NMR studies of pH-induced transport of carboxylic acids across phospholipid vesicle membranes.

Proton NMR spectroscopy was used to demonstrate that transmembrane pH gradients across single-bilayer vesicle membranes effect the transport and concentration of carboxylic acids. The results obtained indicate that this transport occurs $\text{via}$ selective permeation of the membrane by the protonated (uncharged) form of the acid.     

An NMR method for the study of proton transport across phospholipid vesicles

We have identified [1-¹⁴C]-oxindole-3-acetic acid as a catabolic product of [1-¹⁴C]-indole-3-acetic acid metabolism in Zea mays seedlings. The isolation, and chemical and mass spectral characterization of oxindole-3-acetic acid from corn kernel tissue is described together with data suggesting oxindole-3-acetic acid to be a major catabolic product of indole-3-acetic acid.     

Determination of catecholamine permeability coefficients for passive diffusion across phospholipid vesicle membranes

Summary A convenient catecholamine transport assay has been developed which permits continuous, instantaneous monitoring of transmembrane flux. Epinephrine transport has been examined by spectrophotometrically monitoring adrenochrome formation resulting from the passive diffusion of catecholamine into unilamellar phospholipid vesicles containing entrapped potassium ferricyanide. Ferricyanide oxidation of epinephrine under the conditions employed is fast compared to membrane transport, which obviates the need for intravesicular concentration or volume determinations. Epinephrine transport data over a pH 6 to 7 range have been fitted to an integrated rate equation from which a permeability coefficient for neutral epinephrine of 2.7±1.5×10^–6 cm/sec has been obtained.     

A kinetic analysis of electron transport across chromaffin vesicle membranes

Some types of secretory vesicles, such as the chromaffin vesicles of the adrenal medulla, have cytochrome b561 which is believed to mediate the transfer of electrons across the vesicle membrane. To characterize the kinetics of this process, we have examined the rate of electron transfer from ascorbate trapped within chromaffin vesicle ghosts to external ferricyanide. The rate of ferricyanide reduction saturates at high ferricyanide concentrations. The reciprocal of the rate is linearly related to the reciprocal of the ferricyanide concentration. The internal ascorbate concentration affects the y intercept of this double-reciprocal plot but not the slope. These observations and theoretical considerations indicate that the slope is associated with a rate constant k1 for the oxidation of cytochrome b561 by ferricyanide. The intercept is associated with a rate constant k0 for the reduction of cytochrome b561 by internal ascorbate. From k0 and standard reduction potentials, the rate constant k-0 for the reduction of internal semidehydroascorbate by cytochrome b561 can be calculated. Under conditions prevailing in vivo, this rate of semidehydroascorbate reduction appears to be much faster than the expected rate of semidehydroascorbate disproportionation. This supports the hypothesis that cytochrome b561 functions in vivo to reduce intravesicular semidehydroascorbate thereby maintaining intravesicular ascorbic acid.     

Nuclear magnetic resonance studies of cation transport across vesicle bilayer membranes.

We analyze an increasingly popular NMR method analogous to the black lipid membrane (BLM) isotopic tracer experiment for the study of mediated cation transport but involving the preparation of vesicles with an environment asymmetric in that paramagnetic metal ions are present only outside the vesicles. This asymmetry is manifest in the NMR spectrum as two distinct resonances for magnetic nuclei in outside and inside lipid headgroups. As mediated transport begins and for the paramagnetic metal ions enter the vesicles, the inner headgroup resonance line shifts and changes shape with a time course containing much information on the actual ion transport mechanism. Processes by which the ions enter the vesicles one or a few at a time (such as via a diffusive carrier) are easily distinguishable from those by which the ions enter in large bursts (such as by pore activation). The limiting case where intervesicular mediator exchange is slow relative to cation transport (the situation for integral membrane proteins) is treated analytically. Computer simulated curves indicate conditions necessary for certain changes in the line shape which are analogous to the "current jumps" observed in BLM conductance studies. The theory derived allows estimates of the average number of ions entering the first few bursts, how often the bursts occur, and how they depend on the concentration of the mediating species in the vesicular membrane. Preliminary experimental spectra illustrating some of the various possible line shape behaviors are presented.     

