Search PubMed⌕ Search

Biomedical subjects

D W Deamer

Publications and source records attributed to D W Deamer.

At least 55 records · Page 3Linked to original sources
PubMed1986

Enzymatic reconstitution of brain membrane and membrane opiate receptors.

A new method using lysophosphatide and acyl-CoA as detergents has been used to solubilize the rat brain opiate receptor. After solubilization, lysophosphatide and acyl-CoA can be almost completely removed by an enzymatic reaction that uses an acyltransferase from rat liver microsomes and reconstitutes the solubilized receptor in membranous vesicles. Morphological studies performed with negative staining and freeze-fracture electron microscopy revealed that the general appearance and intramembrane particle distribution of fracture faces in the reconstituted membrane are similar to those of the native membrane; this indicates that hydrophobic protein components of the original membrane were incorporated during reconstitution. Reconstituted membrane, however, contained higher levels of phosphatidylcholine and lower levels of cholesterol. The activities of the membrane-bound enzymes Na+, K+-ATPase and Ca2+, Mg2+-ATPase in the reconstituted system were 24 and 3%, respectively, those of the native membrane. Although binding of opiate ligands to the reconstituted membrane was stereospecific and saturable, higher concentrations of some of the unlabeled ligands were required to inhibit binding of the radiolabeled ligands. These changes in receptor characteristics are likely due to changes in lipid composition, physical state, and/or distribution of the lipids in the reconstituted membrane bilayer. This conclusion is supported by an increase in the affinity of opiate ligands for reconstituted membrane after adjustment of the latter's lipid composition to match more closely that of the original membrane. This was accomplished by treatment with phospholipid exchange protein to remove the excess phosphatidylcholine and by incorporation of cholesterol into the reconstituted membrane.

Acyltransferases↗
PubMed1986

Liposome-mediated delivery of antibody to a Drosophila cell line.

Large, unilamellar vesicles composed of equimolar amounts of acidic phosopholipids and phosphatidylethanolamine were able to deliver fluorescent dye [5(6)-carboxyfluorescein] or a monoclonal antibody directed against intermediate-filament proteins to a Drosophila cell line (Kc cells). Millimolar Ca2+ or protamine sulfate in microgram quantities triggered rapid, synchronous delivery of either solute. Delivery required a specific lipid composition: liposomes composed of 1:1 mole ratios of phosphatidylethanolamine:phosphatidylserine were able to deliver their contents, but not if phosphatidylcholine was substituted for phosphatidylethanolamine. Light microscopic observation of Kc cells incubated with free dye or antibody alone showed very little uptake, a result indicating that encapsulation within liposomes is a prerequisite for substantial delivery. Moreover, the stability of adhering vesicles in the absence of calcium or protamine sulfate, the lipid specificity, and the rapid onset of intracellular fluorescence after triggering suggest that vesicle-cell fusion is the predominant mode of solute uptake. Fusion of liposomes with the cell membrane was confirmed by freeze-fracture electron microscopy, which showed liposome vesicles first adhering to cell surfaces, then undergoing fusion when calcium or protamine sulfate was added.

Animals↗
PubMed1985

The effect of general anesthetics on the proton and potassium permeabilities of liposomes.

The pump-leak hypothesis of general anesthesia proposes that anesthetics act by increasing the functional proton permeability of membranes, particularly those of synaptic vesicles. Since transmembrane proton gradients are required for neurotransmitter accumulation, decay of such gradients by an uncompensated anesthetic-induced leak would result in loss of neurotransmitter from the vesicles, followed by synaptic block and anesthesia. We have tested this hypothesis by determining the effect of four different general anesthetics on the relative permeabilities of liposome membranes to protons and potassium ions. In all cases, physiologically relevant levels of anesthetics caused a 200 to 500 percent increment in ionic permeability. There was no marked preference for protons, suggesting that the anesthetics did not induce a leak specific for this ionic species. Instead the anesthetics appeared to produce a more general defect available to both protons and potassium ions which resulted in a functional increment in proton permeability. These observations were compared with available data on proton transport rates by synaptic vesicle ATPase enzymes. The magnitude of the anesthetic-induced leak could not be compensated by the ATPase, which is only capable of a 40 percent increase in rate when uncoupled. We consider these results to be consistent with the pump-leak hypothesis.

Anesthesia, General↗
PubMed1985

A novel method for encapsulation of macromolecules in liposomes.

