Kinetics of proton exchange of phosphatidylethanolamine in phospholipid vesicles.

The rate of proton exchange of the amino protons of phosphatidylethanolamine (PE) in sonicated mixed phospholipid vesicles has been determined by NMR spectroscopy. The rate of exchange increases with increasing pH and phosphate concentration. In the absence of buffer the dominant exchange process is an intrasurface reaction in which NH2 groups react via water with NH3+ groups on the outer surface. Addition of cholesterol reduces the rate constant for intrasurface exchange. The experiments are evidence that such reactions could be dominant in proton transport in and to membrane surfaces.     

Folding kinetics of an alpha helical membrane protein in phospholipid bilayer vesicles

We report a detailed kinetic study of the folding of an alpha-helical membrane protein in a lipid bilayer environment. SDS denatured bacteriorhodopsin was folded directly into phosphatidylcholine lipid vesicles by stopped-flow mixing. The folding kinetics were monitored with millisecond time resolution by time-resolving changes in protein fluorescence as well as in the absorption of the retinal chromophore. The kinetics were similar to those previously reported for folding bacteriorhodopsin in detergent or lipid micelles, except for the presence of an additional apoprotein intermediate. We suggest this intermediate is a result of the greater internal two-dimensional pressure present in these lipid vesicles as compared to micelles. These results lay the groundwork for future studies aimed at understanding the mechanistic origin of the effect of lipid bilayer properties on protein folding. Furthermore, the use of biologically relevant phosphatidylcholine lipids, together with a straightforward rapid mixing process to initiate the folding reaction, means the method is generally applicable, and thus paves the way for an improved understanding of the in vitro folding of transmembrane alpha-helical proteins.     

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.     

A model for the resistance of the proton channel formed by the proteolipid of ATPase

The conductance observed for single proton channels formed by proteolipids of ATPase (Schindler and Nelson 1982) is rationalized in terms of a hydrogen bonded network model. A simple algebraic expression for the resistance predicted for such a model is presented and the results are compared to the experimental observations. The comparision suggests that the conduction involves a series of bound water molecules and perhaps amino acid side groups.     

Interaction of mitochondrial malate dehydrogenase monomer with phospholipid vesicles.

The association between bovine and porcine mitochondrial malate dehydrogenase (EC 1.1.1.37) and phospholipid vesicles was investigated. At concentrations at which malate dehydrogenase exists as a dimer, entrapment within the aqueous compartment but not binding of the 14C-labelled enzyme was observed. The dissociated enzyme was labile to moderate heat and to p-chloromercuribenzoate, but in both cases inactivation was decreased by incubation with suspensions of charged phospholipid vesicles. This suggested an interaction between enzyme subunits and phospholipid, and this was confirmed by direct binding measurements and by studies that followed changes in the fluorescein-labelled enzyme. The circular-dichroism spectra of the enzyme indicated a high alpha-helix content, and suggested that a small conformational change occurred when the enzyme dissociated. Fluorescence data also suggested less-rigid molecules after dissociation. A possible mechanism, based on the flexibility of enzyme monomer and its interaction with phospholipids, by which mitochondrial matrix enzymes are specifically localized in cells, is discussed.     

Reconstitution in phospholipid vesicles of calcium-activated potassium channel from outer renal medulla

Summary A barium-sensitive Ca-activated K⁺ channel in the luminal membrane of the tubule cells in thick ascending limb of Henle's loop is required for maintenance of the lumen positive transepithelial potential and may be important for regulation of NaCl reabsorption. In this paper we examine if the K⁺ channel can be solubilized and reconstituted into phospholipid vesicles with preservation of its native properties. The K⁺ channel in luminal plasma membrane vesicles can be quantitatively solubilized in CHAPS at a detergent/protein ratio of 3. For reconstitution, detergent is removed by passage over a column of Sephadex G 50 (coarse). K⁺-channel activity is assayed by measurement of⁸⁶Rb⁺ uptake against a large opposing K⁺ gradient. The reconstituted K⁺ channel is activated by Ca²⁺ in the physiological range of concentration (K_1/22×10^–7 m at pH 7.2) as found for the K⁺ channel in native plasma membrane vesicles and shows the same sensitivity to inhibitors (Ba²⁺, trifluoperazine, calmidazolium, quinidine) and to protons. Reconstitution of the K⁺ channel into phospholipid vesicles with full preservation of its native properties is an essential step towards isolation and purification of the K⁺-channel protein.Titration with Ca²⁺ shows that most of the active K⁺ channels in reconstituted vesicles have their cytoplasmic aspect facing outward in contrast to the orientation in plasma membrane vesicles, which requires also addition of Ca²⁺ ionophore in order to observe Ca²⁺ stimulation. The reconstituted K⁺ channel is highly sensitive to tryptic digestion. Brief digestion leads to activation of the K⁺ channel in absence of Ca²⁺, to the level of activity seen with saturating concentrations of Ca²⁺. This tryptic split is located in a cytoplasmic aspect of the K⁺ channel that appears to be involved in opening and closing the K⁺ channel in response to Ca²⁺ binding.     

