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Biomedical subjects

A Seelig

Publications and source records attributed to A Seelig.

52 records · Page 3Linked to original sources
PubMed1987

Local anesthetics and pressure: a comparison of dibucaine binding to lipid monolayers and bilayers.

The binding of the local anesthetic dibucaine to monolayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was studied with a Langmuir trough at pH 5.5 (22 degrees C, 0.1 M NaCl). At this pH value only the charged form of the local anesthetic exists in solution. Charged dibucaine was found to be surface active and to penetrate into the lipid monolayer, with the hydrophobic part of the molecule being accommodated between the fatty acyl chains of the lipid. The dibucaine intercalation could be quantitated by measuring the expansion of the film area, delta A, at constant surface pressure, pi. At a given surface pressure, delta A increased with increasing dibucaine in the buffer phase. On the other hand, keeping the dibucaine concentration constant, the area increase, delta A, was strongly dependent on the surface pressure. The area increase, delta A, was large at low surface pressure and decreased with increasing surface pressure. A plot of the relative change in surface area, delta A/A, versus the surface pressure yielded straight lines in the pressure range of 25-36 mN/m for five different concentrations. The delta A/A vs. pi isotherms intersected at pi = 39.5 +/- 1 mN/m with delta A = O, indicating that charged dibucaine apparently can no longer penetrate into the monolayer film. By making judicial assumptions about the area requirement of dibucaine the monolayer expansion curves could be transformed into true binding isotherms. Dibucaine binding isotherms were constructed for different monolayer pressures and were compared to a bilayer binding isotherm measured under similar conditions with ultraviolet spectroscopy. The best agreement between monolayer and bilayer binding data was obtained for a monolayer held at a pressure of 30.7 to 32.5 mN/m, which can thus be considered as the bilayer-monolayer equivalence pressure. It is further suggested from this analogy that the binding of dibucaine does not change the internal pressure in the bilayer phase, at least not in the concentration range of physiological interest (0-2 mM dibucaine) but induces a lateral expansion. At higher molar ratios of cationic dibucaine to lipid, chi b, in the monolayer (chi b greater than 0.20) the area increase is larger than would be expected from the molecular dimensions of dibucaine. This is probably due to charge repulsion effects, which at still higher molar ratios (chi b greater than 0.6) lead to a micellisation. The pressure dependence of the intercalation of cationic dibucaine into lipid membranes may also be of relevance for the phenomenon of pressure reversal in anesthesia.

Dibucaine↗
PubMed1985

Phospholipid composition and organization of cytochrome c oxidase preparations as determined by 31P-nuclear magnetic resonance.

The molecular organization as well as the composition of the phospholipids in cytochrome c oxidase preparations (bovine heart) were investigated by 31P-nuclear magnetic resonance. In the so-called 'lipid-rich' preparation the lipids were found to form a fluid bilayer around the enzyme since the 31P-NMR spectrum was characteristic of a fast, axially symmetric motion of the phosphate groups with a chemical shift anisotropy of delta sigma = -45 ppm. In contrast, the 'lipid-depleted' cytochrome c oxidase gave rise to a broader spectrum where the motion of the phospholipids was no longer axially symmetric. Nevertheless, the total width of the spectrum was still considerably narrower than observed for immobilized phospholipids in solid crystals. Both enzyme preparations were dissolved in 1% detergent solution and used for high-resolution 31P-NMR spectroscopy. Narrow lines of about 20 Hz linewidth were obtained for both types of enzyme preparations, and well-resolved resonances could be assigned to cardiolipin, phosphatidylethanolamin and phosphatidylcholine. The major differences between lipid-rich and lipid-depleted cytochrome c oxidase were the absolute amount of phospholipid associated with the protein and the relative contribution of the individual lipid classes to the 31P-NMR spectrum. For lipid-rich cytochrome c oxidase about 130 molecules phospholipid were bound per enzyme (approx. 11 cardiolipins, 54 phosphatidylethanolamines and 64 phosphatidylcholines). For lipid-depleted cytochrome c oxidase only 6-18 lipids were bound per enzyme (1 or 2 cardiolipins, 3-8 phosphatidylethanolamines and 2-8 phosphatidylcholines). In contrast to earlier suggestions that cardiolipin is the only remaining lipid in lipid-depleted cytochrome c oxidase, the 31P-NMR studies demonstrate that all three lipids remain associated with the protein.

Animals↗
PubMed1981

Copper and manganese electron spin resonance studies of cytochrome c oxidase from Paracoccus denitrificans.

