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C D Stubbs

Publications and source records attributed to C D Stubbs.

51 records · Page 3Linked to original sources
PubMed1988

Ethanol-induced modifications to membrane lipid structure: effect on phospholipase A2-membrane interactions.

Chronic ethanol intoxication leads to the development of a resistance to lipid disordering by ethanol, a phenomenon known as "membrane tolerance". In the absence of the added ethanol, the lipid order, as measured by ESR and fluorescence techniques, does not necessarily change as a result of chronic ethanol ingestion (as in liver microsomes, for example). This suggests that the spectroscopic techniques detect tolerance somewhat indirectly, in that the modification responsible may reside in a region distinct from that being probed and also raises the question of whether membrane tolerance is necessarily associated with an alteration in the membrane lipid structure. Here we show that liver microsomes from rats treated chronically with ethanol are rendered relatively resistant to the hydrolytic action of exogenous phospholipase A2, compared to preparations from control animals. This resistance persists in reconstituted lipid vesicles prepared from extracted phospholipids. Since the same substrate (1-palmitoyl-2-N-(4-nitrobenzo-2-oxa-1,3-diazole)amino caproylphosphatidylcholine) was used in both membranes from ethanol-treated animals and controls, the modification appears to reside in the structure and/or organization of the membrane. Further evidence that the lipid structure is modified by chronic ethanol treatment is provided by the observation that perturbance of the membrane structural integrity by increasing levels of oleic acid led to a progressive loss of the ethanol-induced relative resistance to hydrolysis by phospholipase A2. The results of this study support the idea that membrane tolerance involves a modification to lipid structure probably at the bilayer surface. The use of exogenous phospholipase A2 provides a new method for probing the structural modifications induced by chronic ethanol ingestion.

Animals↗
PubMed1987

Modulation of membrane protein function by bilayer lipids.

In many instances, the composition of fatty acyl groups of membrane phospholipids can be modified to achieve a range of fatty acyl unsaturation without any detectable change in bulk membrane fluidity. At the same time, the function of membrane proteins may be considerably altered, raising questions concerning the property of the lipids that brings about this altered protein function. There is some evidence that the lipids may be laterally distributed in a heterogeneous manner throughout the membrane, and changes in this distribution could be responsible for the effects on proteins. There is also increasing evidence for specific interactions between individual molecular species and membrane proteins that may also modulate membrane protein function.

Animals↗
PubMed1986

The effects of dietary (n-3) fatty acid supplementation on lipid dynamics and composition in rat lymphocytes and liver microsomes.

Rats were fed diets devoid of (n-3) fatty acids (olive oil supplementation) or high in (n-3) fatty acids (fish oil supplementation) for a period of 10 days. In spleen lymphocytes and liver microsomes derived from animals fed fish oil diets, relatively high levels of (n-3) eicosapentaenoic (20:5), docosapentaenoic (22:5) and docosahexaenoic acids (22:6) were obtained compared to minimal levels when fed the olive oil diet. When the average lipid motional properties were examined by measuring the fluorescence anisotropy of diphenylhexatriene, no significant different was found between intact liver microsomes from animals fed the two diets. However, when lipid motion was examined in vesicles of phosphatidylcholine, isolated from the microsomes from fish oil fed animals (21.4% (n-3) fatty acids), the fluorescence anisotropy was significantly less than the corresponding phosphatidylcholine from olive oil fed animals (5.6% (n-3) fatty acids), indicating a more disordered or fluid bilayer in the presence of higher levels of (n-3) fatty acids. Phosphatidylethanolamine (n-3) fatty acids were also elevated after fish oil supplementation (41.3% of total fatty acids), compared to the level after olive oil supplementation (21.4%). The major effect of the fish oil supplementation was a replacement of (n-6) arachidonic acid by the (n-3) fatty acids and when this was 'modeled', using liposomes of synthetic lipids, 1-palmitoyl-2-arachidonyl(n-6) or docosahexaenoyl(n-3)-phosphatidylcholine, significant differences in lipid motional properties were found, with the docosahexaenoate conferring a more disordered or fluid lipid environment. Thus it appears that although lipid order/fluidity can be significantly decreased by increases in the highly unsaturated (n-3) fatty acid levels, alterations in membrane domain organization and/or phospholipid molecular species composition effectively compensated for the changes, at least as far as average lipid motional properties in the intact membranes was concerned.