Interaction of local anesthetics with small phospholipid vesicles investigated by proton NMR spectroscopy

The effects of the local anesthetics tetracaine, procaine (both charged at pH 6), and benzocaine (uncharged) on phospholipid liposomes have been investigated by 500 MHz ¹H NMR Spectroscopy. All the drugs reverse the Pr³⁺ induced shifts of phospholipid resonances in the same sequence as they are shifted by addition of Pr³⁺: choline POCH₂- > choline-CH₂N > choline-N(CH₃)₃ > glycerol > glycerol > acyl C₂ > acyl C₃. The drug effects result from incorporation of positive charges (tetracaine and procaine) and from the induction of a conformational change of the phospholipid head group via an action on the lipid glycerol backbone (benzocaine). From titration experiments with tetracaine on liposomes containing Pr³⁺ inside and outside is derived that the drug passes the bilayer by transverse diffusion. Tetracaine partitions outside/inside at a ratio of 21. Changes in linewidths of the drug resonances when incorporated into the liposomes allow the conclusion that the tetracaine molecule is located in an elongated way between the lipid acyl chains with its nitrogen group near the glycerol backbone. Benzocaine, showing strong effects on the line shapes of the protons on C₂ and C₃ of the lipid acyl chains is also located near the glycerol backbone, the region with the strongest hydrophobic forces.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 30), Cardiology.     

Fusion of phospholipid vesicles reconstituted with cytochromec oxidase and mitochondrial hydrophobic protein

Summary Reconstituted cytochrome oxidase liposomes were fused with liposomes reconstituted with mitochondrial hydrophobic protein, which acts as a membrane-bound uncoupler of cytochrome oxidase. Fusion was assayed by the loss of respiratory control of cytochrome oxidase as measured by the increased rate of ascorbate oxidation induced by hydrophobic protein when both proteins shared the same vesicles. Fusion was dependent on the presence of phosphatidylserine in the liposomes and Ca⁺⁺ in the aqueous medium. Phosphatidylcholine-phosphatidylserine liposomes required higher concentrations of phosphatidylserine and Ca⁺⁺ than did phosphatidylethanolamine-phosphatidylserine liposomes. Cytochrome oxidase vesicles containing high concentrations of phosphatidylserine showed little or no respiratory control, while those with lower concentrations showed high respiratory control; respiratory control could be induced by fusing cytochrome oxidase vesicles containing high phosphatidylserine with protein-free liposomes containing low phosphatidylserine concentration. If cytochrome oxidase vesicles and hydrophobic protein vesicles were prefused separately for 15 min, they lost the ability to fuse upon being subsequently mixed together. The reconstituted vesicles had diameters of about 200 Å; fusion yielded vesicles with diameters in excess of 1000 Å.     

Spontaneous reactivation of covalently labeled proton adenosinetriphosphatase

Bovine heart mitochondrial adenosinetriphosphatase selectively labeled by [14C]-N,N'-dicyclohexylcarbodiimide or [14C]-7-chloro-4-nitro-2,1,3-benzoxadiazole was used together with other components to form reconstituted submitochondrial particles. When assayed for ATP hydrolysis under normal hydrolysis condition, these labeled submitochondrial particles were found to increase slowly in specific activity with preincubation time, without losing the covalent label. But when assayed for oxidative phosphorylation, the ratio of the specific activity of the same labeled particles to that of the control particles was higher and was unaffected by preincubation. If the labeled particles had been treated by a simulated procedure for oxidative phosphorylation measurement before the ATPase assay, their specific activities for ATP hydrolysis were also found to be higher and unaffected by preincubation. These observations are difficult to reconcile with the alternating three-site model for proton adenosinetriphosphatase or any model which requires the sequential action of three identical sites for ATP hydrolysis and synthesis. A new model with one active and two latent interacting sites is proposed for interpreting the present data.     

Observation of the phosphatidyl ethanolamine amino proton magnetic resonance in phospholipid vesicles: inside/outside ratios and proton transport.

The amino proton resonance of phosphatidyl ethanolamine in sonicated mixed phospholipid vesicles is observed 3.3 ppm downfield from H₂O. Above pH ～ 5 it is broadened beyond detectability as a result of exchange with H₂O protons. In low salt, resonances of amino protons inside the vesicles appear to persist as the pH is raised, while those on the outside disappear. Solvent catalized proton conduction along the surface is proposed, with an effective -NH₂ to -NH₃ transfer rate of about 8 × 10⁵ sec^?1 at 25°C.     

