Proton conduction in lecithins. I. Electrical properties in the dry state

The electrical conduction of lecithins in the dry state was studied by dielectric spectroscopy using a wide frequency range and different temperatures. From the frequency dependence three impedances were determined which can be related to bulk, electrode and diffusion limited processes. The qualitative picture of the conduction does not change with temperature, it is independent of the nature of the long aliphatic chains and the phase of the lipids. The activation energies of bulk conduction are in agreement with direct current measurements. It is concluded that charge transport occurs in the polar head group regions with the possible involvement of the head group rotational movements. The charge carriers are considered to be ionic in nature, most probably protons.     

Proton conduction in lecithins. III. Nature of charge carriers

The nature of charge carriers was studied in hydrated lecithin using different techniques. The current-voltage characteristics confirmed the ionic nature of conduction. Attempts to determine the charge mobility were unsuccessful. From measurements of the thermovoltage at different hydration levels the charge carrier was found to be positive. The value of the thermo electromotive force (thermo e.m.f.) depends on the phase state of the lipid but is independent of its water content. The mechanism of charge generation is discussed. The activation energy of conduction does not reflect the phase change directly. Its dependence on water content is attributed to charge mobility which involves the motion of the head groups. Electrolysis experiments were carried out and the amount of hydrogen evolved was determined by gas chromatography. A proton mechanism of conduction is suggested.     

Electrical conduction in collagen. II. Some aspects of hydration

Determinations of the amount of bound water in hydrated proteins yield strongly diverging values. The cause of this is the continuity of the transition from bound to free water, and the diffeernt sensitivities to water structure of the measuring techniques. Only the methods that aim at the determination of the amount of water, whose phase remians unchanged duing freezing, yield similar values. The value for collagen as deduced from conductivity data is about 50% water of the dry weight. It is believed that this water interacts with adsorptive groups on the macromolecules, whereas the freezable water occurs in capillaries.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Signal transduction in the photoactive yellow protein. II. Proton transfer initiates conformational changes

Molecular dynamics simulation techniques, together with semiempirical PM3 calculations, have been used to investigate the effect of photoisomerization of the 4-hydroxy-cinnamic acid chromophore on the structural properties of the photoactive yellow protein (PYP) from Ectothiorodospira halophila. In this bacteria, exposure to blue light leads to a negative photoactic response. The calculations suggest that the isomerization does not directly destabilize the protein. However, because of the isomerization, a proton transfer from a glutamic acid residue (Glu46) to the phenolate oxygen atom of the chromophore becomes energetically favorable. The proton transfer initiates conformational changes within the protein, which are in turn believed to lead to signaling.     

Proton transfer along the external proton channel of bacteriorhodopsin

The process of proton transfer along a proton channel is considered using bacteriorhodopsin as a model system, for which a large body of experimental data is available. The possible amino acid composition of the external proton half-channel of bacteriorhodopsin and the stepwise scheme of proton transfer consistent with experimental data are proposed. The rate of proton transfer between fixed centers is assessed for certain regions of this channel for which spectroscopic data are available.     

Proton transfer in a Thr200His mutant of human carbonic anhydrase II

Human carbonic anhydrase II (HCA II) has a histidine at position 64 (His64) that donates a proton to the zinc-bound hydroxide in catalysis of the dehydration of bicarbonate. To examine the effect of the histidine location on proton shuttling, His64 was replaced with Ala and Thr200 replaced with histidine (H64A-T200H HCAII), effectively relocating the proton shuttle residue 2 A closer to the zinc-bound hydroxide compared to wild type HCA II. The crystal structure of H64A-T200H HCA II at 1.8 A resolution shows the side chain of His200 directly hydrogen-bonded with the zinc-bound solvent. Different proton transfer processes were observed at pH 6 and at pH 8 during the catalytic hydration-dehydration cycle, measured by mass spectrometry as the depletion of 18O from C18O2 by H64A-T200H HCA II. The process at pH 6.0 is attributed to proton transfer between the side chain of His200 and the zinc-bound hydroxide, in analogy with proton transfer involving His64 in wild-type HCA II. At pH 8.0 it is attributed to proton transfer between bicarbonate and the zinc-bound hydroxide, as supported by the dependence of the rate of proton transfer on bicarbonate concentration and on solvent hydrogen isotope effects. This study establishes that a histidine directly hydrogen-bonded to the zinc-bound hydroxide, can adopt the correct distance geometry to support proton transfer     

