United States--Australia workshop on membrane biophysics.
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Amino Acid Sequence↗
Biomedical subjects
D W Deamer
Publications and source records attributed to D W Deamer.
Evidence that sensitivity to steroid anesthetics appears late in evolution.
The effects of pregnanolone, a steroid anesthetic, were compared with diethyl ether and short chain alkanols in 21 aquatic species from 7 phyla. Loss of righting reflex and escape response were used as indicators of anesthesia. All organisms were anesthetized by diethyl ether and short chain alkanols, but pregnanolone affected only organisms belonging to the phylum Chordata. It is probable that pregnanolone exerts its effect on the gamma-aminobutyric acid (GABA) receptor. Because many invertebrates do possess GABA receptors, our results suggest that a binding site at which steroid binding causes organismal anesthesia appeared early in chordate evolution on a previously existing GABA receptor. The results also appear to exclude a primary lipid bilayer site for steroid anesthetic action.
Alcohols↗
Biogenesis as an evolutionary process [letter].
The earliest fossil stromatolites present evidence of a complex ecosystem of photosynthetic organisms. Because the origin of present life can be dated within a few hundred million years prior to these fossils, their complexity poses a problem. A heuristic model outlines the first radiation leading to the universal ancestor.
Biological Evolution↗
Proton conductance by the gramicidin water wire. Model for proton conductance in the F1F0 ATPases?
The gramicidin channel contains a single strand of water molecules associated through hydrogen bonds. Previous work has shown that channels of similar size are formed by association of transmembrane alpha helices of synthetic leucine-serine peptides. Both types of channels translocate protons with considerable selectivity relative to other cations, and it has been proposed that the selectivity arises by proton "hopping" along hydrogen-bonded chains of water, whereas other cations must cross by ordinary diffusion processes. It is possible that a similar mechanism underlies proton transport in the Fo subunit of the F1F0 ATP synthase. Using the gramicidin channel as a model, we have tested whether a single strand of water is kinetically competent to translocate protons at a rate sufficient to support known rates of ATP synthesis. We found that the gramicidin channel saturates at approximately 530 pS of protonic current in 4 M HCl, more than sufficient for typical ATP synthesis rates. It follows that proton diffusion to a putative channel in Fo, rather than the channel itself, may limit ATP synthesis rates.
Gramicidin↗
Sensitivity to anesthesia by pregnanolone appears late in evolution.
The anesthetic effect of certain steroids in vertebrate systems is well documented, but it is not known if these compounds can cause anesthesia in simpler organisms. To answer this question, ether, short-chain alkanols, and pregnanolone were tested for their ability to induce anesthesia in 20 aquatic species from 7 phyla. Organisms were placed in water containing clinical concentrations of anesthetic. Loss of righting reflex and escape response were used as indicators of anesthesia. All organisms tested responded to ether and short-chain alkanols, but pregnanolone affected only organisms belonging to the phylum Chordata. It is probable that pregnanolone exerts its effect on the gamma amino butyric acid (GABA) receptor, and many invertebrates do possess GABA receptors. These results suggest that a steroid anesthetic binding site appeared early in chordate evolution on a previously existing GABA receptor. Further, this experiment appears to exclude lipid bilayer sites for steroid anesthetic action.
1-Butanol↗
Light-dependent pH gradients are generated in liposomes containing ferrocyanide.
Ferrocyanide solutions absorb light energy in a photochemical reaction that causes cyanide radicals to be lost from the iron complex. Because cyanide is a weak acid (pKa = 9.2), at pH ranges between 6 and 9 it associates with protons, thereby causing the pH of its solutions to increase. The pH change is proportional to the photon flux and concentration of ferrocyanide. For instance, the pH of an unbuffered 1.0 mM potassium ferrocyanide solution increases from 6 to 9 within seconds after illumination. We have encapsulated ferrocyanide in liposomes, and demonstrated that light-dependent pH gradients were readily produced, alkaline inside. This preparation offers a convenient system for generating pH gradients across lipid bilayer membranes. It also provides a useful model for simulating primitive bioenergic systems relevant to light energy transduction by early forms of life.
Arylsulfonates↗
Steady-state catecholamine distribution in chromaffin granule preparations: a test of the pump-leak hypothesis of general anesthesia.
The molecular mechanism of general anesthesia is not understood. Possible modes of action include binding at a protein site, such as a receptor or channel, or physical effects on membrane lipid properties. The pump-leak hypothesis suggests that anesthetics perturb the bilayer of synaptic vesicles, thereby increasing ionic permeability. This results in decay of proton gradients required for transport and accumulation of neurotransmitters. The subsequent loss of neurotransmitters from synaptic vesicles reduces the efficiency of synaptic transmission and results in the anesthetized state. We have determined the effects of general anesthetics on certain parameters of enzyme activity and membrane permeability relevant to the pump-leak hypothesis. We used chromaffin granules as a convenient model system and focused on clinically relevant anesthetic concentrations (ED50), quantitative measurements of permeability changes, and the kinetics of gradient decay. General anesthetics at ED50 have little or no effect on the proton-transport ATPase activity, but do cause modest increments in proton permeability that change the catecholamine distribution in actively pumping chromaffin granule preparations. We found that pH gradients do not collapse entirely under these conditions and that only a fraction of total catecholamine is lost from the chromaffin granules. When total collapse is induced by other means, efflux of catecholamines occurs with a half-time near 30 min. These results suggest that if the pump-leak hypothesis is valid, then very small losses of catecholamines must be sufficient to induce anesthesia. We conclude that the weight of evidence favors other mechanisms, notably direct binding of anesthetics to sensitive proteins.
