为CoLH2 ·2H2 O和NiLH2 ·2H2 O。采用紫外———可见光谱研究了配合物在水溶液中的结构特征 ,并对所得结果进行了讨论。     

微过氧化物酶-11在SiO2纳米粒子修饰玻碳电极上的电化学研究

采用简单的方法将微过氧化物酶-11（MP-11）固定到二氧化硅（SiO2）纳米粒子的表面,并且制备成MP-11/SiO2/GC修饰电极,运用循环伏安法研究MP-11/SiO2/GC电极上MP-11的电化学行为。结果表明：MP-11在玻碳修饰电极上发生了直接的、可逆的2个电子、1个质子的电化学反应。MP-11/SiO2/GC修饰电极可以对氧气进行电催化反应,并且该电催化过程受扩散控制,这样该修饰电极有希望在酶生物燃料电池中作阴极使用。另外,MP-11/SiO2/GC修饰电极对过氧化氢（H2O2）的电催化反应表现出传感器的性能,在线性范围内,信噪比为3时,最低检出限为0.22mmol/L,米氏常数为0.13mmol/L,说明SiO2载体上的酶MP-11与底物H2O2的亲和力较大,对H2O2电催化反应效率高。因此,未来MP-11/SiO2/GC修饰电极也有可能在H2O2传感器中得到广泛的应用。     

2-(2,5-二羟基苯基)-4(3H)-喹唑啉酮质子转移及七元瓜环限制作用的理论研究

在B3LYP/6-311++G(d)和ONIOM(B3LYP/6-311++g(d):PM3)水平上,研究了2-(2,5-二羟基苯基)-4(3H)-喹唑啉酮(DHPQ)及其在七元瓜环(CB7)限制下的双质子转移过程,确定了DHPQ分子各互变异构体的结构、能量以及基态质子转移反应的过渡态.通过对结构变化和能量差异的分析,探讨了DHPQ中的双质子转移机理,考察了质子转移的纳米笼效应以及该笼效应对质子转移热力学性质的影响,同时研究纳米腔与DHPQ的几何匹配关系以及水溶剂对包合物质子转移的影响.计算结果表明:DHPQ是采取分步转移机理,纳米腔的限制对质子转移过程产生一个很大的能垒,抑制分子内质子转移过程,水溶剂的引入促进Path 2,抑制Path 1.