A 31P- and 2H-NMR study on lecithins in liquid crystalline polyoxyethylene detergents

Phosphatidylcholines were incorporated into hexagonal liquid cyrstalline mixtures of the non-ionic detergents Triton X-100 and octaethyleneglycoldodecylether with D₂O. It is shown by nuclear magnetic resonance (NMR) that the phospholipids adopt the hexagonal liquid crystalline structure of the detergent host lattice. The anisotropic motion of the phospholipid headgroups seems to be unaffected, whereas the acyl chains are disordered. Increasing phospholipid concentration leads to separation of a lamellar phase. The lamellar structure is also preferred at elevated temperatures. Phosphatidylcholines with saturated acyl chains undergo a transition from the hexagonal liquid crystalline to an ordered lamellar state. The shape of the ³¹P-NMR signals suggests that pure gel phase phospholipid separates out. The headgroup region of this gel phase phospholipid becomes immobilized after a few weeks of storage below the transition temperature as judged from ³¹P-NMR. At the same time ²H-NMR exhibits a new signal from D₆₂O undergoing slow isotropic motion. This behavior bears resemblance to the formation of a coagel in fatty acid-water systems.     

Proton NMR bandshape studies of lamellar liquid crystals and gel phases containing lecithins and cholesterol.

Proton NMR spectra for gel and liquid crystalline samples, composed of dimyristoyl and/or dipalmitoyl lecithin, cholesterol and water, can be consistently interpreted in terms of mesophase symmetry and molecular diffusion according to a model proposed by Wennerstrom (Wennerstrom, H. (1973) Chem. Phys. Lett. 18, 41-44). It is shown by computer simulation that the characteristic "super-lorentzian" bandshape of the lamellar mesophase can be described by the superposition of three gaussian curves. The NMR signal of the gel phase can be simulated by the superposition of two gaussian curves with widths at half height of 2.5 kHz and 19 kHz. An upper limit of the lateral diffusion coefficient of the lecithin molecules in the gel phase is calculated to be about 5-10(-15) m-2/s. It is therefore concluded that the static intermolecular dipolar couplings average to zero in the lamellar mesophase. An estimation of the order parameter of the liquid crystalline phase is made from experimental data and a calculated "rigid lattice" linewidth. A two phase system is shown to exist in the temperature range 28-34 degrees C for a mesophase of a mixture of dimyristoyl and dipalmitoyl lecithin. The presence of cholesterol results in enhanced lateral diffusion of the lecithin molecules at temperatures below the Chapman transition point.     

Proton transfer pathways in the mutant His-64-Ala of human carbonic anhydrase II

We have investigated the possible proton transfer pathways from the surface of the protein to the zinc-bound water molecule in the mutant His-64-Ala of human carbonic anhydrase II. Starting with an input of known crystallographic structures of the mutant, we model the proton pathways as hydrogen-bonded networks of proton conducting groups and bound solvent molecules. No proton path is detected in the mutant, in close agreement with the experimental observation of a 20-fold decrease in its catalytic efficiency compared to the wild-type enzyme. We also investigate in detail changes in hydration structure at the active site of the mutant and the resulting proton paths in the presence of an exogenous proton donor 4-methylimidazole (4-MI). The proton transfer pathways thus detected are correlated to the observed chemical rescue of catalytic activity by 4-MI.     