The two-subunit cytochrome c oxidase from Paracoccus denitrificans contains two heme a groups and two copper atoms. However, when the enzyme is isolated from cells grown on a commonly employed medium, its electron paramagnetic resonance (EPR) spectrum reveals not only a Cu(II) powder pattern, but also a hyperfine pattern from tightly bound Mn(II). The pure Mn(II) spectrum is observed at -40 degrees C; the pure Cu(II) spectrum can be seen with cytochrome c oxidase from P. denitrificans cells that had been grown in a Mn(II)-depleted medium. This Cu(II) spectrum is very similar to that of cytochrome c oxidase from yeast or bovine heart. Manganese is apparently not an essential component of P. denitrificans cytochrome c oxidase since it is present in substoichometric amounts relative to copper or heme a and since the manganese-free enzyme retains essentially full activity in oxidizing ferrocytochrome c. However, the manganese is not removed by EDTA and its EPR spectrum responds to the oxidation state of the oxidase. In contrast, manganese added to the yeast oxidase or to the manganese-free P. denitrificans enzyme can be removed by EDTA and does not respond to the oxidation state of the enzyme. This suggests that the manganese normally associated with P. denitrificans cytochrome c oxidase is incorporated into one or more internal sites during the biogenesis of the enzyme.

Animals↗
PubMed1978

Lipid-protein interaction in reconstituted cytochrome c oxidase/phospholipid membranes.

Deuterium and 31P nuclear magnetic resonance have been employed in an investigation of the effect of cytochrome c oxidase (EC 1.9.3.1) on the structure of lecithin bilayers. Cytochrome c oxidase was isolated from beef heart mitochondria in lipid-free form and reconstituted as a functional enzyme in bilayers composed of synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Two separate reconstitution experiments were performed in which the lipid was selectively deuterated either at the C-5' or at the C-14' segment of the palmitic acyl chain. The phospholipid-to-protein ratio of both reconstituted complexes was 0.74 (mg/mg), corresponding to about 200 molecules lipid per molecule cytochrome c oxidase. The deuterium quadrupole splitting deltanuQ, and the phosphorus chemical shielding anisotropy, deltasigma, of the cytochrome c oxidase-phospholipid recombinants were measured as a function of temperature and compared to the results obtained for the pure lipid membrane without protein for the pure lipid membrane without protein. deltanuQ and deltasigma are highly sensitive to the structural organization of the lipid membrane and these measurements demonstrate that the incorporation of cytochrome c oxidase into phosphatidylcholine bilayers leads to a more disordered conformational state of the lipids. This result can be explained by a rapid exchange between lipids in direct contact with hydrophobic protein and those further away from it (exchange rate greater than 10(4) Hz). The irregular protein surface is sensed by all lipid molecules and induces a more disordered bilayer structure. In contrast to previous interpretations, our measurements do not suggest a special type of boundary lipid.

Electron Transport Complex IV↗
PubMed1977

Effect of a single cis double bond on the structures of a phospholipid bilayer.

The ordering of the hydrocarbon chains and the rates of lipid motion are two independent parameters characterizing the structure and the dynamics, respectively, of a bilayer membrane. In this work, deuterium magnetic resonance has been used to elucidate the influence of a single cis double bond on the hydrocarbon chain ordering of a phospholipid bilayer. 1-Palmitoyl-2-oleoyl-3-sn-phosphatidylcholine was specifically deuterated at various segments of the palmitic acyl chain and at the 9, 10 position of the oleic acyl chain, and the segmental order parameters were deduced from the quadrupole splittings of the unsonicated bilayer phases. The shape of the order profile of the palmitic acyl chain is similar to that observed for the corresponding fully saturated membrane, but the magnitude of the order parameters is distinctly smaller in the unsaturated system. This demonstrates that the presence of a double bond in a membrane causes a more disordered conformation of the hydrocarbon chains. Considering the relative flexibility within the palmitic acyl chain, the deuterium resonance data indicate a local stiffening of those segments which are located in the vicinity of the double bond. The membrane fluidity was investigated using a nitroxide-labeled stearic acid spin probe. The smaller electron paramagnetic resonance line width in bilayers of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine demonstrates an increased fluidity compared to bilayers of 1,2-dipalmitoyl-3-sn-phosphatidylcholine.

Magnetic Resonance Spectroscopy↗
PubMed1975

Bilayers of dipalmitoyl-3-sn-phosphatidylcholine. Conformational differences between the fatty acyl chains.

Dipalmitoyl-3-sn-phosphatidylcholine is specifically deuterated at the C-2 position of the fatty acyl chains. Using deuterium magnetic resonance it is then possible to probe the chain conformation in the vicinity of the polar head groups. Three separate quadrupole splittings are observed for bilayers of 1,2[2'-2H2]palmitoyl-3-sn-phosphatidylcholine, indicating that the two chains behave differently. The synthesis of phosphatidylcholines each deuterated in only one chain allows the assignment of the three resonances. It is concluded that the beginnings of the two chains have orientations parallel and perpendicular to the bilayer normal. The data further suggest the possibility of two long-lived conformations of the glycerol constituent.

Binding Sites↗