Reaction centers fromRhodopseudomonas sphaeroides in reconstituted phospholipid vesicles. II. Light-induced proton translocation

Unidirectional light-dependent proton translocation was demonstrated in a suspension of reconstituted reaction center (RC) vesicles supplemented with cytochromec and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ₀), a lipid-and water-soluble quinone. Proton translocation was detected only at alkaline pH. The pH dependence can be accounted for by the slow redox reaction between the reduced quinone (UQ₀H₂) and oxidized cytochromec. This conclusion is based on (i) the pH dependence of partial reactions of the reconstituted proton translocation cycle, measured either optically or electrometrically and (ii) titration studies with cytochromec and UQ₀. At 250 and 25 µM UQ₀ and cytochromec, respectively, maximal proton translocation was observed at pH 9.6. This pH optimum can be extended to a more acidic pH by increasing the concentration of the soluble redox mediators in the reconstituted cyclic electron transfer chain. At the alkaline side of the pH optimum, proton translocation appears to be limited by electron transfer from the endogenous primary to the secondary quinone within the RCs. The light intensity limits the reconstituted proton pump at the optimal pH. The results are discussed in the context of a reaction scheme for the cyclic redox reactions and the associated proton translocation events.Abbreviations RC reaction center - UQ₀/UQ₀H₂ oxidized and reduced form of 2,3-dimethoxy-5-methyl-1,4-benzoquinone - D/D⁺ reduced and oxidized form of the primary electron donor of the RCs - CCCP carbonylcyanide-trichloromethoxy phenylhydrazone - UQ_A/UQ _A ^– oxidized and semiquinone form of the primary electron acceptor of the RCs - UQ_B/UQ _B ^– /UQ_BH₂ oxidized, semiquinone, and reduced form of the secondary electron acceptor of the RCs - LDAO lauryldimethylamine-N-oxide During the course of this study K.J.H. was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). This research was supported by grants from the National Institutes of Health (EY-02084) and from the Office of Naval Research (ONR-NOOO 14-79-C 0798) to M. Montal.     

Interaction of the Saccharomyces cerevisiae alpha-factor with phospholipid vesicles as revealed by proton and phosphorus NMR

Proton and phosphorus-31 nuclear magnetic resonance (1H and 31P NMR) studies of the interaction between a tridecapeptide pheromone, the alpha-factor of Saccharomyces cerevisiae, and sonicated lipid vesicles are reported. 31P NMR studies demonstrate that there is interaction of the peptide with the phosphorus headgroups, and quasielastic light scattering (QLS) studies indicate that lipid vesicles increase in size upon addition of peptide. Previous solution (aqueous and DMSO) studies from this laboratory indicate that alpha-factor is highly flexible with only one long-lived identifiable structural feature, a type II beta-turn spanning the central portion of the peptide. Two-dimensional (2D) 1H nuclear Overhauser effect spectroscopy (NOESY) studies demonstrate a marked ordering of the peptide upon interaction with lipid, suggesting a compact N-terminus, in addition to a stabilized beta-turn. In contrast to our results in both solution and lipid environment, Wakamatsu et al. [Wakamatsu, K., Okada, A., Suzuki, M., Higashijima, T., Masui, Y., Sakakibara, S., & Miyazawa, T. (1986) Eur. J. Biochem. 154, 607-615] proposed a lipid environment conformation, on the basis of one-dimensional transferred NOE studies in D2O, which does not include the beta-turn.     

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.     

Encapsulation of hemoglobin in phospholipid vesicles

Hemoglobin has been encapsulated in phospholipid vesicles by extrusion of hemoglobin/lipid mixtures through polycarbonate membranes. This technique avoids the use of organic solvents, sonication, and detergents which have proven deleterious to hemoglobin. The vesicles are homogeneous, with a mean size of 2400 A as determined by photon correlation spectroscopy. The encapsulated hemoglobin binds oxygen reversibly and the vesicles are impermeable to ionic compounds. Hemoglobin encapsulated in egg phosphatidylcholine vesicles converts to methemoglobin within 2 days at 4 degrees C. By contrast, when a mixture of dimyristoyl phosphatidylcholine, cholesterol and dicetyl phosphate is used there is no acceleration in methemoglobin formation, and the preparation is stable for at least 14 days at 4 degrees C.     

Characteristics of redox-linked proton ejection in cytochrome c oxidase reconstituted in phospholipid vesicles. New observations support mechanisms different from proton pumping

Experimental observations reveal a number of characteristics of the redox-linked proton ejection from cytochrome c oxidase vesicles, which apparently cannot be explained by a proton pumping activity of the oxidase. These observations seem, on the other hand, to provide useful elements for alternative explanation(s) of the proton ejection. It is proposed here that the process is scalar and not vectorial and can derive from redox-linked rupture of protonated salt-bridges in the oxidase-lipid complex.