Proton cotransport of thiourea in Chlorella fusca

Abstract Active thiourea uptake by Chlorella fusca var. vacuolata was accompanied by a simultaneous uptake of protons. The ratio of uptake of protons: uptake of thiourea was 0.92 (average of 5 experiments). The half maximal rate of proton uptake occurred at a thiourea concentration of 62 μM. Rate of thiourea uptake was highest at pH 5.5 and fell to 20% of the maximal rate as pH was increased to 8.0. It is concluded that thiourea is transported into Chlorella by a proton cotransport system similar to that known for glucose transport.     

1-Acyl-lysolecithin acyltransferase and synthesis of biliary lecithins in rat liver

The role of lysolecithin acyltransferase activities in biliary lecithin formation was investigated, using livers perfused in the presence of labeled palmitoyl-lysolecithin and albumin, overloaded or not with linoleic acid. At the end of liver perfusion, the lecithins extracted from microsomes, mitochondria and plasma membranes displayed the same specific activity. Double-labeled lysolecithin was used to prove that labeled lecithins were synthesized by lysolecithin acylation. In the absence or presence of a linoleic acid overload, the level of lysolecithin incorporation into linoleyl and arachidonyl containing lecithin was identical. Hence fatty acids did not influence phosphatidylcholine synthesis by the acylation pathway. In vitro the rate of linoleyl lecithin synthesis was the same in plasma membranes, mitochondria and microsomes provided the linoleyl-CoA concentration was lower than 30 microM. Taurocholate was essential to the excretion of lecithin synthesized from lysolecithin and stimulated its synthesis. The specific activities of the two lecithin molecular species excreted in bile (linoleyl and arachidonyl) were not significantly different. These results enabled us to evaluate the contribution of the lysolecithin pathway to the synthesis of lecithin in liver and bile: this contribution in bile was less than 2% under the perfusion conditions used.     

Hydrogen bonding and proton transfer between nitro-phenols and lecithin in apolar solvents

The interaction of p-nitrophenol (p-NP), 2,4-dinitrophenol (DNP) (II) and 2,4,6-trinitrophenol (TNP)(III) with dipalmitoyllecithin in apolar solvents has been examined by IR and UV spectroscopy. Addition of any nitrophenol to the solution of lecithin in CCl₄ causes disappearance of broad absorption band of water bound with lecithin phosphate grops (3150–3600 cm^?1), which was accompanied by an insignificant increase of absorption near 3040 and 2800 cm^?1. Association of phenolic groups of (I) with the lecithin was observed by disappearance of the free OH absorption band. In UV spectra of (I), complex formation with lecithin results in a 30 nm red shift of phenol long-wave absorption band and in the appearance of an isosbestic point at 303 nm. In the case of III, addition of the lecithin causes a red shift and strong hyperchromic effect, which is accompanied by the appearance of a new absorption band near 420 nm. It was concluded, that nitrophenols displace a part of water from the polar groups of lipids and form hydrogen bonded complexes or ion-pair structures, depending upon acidic properties of the proton donor.     

Proton NMR assignments of systemin

Complete proton NMR assignments have been made for a synthetic 18-amino acid peptide named systemin, which functions as a wound-induced polypeptide hormone in tomato plants, and three of its derivatives. The wild-type peptide and this synthetic homolog have equivalent activities in their functional roles as systemic inducing signals in tomato plants. Proton NMR studies were carried out to characterize the solution properties of systemin. A variety of homonuclear proton NMR experiments at both 500 and 600 MHz were utilized in making these assignments, which have resulted in additional structural information. Whereas these results provide no evidence for persistence of common secondary (helix, sheet) or tertiary structural elements in the systemin polypeptide, there is evidence for two distinct molecular conformations at the carboxy terminus.     