Animals↗
PubMed1985

Solvent relaxation in lipid bilayers with dansyl probes.

The solvent relaxation properties of the dansyl group attached to two lipids (dansylphosphatidylethanolamine and dansylphosphatidylserine), a fatty acid (dansylundecanoic acid), and two drugs (dansylbenzocaine and dansylpropranolol) were compared in a variety of different lipid systems. Several methods for characterising solvent relaxation were compared in detail for dansylpropranolol in bilayer vesicles of egg phosphatidylcholine. It was shown that the relaxation process is non-monoexponential; nevertheless, for comparative purposes, a model was adopted in which the lifetime associated with the negative exponent in a two exponential decay analysis, obtained at a particular energy on the red edge of emission, was taken as an approximation to a 'solvent relaxation' rate. A negative exponent, indicative of solvent relaxation processes, occurring in the nanosecond time-scale, was found only for dansylpropranolol, dansylPE and dansylundecanoic acid. On addition of the spin probe, 5-doxylstearate, the negative exponent was unaffected in liquid-crystalline phase lipids but was no longer found in gel-phase lipid in the case of dansylpropranolol, while for dansylPE the relaxation time was reduced. On the basis of these types of measurement it was possible to distinguish between different lipid environments using the same probe or between different dansyl environments of the different probes in the same lipid in cases where this would have been difficult or impossible solely on the basis of steady-state or fluorescence lifetime measurements.

Dansyl Compounds↗
PubMed1985

Structural and related functional changes in sarcoplasmic reticulum induced by long-chain fatty acids.

The effect of palmitic and oleic acids on Ca2+-ATPase activity in coupled preparations of sarcoplasmic reticulum isolated from rabbit hind leg muscle have been compared with their effects on vesicles uncoupled with Ca2+ ionophore, A23187. Palmitate at 2 microM X mg protein-1 has no significant effect on enzyme activity and does not uncouple catalytic activity from calcium accumulation within the vesicles. Oleic acid at 1 microM X mg protein-1 uncouples the vesicles, whereas 2 microM X mg protein-1 completely inhibits Ca2+-ATPase activity. Fluorescence anisotropy of diphenylhexatriene is not significantly altered by palmitate, but a large transient increase in motion of the probe is observed with addition of oleic acid. The effects of oleic acid on enzyme activity are not mediated via an effect on the bulk properties of the hydrophobic domain of the membrane lipids.

Animals↗
PubMed1984

The dynamics of lipid motion in sarcoplasmic reticulum membranes determined by steady-state and time-resolved fluorescence measurements on 1,6-diphenyl-1,3,5-hexatriene and related molecules.

Steady-state and time-resolved fluorescence anisotropy measurements were made on 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1-acyl-2-(DPH)-phosphatidylcholine (DPH-PC) incorporated into sarcoplasmic reticulum membranes. The results were analysed in terms of the 'wobbling-in-cone' model. Considerable differences in the fluorescence parameters were found. In particular TMA-DPH and DPH-PC showed a smaller cone angle, relating to the range of acyl chain motion, compared to DPH, taken to be a reflection of a difference in probe locations. The influence of the protein component was also found to restrict DPH motion more than TMA-DPH and DPH-PC. Effectiveness in assessment of perturbation of the membrane by the non-esterified fatty acid, oleic acid again revealed differences. The steady-state anisotropy decreased on addition of oleic acid; a recovery to control values was observed with DPH but not with the other probes. Time-resolved parameters followed the same pattern. The results of this work demonstrated the effectiveness of these three probes in revealing differences in membrane properties, such as protein and fatty acid perturbation of membrane lipid structure and dynamics.

Animals↗
PubMed1983

The effect of concanavalin A on the incorporation of fatty acids into lymphocyte phospholipids.

The incorporation of radioactively labeled palmitic, stearic, oleic, linoleic and arachidonic acid into the phospholipids of lymphocytes was studied. When concanavalin A (Con A) was added a gradual increase in the incorporation was found over the period investigated (0-4 h). Changes were not detected over the first 15 min. The increased incorporation at 4 h was shown to be Con A dose dependent and the Con A concentrations for the maximum effect on fatty acid incorporation (at 4 h) and on [3H]thymidine incorporation (at 72 h) were the same. With the exception of stearic acid all the other fatty acids showed an increased incorporation into phosphatidyl choline on addition of Con A, linoleic and oleic acids also had an increased incorporation into phosphatidyl ethanolamine.