Hemoglobin and alkaline phosphatase were each encapsulated in phosphatidylcholine liposomes using a dehydration-rehydration cycle for liposome formation. In this method, liposomes prepared by sonication are mixed in aqueous solution with the solute desired to be encapsulated and the mixture is dried under nitrogen in a rotating flask. As the sample is dehydrated, the liposomes fuse to form a multilamellar film that effectively sandwiches the solute molecules. Upon rehydration, large liposomes are produced which have encapsulated a significant fraction of the solute. The optimal mass ratio of lipid to solute is approx. 1:2 to 1:3. This method has potential application in large-scale liposome production, since it depends only on a controlled drying and rehydration process, and does not require extensive use of organic solvents, detergents, or dialysis systems.

Alkaline Phosphatase↗
PubMed1985

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.

Animals↗
PubMed1983

Proton-hydroxide permeability of liposomes.

Liposome permeability to net proton-hydroxide flux was compared under two conditions that have produced extreme values in previous studies. In the first [Nichols, J.W. & Deamer, D.W. (1980) Proc. Natl. Acad. Sci. USA 77, 2038-2042] small pH gradients were permitted to decay near pH 7 and permeability coefficients in the range of 10(-4) cm/sec were obtained. In the second [Nozaki, Y. & Tanford, C. (1981) Proc. Natl. Acad. Sci. USA 78, 4324-4328] the decay rate of large pH gradients (approximately equal to 3 pH units) was measured and apparent permeability coefficients in the range of 10(-9) cm/sec were reported. We have repeated the conditions of the latter study and have found that the decay of large pH gradients produces a diffusion potential that limits net proton-hydroxide flux. The permeability coefficients calculated from the flux are therefore underestimated and do not reflect intrinsic proton permeability. With the same liposome preparations under conditions in which small gradients decay so that diffusion potentials are negligible, the measured permeability coefficient is near 10(-4) cm/sec, as reported earlier.

Anions↗
PubMed1982

Purification of acyl CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase.

Acyl coenzyme A:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase (EC 2.3.1.23) is capable of forming lipid bilayer vesicles from its soluble substrates lysophosphatidylcholine (LPC) and oleoyl CoA. This suggested a purification method in which rat liver microsomes are first washed with deoxycholate to increase specific activity of the endogenous acyltransferase approximately fivefold, then solubilized by the detergent effect of excess LPC and oleoyl CoA in 1:1 stoichiometric ratios. As the LPC is converted to phosphatidylcholine by acyl group transfer, the detergent effect is lost and lipid vesicles containing the enzyme activity are produced. Other microsomal proteins are excluded from the vesicles. The vesicles may be separated by density gradient flotation and are found to contain acyltransferase with a specific activity of 9-10 mu mol/mg/min. This reflects a purification of approximately 140-fold, about ten times greater than achieved in previous studies.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1982

Encapsulation of macromolecules by lipid vesicles under simulated prebiotic conditions.

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.

Animals↗
PubMed1981

Selective interaction of D-beta-hydroxybutyrate dehydrogenase with intracellular membranes.

We are investigating the properties of pre-existing membrane structures that may contribute to localization of newly formed polypeptides on target membranes. To this end, D-beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30) was purified from inner membranes of rat liver mitochondria and interacted with three different cellular membranes, as well as with liposomes prepared from membrane lipid extracts. (1) The purified lipid-free enzyme displayed little catalytic activity. Its activity was restored by interaction with rat liver mitochondrial inner membranes or microsomal membranes, but not with rat erythrocyte plasma membrane vesicles. (2) Plasma membranes from which membrane proteins had been partially removed did not reactivate the enzyme, but microsomal membranes treated in a similar manner displayed an increased efficiency of reactivation. (3) The selective reactivation found in the three membrane species was confirmed in liposomes prepared with total lipid extracts of the native membranes. The results suggest that the interaction of exogeneously added enzyme with the membranes is primarily dependent on lipid components or some specific lipid environment on the acceptor membranes.

Animals↗
PubMed1981

Carbon dioxide efflux accompanies release of fertilization acid from sea urchin eggs.