Aspirin,acetaminophen and proton transport through phospholipid bilayers and mitochondrial membranes

Mechanisms of proton transport were investigated in planar phospholipid bilayer membranes exposed to aspirin (acetylsalicylic acid), acetaminophen (4-acetamidophenol), benzoic acid and three aspirin metabolites (salicylic acid, gentisic acid and salicyluric acid). The objectives were to characterize the conductances and permeabilities of these weak acids in lipid bilayer membranes and then predict their effects on mitochondrial membranes. Of the compounds tested only aspirin, benzoate and salicylate caused significant increases in membrane conductance. The conductance was due mainly to proton current at low pH and to weak acid anion current at neutral pH. Analysis of the concentration and pH dependence suggests that these weak acids act as HA₂ ^–-type proton carriers when pH pK and as lipid soluble anions at neutral pH. Salicylate is much more potent than aspirin and benzoate because salicylate contains an internal hydrogen bond which delocalizes the negative charge and increases the permeability of the anion. Model calculations for mitochondria suggest that salicylate causes net H⁺ uptake by a cyclic process of HA influx and A^– efflux. This model can explain the salicylate-induced uncoupling and swelling observed in isolated mitochondria. Since ingested aspirin breaks down rapidly to form salicylate, these results may clarify the mechanisms of aspirin toxicity in humans. The results may also help to explain why the ingestion of aspirin but not acetaminophen is associated with Reye's syndrome, a disease characterized by impaired energy metabolism and mitochondrial swelling.     

Localization of dolichols in phospholipid membranes. An ESR spin label study

We have used ESR methods employing spin-labeled stearates to investigate the effects of dolichol on the motion of lipid molecules in phospholipid membranes of phosphatidylethanolamine and phosphatidylcholine. The ESR spectra show that the presence of dolichol affects the motion of the spin probes at carbon-16, but not at carbon-5. Similar results are obtained with phospholipid membranes comprising only phosphatidylcholine. It is suggested that dolichol molecules are present mainly in the lipid core region of phospholipid membranes.     

Divalent cation-induced interaction of phospholipid vesicle and monolayer membranes

The effects of phospholipid vesicles and divalent cations in the subphase solution on the surface tension of phospholipid monolayer membranes were studied in order to elucidate the nature of the divalent cation-induced vesicle-membrane interaction. The monolayers were formed at the air/water interface. Various concentrations of unilamellar phospholipid (phosphatidylserine, phosphatidylcholine and their mixtures) vesicles and divalent cations (Mg²⁺, Ca²⁺, Mn²⁺, etc.) were introduced into the subphase solution of the monolayers. The changes of surface tension of monolayers were measured by the Wilhelmy plate (Teflon) method with respect to divalent ion concentrations and time.When a monolayer of phosphatidylserine and vesicles of phosphatidylserine/phosphatidylcholine (1 : 1) were used, there were critical concentrations of divalent cations to produce a large reduction in surface tension of the monolayer. These concentrations were 16 mM for Mg²⁺, 7 mM for Sr²⁺, 6 mM for Ca²⁺, 3.5 mM for Ba²⁺ and 1.8 mM for Mn²⁺. On the other hand, for a phosphatidylcholine monolayer and phosphatidylcholine vesicles, there was no change in surface tension of the monolayer up to 25 mM of any divalent ion used. When a phosphatidylserine monolayer and phosphatidylcholine vesicles were used, the order of divalent ions to effect the large reduction of surface tension was Mn²⁺ > Ca²⁺ > Mg²⁺ and their critical concentrations were in between the former two cases. The threshold concentrations also depended upon vesicle concentrations as well as the area/molecule of monolayers. For phosphatidylserine monolayers and phosphatidylserine/phosphatidylcholine (1 : 1) vesicles, above the critical concentrations of Mn²⁺ and Ca²⁺, the surface tension decreased to a value close to the equilibrium pressure of the monolayers within 0.5 h.This decrease in surface tension of the monolayers is interpreted partly as the consequence of fusion of the vesicles with the monolayer membranes. The     

The proton gradient across mycoplasma membranes

The proton gradient across mycoplasma membranes was determined by using different probes which distribute between the intracellular space and the suspension medium in response to a transmembrane proton gradient. The intracellular pH of intact glycolyzing mycoplasmas was generally more alkaline than the extracellular medium: pH_ext=7 and pH_int=7.4; hence, pH=0.4. The size of the proton gradient depended upon the extracellular pH. Without nutrient substrate, the mycoplasmas were unable to maintain a transmembrane proton gradient, i.e., pH approximated O.N, N-dicyclohexylcarbodiimide, an inhibitor of membrane-bound ATPase, carbonyl cyanide-m-chlorophenyl hydrazone, a proton conductor, and gramicidin, an antibiotic forming cation conduction channels across membranes, strongly affected and even abolished the proton gradient across mycoplasma membranes. These substances also impaired the metabolic activity and viability of mycoplasmas.     

