Oxidation of thiols in the Ca2+-ATPase of sarcoplasmic reticulum microsomes

We recently showed that oxidative stress impairs the function of the sarcoplasmic reticulum to transport and retain calcium. Inhibition results primarily from oxidation of one or more thiol groups in the Ca2+-ATPase. We now report that thiol oxidation does not result in disulfide formation. Oxidative inhibition of Ca2+-ATPase activity was not reversed by dithiothreitol. Also, arsenite, which crosslinks dithiols, only mildly inhibited Ca2+-ATPase activity and protected against inhibition by peroxydisulfate. These data suggest the thiols susceptible to oxidation are not spatially close enough to form a disulfide. Furthermore, these thiols appear to be involved in some aspect of phosphoenzyme formation. ATP, in the presence of calcium and magnesium, protected against inhibition of Ca2+-ATPase activity by both oxidants and thiol-binding agents. Both inhibitors also decreased binding of the nucleotide analogue TNP-AMP after phosphorylation by Pi. Dithiothreitol and arsenite were protective. In conclusion, reversible redox regulation of the Ca2+-ATPase of sarcoplasmic reticulum by thiol-disulfide exchange does not occur. However, some other mechanism of redox regulation may operate because the enzyme is sensitive to oxidants, thiol-binding agents and activity can be enhanced by prolonged exposure to dithiothreitol.     

Proton permeation of lipid bilayers

Proton permeation of the lipid bilayer barrier has two unique features. First, permeability coefficients measured at neutral pH ranges are six to seven orders of magnitude greater than expected from knowledge of other monovalent cations. Second, proton conductance across planar lipid bilayers varies at most by a factor of 10 when pH is varied from near 1 to near 11. Two mechanisms have been proposed to account for this anomalous behavior: proton conductance related to contaminants of lipid bilayers, and proton translocation along transient hydrogen-bonded chains (tHBC) of associated water molecules in the membrane. The weight of evidence suggests that trace contaminants may contribute to proton conductance across planar lipid membranes at certain pH ranges, but cannot account for the anomalous proton flux in liposome systems.Two new results will be reported here which were designed to test the tHBC model. These include measurements of relative proton/potassium permeability in the gramicidin channel, and plots of proton flux against the magnitude of pH gradients. (1) The relative permeabilities of protons and potassium through the gramicidin channel, which contains a single strand of hydrogenbonded water molecules, were found to differ by at least four orders of magnitude when measured at neutral pH ranges. This result demonstrates that a hydrogen-bonded chain of water molecules can provide substantial discrimination between protons and other cations. It was also possible to calculate that if approximately 7% of bilayer water was present in a transient configuration similar to that of the gramicidin channel, it could account for the measured proton flux. (2) The plot of proton conductance against pH gradient across liposome membranes was superlinear, a result that is consistent with one of three alternative tHBC models for proton conductance described by Nagle elsewhere in this volume.     

Platelet aggregation is inhibited by long chain acyl-CoA

Oleoyl coenzyme A and other acyl-CoA derivatives inhibited ADP or thrombin-induced aggregation of platelets. Arachidonic acid-induced aggregation was also inhibited, but not the slower aggregation caused by 1-oleoyl-2-acetylglycerol or tetradecanoyl-phorbol-13-acetate. Coenzyme A and free fatty acids had little or no effect, and transfer of labeled oleate from oleoyl Co-A to other lipid classes was not detected. Because acyl Co-A compounds have recently been shown to modulate protein kinase C activity, acyl Co-A may provide a useful tool for investigating activation sequences in platelets and other membranes.     

Encapsulation of macromolecules by lipid vesicles under simulated prebiotic conditions

Summary Phospholipid vesicles (liposomes) were subjected to dehydration-hydration cycles in the presence of 6-carboxyfluorescein or salmon sperm DNA. We found that the vesicles fused into multilamellar structures during dehydration with solutes trapped between the lamellae. Upon rehydration the lamellae swelled and formed large vesicular structures containing solute. This model can be used to study encapsulation of macromolecules by lipid membranes to form protocellular structures under prebiotic conditions.     

Alcohol effects on lipid bilayer permeability to protons and potassium: relation to the action of general anesthetics

Past work has shown that general anesthetics perturb the membranes of isolated synaptic vesicles, thereby increasing permeability to protons and inhibiting the ability of the vesicles to take up catecholamines. It has been proposed that such effects may produce anesthesia through inhibition of synaptic transmission. The mechanisms of perturbation is unknown. Two possible explanations include alterations of dielectric constant or production of defects as anesthetics partition into the bilayer phase. In order to choose between these alternatives, we measured the effect of nine alcohols and two alkanes on liposome permeability to protons and potassium. Ionic permeability was increased by alcohols and alkanes to similar degrees, thereby ruling out direct effects on the membrane dielectric constant caused by partitioning of anesthetics into the bilayer. Other experiments confirmed earlier reports that the enhanced permeability caused by anesthetics is not specific for protons. We conclude that these membrane perturbants act by increasing the number of transient, ion-conducting defects normally present in the bilayer structure.     

pH-dependent fusion of liposomes using titratable polycations

Polylysine promoted extensive membrane mixing of liposomes only if the buffer pH was below the pKa of the lysyl residues. This observation suggested that fusion could be regulated in a physiological pH range if the homopolymer of L-histidine was substituted as fusogen. Microgram quantities of polyhistidine were added to liposomes composed of soybean phospholipids, or to defined phospholipid-cholesterol mixtures which simulate the lipid composition of plasma membranes. A quantitative resonance energy transfer assay determined the extent of lipid phase mixing related to fusion. No fusion was detected at pH 7.4, but when the pH was lowered to 6.5 or below, fusion was rapid and substantial. The extent of membrane mixing increased with progressive acidification of the vesicle-fusogen suspension. The charge density of each polyhistidine molecule, not the total cationic charge per vesicle, influenced the extent of fusion. The kinetics of the fusion reaction were rapid, as membrane mixing was completed within 1 min. If the vesicle suspension was acidified before fusogen addition, the rate of membrane mixing slowed 4-fold. This, as well as a slight increase in light scattering noted whenever polyhistidine was added at pH 7.4, suggests an enhancement of fusion kinetics by preaggregation of vesicles at neutral pH. The lipid composition, regulation of membrane mixing by pH in a physiological range, and rapid kinetics suggest that this model of liposome fusion may be pertinent to understanding some biological fusion events.     

Role of amphiphilic compounds in the evolution of membrane structure on the early earth

A variety of amphiphilic compounds have the capacity to self-assemble into membranous structures in the form of bilayers. The earliest cellular organisms must have incorporated such compounds into boundary membranes, and this review discusses amphiphilic components of the prebiotic environment which would be candidates. One possible source is organic material carried to the earth's surface by meteoritic infall. To test this, we have extracted and analysed non-polar substances from the Murchison carbonaceous chondrite, and found that at least some of the components can produce boundary structures which resemble membranes. This observation suggests that membranous boundary structures were present on the early earth, and available to participate in the origin and evolution of the first cellular forms of life.