Adrenal Glands↗
Amphiphilic components of the Murchison carbonaceous chondrite: surface properties and membrane formation.
We have investigated physicochemical properties of amphiphilic compounds in carbonaceous meteorites. The primary aim was to determine whether such materials represent plausible sources of lipid-like compounds that could have been involved as membrane components in primitive cells. Samples of the Murchison CM2 chondrite were extracted with chloroform-methanol, and the chloroform-soluble material was separated by two-dimensional thin layer chromatography. Fluorescence, iodine stains and charring were used to identify major components on the plates. These were then scraped and eluted as specific fractions which were investigated by fluorescence and absorption spectra, surface chemical methods, gas chromatography-mass spectrometry, and electron microscopy. Fraction 5 was strongly fluorescent, and contained pyrene and fluoranthene, the major polycyclic aromatic hydrocarbons of the Murchison chondrite. This fraction was also present in extracts from the Murray and Mighei CM2 chondrites. Fraction 3 was surface active, forming apparent monomolecular films at air-water interfaces. Surface force measurements suggested that fraction 3 contained acidic groups. Fraction 1 was also surface active, and certain components could self-assemble into membranous vesicles which encapsulated polar solutes. The observations reported here demonstrate that organic compounds plausibly available on the primitive Earth through meteoritic infall are surface active, and have the ability to self-assemble into membranes.
Australia↗
Proton flux mechanisms in model and biological membranes.
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Electrochemistry↗
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.
Acyl Coenzyme A↗
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.
Alcohols↗
The chemical logic of a minimum protocell.
Traditional schemes for the origin of cellular life on earth generally suppose that the chance assembly of polymer synthesis systems was the initial event, followed by incorporation into a membrane-enclosed volume to form the earliest cells. Here we discuss an alternative system consisting of replicating membrane vesicles, which we define as minimum protocells. These consist of vesicular bilayer membranes that self-assemble from relatively rare organic amphiphiles present in the prebiotic environment. If some of the amphiphiles are primitive pigment molecules asymmetrically oriented in the bilayer, light energy can be captured in the form of electrochemical ion gradients. This energy could then be used to convert relatively common precursor molecules into membrane amphiphiles, thereby providing an initial photosynthetic growth process, as well as an appropriate microenvironment for incorporation and evolution of polymer synthesis systems.
Cells↗
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 hydrogen-bonded 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.
Biological Transport↗
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.
Animals↗
Oxidative stress impairs the function of sarcoplasmic reticulum by oxidation of sulfhydryl groups in the Ca2+-ATPase.
Sarcoplasmic reticulum (SR) microsomes were oxidized by exposure to peroxydisulfate, hydrogen peroxide, or iron/ascorbate or by extended storage. The decline in Ca2+-ATPase activity, Ca2+ transport, and the increase in Ca2+ permeability which occurred under these conditions did not appear to result from lipid oxidation because these functional changes were not correlated with the amount of thiobarbituric acid-reactive lipid. Consistent with this interpretation, lipid antioxidants did not prevent the decline in SR function. This suggests that inhibition was independent of lipid oxidation. Instead, oxidation directly inhibited the Ca2+-ATPase. The decline in enzyme activity may be due to oxidation of SH groups, as suggested by the ability of reducing agents to prevent inhibition, the decline in sulfhydryl content of oxidized SR, and the ability of sulfhydryl-binding agents to inhibit Ca2+-ATPase. Inhibition was not primarily due to crosslinking of the Ca2+-ATPase, because sodium dodecyl sulfate-polyacrylamide gels of normal and oxidized SR showed that the area of the Ca2+-ATPase band was not correlated with the Ca2+-ATPase activity. Inhibition of the Ca2+-ATPase by oxidative stress is relevant to models of cellular dysfunction in which toxicity is caused by a rise in intracellular calcium.
Animals↗
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.
Biological Evolution↗
Proton permeation through model membranes.
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Coloring Agents↗
Calcium efflux from sarcoplasmic reticulum microsomes due to oxidation and sulfhydryl-binding agents.
Calcium permeability of sarcoplasmic reticulum (SR) microsomes was measured after aging or after exposure to peroxydisulfate or to sulfhydryl-binding agents. Under conditions where the Ca2+-ATPase was active, the maximum net release of Ca2+ was not significantly different between control and oxidized SR. However, when calcium uptake was prevented by EGTA or apyrase, the Ca2+ permeability of oxidized microsomes was 2 to 3 times greater than control of low (10(-9), 10(-7) M) but not high (10(-6) M) levels of external calcium. The observation that vesicles preincubated with 5 mM dithiothreitol loaded up to 3 times as much calcium and had a slightly lower calcium permeability coefficient than control vesicles suggested that sulfhydryl oxidation might modulate calcium flux. This hypothesis was tested by exposing to sulfhydryl-binding agents: silver, arsenite, and p-chloromercuriphenylsulfonic acid. Sulfhydryl-binding agents initiated a rapid release of calcium from microsomes, and release was halted by dithiothreitol. Inhibition of calcium transport could not entirely account for the apparent increase in permeability because the calcium permeability of SR treated with sulfhydryl-binding agents was 5 times greater than that of SR exposed to Ca2+-ATPase inhibitors. These results suggest that oxidation may increase the calcium permeability of SR by allowing calcium loss through a channel that can be gated by sulfhydryl oxidation.
4-Chloromercuribenzenesulfonate↗