Microinjection in combination with microfluorimetry to study proton diffusion along phospholipid membranes

Proton diffusion along the surface of a planar bilayer lipid membrane was measured by means of acid/base injection with a micropipette and recording of the kinetics of fluorescence changes of fluorescein-labelled lipid on the surface. The dimensionality of the process was assayed by fitting the kinetic curves with two-dimensional (2D) or three-dimensional (3D) diffusion equations. In agreement with Serowy et al. (Biophys J 84:1031-1037, 2003), lateral proton diffusion proceeded via bulk phase by means of buffer molecules as proton carriers (D = 600 microm2/s) under the conditions of 1 mM buffer in the solution. Introduction of proton binding sites on the membrane surface led to the appearance of a considerable contribution of two-dimensional proton diffusion on the membrane surface with D = 1,100 mum(2)/s. The system described can be used to study the dependence of the proton diffusion rate on the phospholipid and protein composition of the membrane.     

Electron transfer across posterior pituitary neurosecretory vesicle membranes

Secretory vesicles from the neurohypophysis have a transmembrane electron carrier very similar to that found in adrenal medullary chromaffin granules. Two different tests show that ascorbic acid contained in the vesicles will reduce an external electron acceptor. First, reduction of cytochrome c or ferricyanide in the medium by a neurosecretory vesicle suspension can be followed spectrophotometrically. Second, the membrane potential (inside positive) generated by electron transfer can be monitored using the membrane potential-sensitive optical probe Oxonol VI. As in chromaffin granules, this electron transfer is probably mediated by cytochrome b561. It may function to regenerate internal ascorbic acid and to provide reducing equivalents needed by the intravesicular amidating enzyme.     

Electrogenic proton transport in epithelial membranes

Summary Certain polar epithelial cells have strong transport capacities for protons and can be examinedin vitro as part of an intact epithelial preparation. Recent studies in the isolated turtle bladder and other tight urinary epithelia indicate that the apical membranes of the carbonic anhydrase-containing cell population of these tissues contain an electrogenic proton pump which has the characteristics of a proton-translocating ATPase. The translocation of protons is tightly coupled to the energy of ATP hydrolysis. Since the pump translocates protons without coupling to the movement of other ions, it may be regarded as an ideal electrogenic pump. The apparent simplicity of the functional properties has led to extensive studies of the characteristics of this pump and of the cellular organization of the secondary acid-base flows in the turtle bladder. Over a rather wide range of electrochemical potential gradients for protons ( ) across the epithelium, the rate of H⁺ transport is nearly linear with . The formalisms of equivalent circuit analysis and nonequilibrium thermodynamics have been useful in describing the behavior of the pump, but these approaches have obvious limitations. We have attempted to overcome some of these limitations by developing a more detailed set of assumptions about each of the transport steps across the pump complex and to formulate a working model for proton transport in the turtle bladder that can account for several otherwise unexplained experimental results. The model suggests that the real pump is neither a simple electromotive force nor a constant current source. Depending on the conditions, it may behave as one or the other.     

Retinol transfer across and between phospholipid bilayer membranes

The transfer of retinol across and between bilayer membranes was studied in vitro using unilamellar liposomes and erythrocytes. Transmembrane movement of retinol in phospholipid bilayer membranes was a spontaneous and rapid process with a halflife of less than 30 s. Retinol transfer between liposomes and between liposomes and erythrocytes was also a spontaneous and rapid process with a halflife of less than 10 min. The results suggest that retinol transport in the cell might not need the participation of specific transfer proteins.     

Study of water permeability through phospholipid vesicle membranes by O NMR

Vesicle suspensions of up to 5 % egg lecithin and 2.5 % cholesterol have been found to have no effect on the NMR relaxation times of ¹⁷O from water. Addition of 1–5 mM Mn²⁺ to an equimolar vesicle suspension of egg lecithin and cholesterol permitted resolution of the free induction decay into two exponential components, a fast one arising from the external water and a slow one arising from the intravesicular fluid. From the rates of relaxation the mean life time of the water molecules within the vesicles was calculated to be 1±0.1 ms at 22°C. The size of the vesicle was estimated from electron micrographs to be about 500 Å in diameter. These data yield an equilibrium water permeability, P_w, of about 8 μs⁻¹ for the vesicle membranes. From the temperature dependence of P_w an activation energy of 12±2 kcal/mol was obtained. The longitudinal relaxation time (T₁) of water within vesicles remained the same as in pure water.