Animals↗
PubMed1981

Effect of double bonds on the dynamic properties of the hydrocarbon region of lecithin bilayers.

The motional properties of the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene have been assessed in liposomes of various lecithins: dipalmitoyl-phosphatidylcholine (DPPC), dioleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1- palmitoyl-2-linoleoylphosphatidylcholine (PLPC), and 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC). The temperature dependence of the steady-state fluorescence anisotropy was determined within the range of 10-50 degrees C. Nanosecond measurements were also made and the results analyzed by a model of wobbling diffusion confined within a cone. In this treatment the cone angle (theta c) obtained relates to the degree of order of the fatty acyl chains, and the wobbling diffusion constant (Dw) relates to the rate of motion of the hydrocarbon chains. Theta c was increased (order decreased) and Dw increased (rate increased) for the first double bond (POPC) as compared to the fully saturated DPPC. In all four unsaturated lecithins the temperature dependences of theta c and Dw were similar: theta c increased by about 4-5 degrees, and Dw increased by a factor of 1.5 per 10 degrees C. DPPC in the liquid-crystalline phase also showed a similar trend. The absolute values, on the other hand, showed some differences: for theta c, DOPC (78 degrees at 37 degrees C) greater than PAPC (75 degrees) greater than POPC (70 degrees) congruent to PLPC (69 degrees) greater than DPPC (20 degrees), and for Dw, PAPC (0.28 ns-1) greater than DOPC (0.22 ns-1) congruent to PLPC (0.24 ns-1) greater than POPC (0.21 ns-1) greater than DPPC (0.05 ns-1). The differences among the unsaturated lecithins are relatively small suggesting that the first double bond introduced into a lecithin plays the most important role at physiological temperatures. In 1:1 mixtures of the disaturated lecithin, DPPC, with the unsaturated lecithins, the temperature of the phase transition of the former was lowered and broadened, by approximately the same amount, irrespective of the differences in unsaturation.

Chemical Phenomena↗
PubMed1980

Incubation of exogenous fatty acids with lymphocytes. Changes in fatty acid composition and effects on the rotational relaxation time of 1,6-diphenyl-1,3,5-hexatriene.

Lymphocytes were incubated with various long chain fatty acids bound to albumin. Incubation for 20 h with unsaturated fatty acids resulted in uptake into the neutral lipids and phospholipids. The addition of concanavalin A enhanced the uptake. With the unsaturated fatty acids the rotational relaxation time of 1,6-diphenyl-1,3,5-hexatriene (DPH) was decreased, while with saturated fatty acids there was only a very small effect. The effect on the rotational relaxation times with unsaturated fatty acids was found to be due to the formation of lipid droplets in the cytoplasm. When plasma membrane free of lipid droplets was prepared, there was no effect on the rotational relaxation time despite incorporation of either linoleate or palmitate into the membrane phospholipids. Phospholipid liposomes prepared from plasma membranes obtained from cells which had been cultured with and without exogenous linoleate gave identical rotational relaxation times regardless of the history of the cells from which they were obtained. It is concluded that although the fatty acid composition of lymphocyte plasma membrane can be modified by exogenous fatty acids, there is little effect on the degree of order of the membrane phospholipid fatty acyl chains as monitored by the effects on the rotational relaxation time of DPH.

Animals↗
PubMed1979

The isolation and analysis of the luminal plasma membrane of calf urinary bladder epithelium.

The luminal plasma membrane of calf urinary bladder epithelium (urothelium) has been isolated by a method designed to preserve enzymic activity as well as structural integrity. The yield was about 80 micrograms per calf bladder. Low levels of 5' nucleotidase, Mg2+-ATPase and (Na+ + K+)-ATPase activities were found in the luminal membrane fraction. Cerebroside was the major lipid present and dodecyl sulphate gel electrophoresis revealed a complex protein and glycoprotein composition in the whole membrane. A membrane fraction consisting of only the plaque areas was shown to have a simpler protein composition with major polypeptides of apparent Mr 12 000 and 22 000. These may associate to form a 30 000 apparent Mr complex which could represent the individual 'particles' of the dodecameric subunits seen by electron microscopy in the plaque regions.

Animals↗