Proton release from water oxidation by photosystem II: similar stoichiometries are stabilized in thylakoids and PSII core particles by glycerol

During the four-stepped catalytic cycle of water oxidation by photosystem II (PSII) molecular oxygen is released in only one of the four reaction steps whereas the release of four protons is distributed over all steps. In principle, the pattern of proton production could be taken as indicative of the partial reactions with bound water. In thylakoids the extent and rate of proton release varies as function of the redox transition and of the pH without concomitant variations of the redox pattern. The variation has allowed to discriminate between deprotonation events of peripheral amino acids (Bohr effects) as opposed to the chemical deprotonation of a particular redox cofactor, and of water. In contrast, in thylakoids grown under intermittent light, as well as in PSII core particles the pattern of proton release is flat and independent of the pH. This has been attributed to the lack in these materials of the chlorophyll a,b-binding (CAB) proteins. We now found that a thylakoid-like, oscillatory pattern of proton release was restored simply by the addition of glycerol which modifies the protein–protein interaction. Being a further proof for the electrostatic origin of the greater portion of proton release, this effect will serve as an important tool in further studies of water oxidation.     

Proton extrusion in seed coats of Phaseolus vulgaris L.

Abstract. The present investigations were designed to identify proton pumps in seed coats of Phaseolus vulgaris L. Vacated seed-coat halves were exposed to bathing solutions with indicators for proton pump action and the pH changes in the media were measured. Fusicoccin increased the rate of proton extrusion from the seed coats. Orthovanadate and abscisic acid retarded the proton extrusion evoked by fusicoccin. Abolition of the proton extrusion by parachloromercuriphenylsulphonic acid was partially reversed by diethioerythritol. The extrusion was stimulated by high osmolarities (100 mol m⁻³ sorbitol), potassium ions (100 mol m⁻³ KCI) and light. Old seed coats reacted more rapidly to fusicoccin treatments than young ones. Proton pumping in seed coats and cotyledons showed differential responses to fusicoccin, K⁺ and sucrose. In contrast to seed coats, medium acidification by cotyledons was prohibited by addition of sucrose. The significance of proton pumps for photosynthate transfer in vivo is discussed.     

质子漏及其在基础代谢中的作用

“质子漏”是指电子传递链跨膜泵出的质子通过不涉及ATP合成的途径而跨膜扩散流回基质的过程,它的出现形成了由呼吸链驱动的质子泵出和质子回漏的无效循环通路.质子漏的耗氧在呼吸速率中占有重要的比重,对细胞呼吸有很强的控制作用,可以调节能量偶联系数,同时质子漏也是重要的产热过程,它承担了基础代谢产热的20%～30%.质子漏的生理功能有产热、增加代谢调节潜能、清除有害自由基和调节碳流等.     

Proton conductance caused by long-chain fatty acids in phospholipid bilayer membranes

Summary Mechanisms of proton conductance (G _H) were investigated in phospholipid bilayer membranes containing long-chain fatty acids (lauric, myristic, palmitic, oleic or phytanic). Membranes were formed from diphytanoyl phosphatidylcholine in decane plus chlorodecane (usually 30% vol/vol). Fatty acids were added either to the aqueous phase or to the membrane-forming solution. Proton conductance was calculated from the steadystate total conductance and the H⁺ diffusion potential produced by a transmembrane pH gradient. Fatty acids causedG _H to increase in proportion to the first power of the fatty acid concentration. TheG _H induced by fatty acids was inhibited by phloretin, low pH and serum albumin.G _H was increased by chlorodecane, and the voltage dependence ofG _H was superlinear. The results suggest that fatty acids act as simple (A^– type) proton carriers. The membrane: water partition coefficient (K _p ) and adsorption coefficient () were estimated by finding the membrane and aqueous fatty acid concentrations which gave identical values ofG _H. For palmitic and oleic acidsK _p was about 10⁵ and was about 10^–2 cm. The A^– translocation or flip-flop rate (k _a ) was estimated from the value ofG _H and the fatty acid concentration in the membrane, assuming that A^– translocation was the rate limiting step in H⁺ transport. Thek _A 's were about 10^–4 sec^–1, slower than classical weak-acid uncouplers by a factor of 10⁵. Although long-chain fatty acids are relatively inefficient H⁺ carriers, they may cause significant biological H⁺ conductance when present in the membrane at high concentrations, e.g., in ischemia, hypoxia, hormonally induced lipolysis, or certain hereditary disorders, e.g., Refsum's (phytanic acid storage) disease.