"Fertilization acid" is released from sea urchin eggs upon fertilization and decreases the pH of the surrounding seawater. In bicarbonate-free artificial seawater flushed with nitrogen gas, the pH shift still occurs but returns to the original value in a few minutes, suggesting that the released acid volatile. A likely candidate for a volatile acid is carbon dioxide released from the eggs. Therefore, the total CO2 content of seawater was measured pre- and post-fertilization and was found to be correlated stoichiometrically with released proton equivalents, leading to the conclusion that fertilization acid is largely carbon dioxide. Manometric analysis of cell extracts and ashed eggs suggest that the carbon dioxide may be stored in the unfertilized egg as an inorganic carbonate.

Animals↗
PubMed1980

Measurement of net proton-hydroxyl permeability of large unilamellar liposomes with the fluorescent pH probe, 9-aminoacridine.

The fluorescent probe 9-aminoacridine was used to measure the rate of decay of experimentally established pH gradients across liposome membranes. From the rate of decay, separate permeability coefficients for protons (PH) and hydroxyls (POH) were calculated and summed to yield the net proton-hydroxyl permeability (Pnet). The net permeability of protons and hydroxyls was found to be approximately 10(-4) cm/s, six orders of magnitude greater than that measured for sodium and pyrophosphate ions under similar conditions. This suggests that protons and/or hydroxyls cross lipid bilayers by a different mechanism than do other monovalent cations and anions. In addition, the measurements provide a standard for net proton-hydroxyl permeability in pure phospholipid bilayers for comparison with biological membranes.

Aminoacridines↗
PubMed1980

Endogenously produced CO2 induces stationary phase in Tetrahymena cultures.

Media concentration of total soluble CO2 increases with culture age of Tetrahymena pyriformis. CO2 is a weak acid and is capable of acidifying intracellular pH (pHi). Changes in pHi have been demonstrated to affect cell metabolism and growth in many systems. For these reasons, we investigated whether the concentrations of CO2 produced in vitro were sufficient to affect cell proliferation and pHi in Tetrahymena. In this study, we used DMO to mimic the weak acid properties of CO2. DMO is freely permeable to membranes in its uncharged form and has a pKa similar to that of CO2/HCO3(-). In addition, it has the advantages of being metabolically inert and non-volatile. At concentrations similar to endogenously produced CO2, DMO acidifies pHi and arrests culture growth. In addition, procedures are described which decrease the media CO2 concentrations in both growing and non-growing cultures. These conditions lead to increased maximum culture density at stationary phase. The data indicate that, under our conditions, accumulation of CO2 in the culture leads to cessation of growth, probably through elimination of transmembrane pH gradients, which are necessary for regulation of metabolism and growth.

Ammonium Chloride↗
PubMed1980

Net proton-hydroxyl permeability of large unilamellar liposomes measured by an acid-base titration technique.

The net proton-hydroxyl permeability of large unilamellar liposomes has been measured by an acid-base pulse titration technique and has been determined to be several orders of magnitude greater than that measured for other monovalent ions. This permeability is relatively insensitive to variations in lipid composition. Proton permeability and hydroxyl permeability vary with pH 6 to 8, and this variation can occur in the absence of alterations in surface charge density resulting from titrations of acidic and basic groups on the lipids. In order to account for the exceptionally high proton-hydroxyl permeability with respect to other monovalent ions, we propose that protons or hydroxyls or both interact with clusters of hydrogen-bonded water molecules in the lipid bilayer, such that they are transferred across the bilayer by rearrangement of hydrogen bonds in a manner similar to their transport in water and ice.

Acid-Base Equilibrium↗
PubMed1980

An enzymatically driven membrane reconstitution from solubilized components.

Acyltransferase activity is present in a variety of membranes species, including liver microsomes. The substrates of this enzyme are lysophosphatides and acyl CoA derivatives. We have found that the detergent effect of these substrates can be used to solubilize rat liver microsomes. If the solubilized fraction in incubated, the acyltransferase acylates the lysophosphatide and thereby degrades the detergent effect so that vesicular membranes re-form. Gel electrophoresis patterns show that the reconstituted membranes contain all of the major protein components of the original microsomes. A marker enzyme for liver microsomes, NADPH-cytochrome c reductase, was present in the reconstituted membranes at 70% of the specific activity in the original microsomes, and freeze-fracture electron microscopy showed intramembrane particles on all fracture faces. The system may provide a useful model for studies particles on all fracture faces. This system may provide a useful model for studies of certain membrane biogenesis reactions that utilize acyltransferase in vivo.

Acyl Coenzyme A↗