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Biomedical subjects

A Seelig

Publications and source records attributed to A Seelig.

17 recordsLinked to original sources
PubMed1992

Interaction of a substance P agonist and of substance P antagonists with lipid membranes. A thermodynamic analysis.

The molecular characteristics of the neuropeptide substance P (SP), its agonist [Sar9,Met-(O2)11]SP, and three of its antagonists [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, [D-Arg1,D-Trp7,9,Leu11]SP, and [D-Pro2,D-Trp7,9]SP were investigated at the air/water interface and when bound to lipid monolayers and bilayers. Measurement of the Gibbs adsorption isotherm showed that the surface areas of SP and its agonist (240 +/- 5 A2 at biologically relevant concentrations) were distinctly larger than those of the antagonists (138 +/- 5 A2) [Seelig, A. (1990) Biochim. Biophys. Acta 1030, 111-118]. The surface activity of the peptides increased in the order [Sar9,Met(O2)11]SP less than SP less than [D-Pro2,D-Trp7,9]SP less than [D-Arg1,D-Trp7,9,Leu11]SP = [D-Arg1,D- Pro2,D-Trp7,9,Leu11]SP and correlated with the respective binding affinities to lipid membranes. The agonist did not insert into neutral and negatively charged bilayers or into densely packed lipid monolayers (at surface pressures greater than 31 mN/m). In contrast, the three antagonists gave rise to a strong binding both to neutral and to charged lipid monolayers and bilayers. The degree of binding was evaluated from the area increase of lipid monolayers upon peptide insertion, and the binding isotherms were analyzed in terms of the Gouy-Chapman theory. At the monolayer-bilayer equivalence pressure of approximately 32 mN/m, the binding can be described by a surface partition equilibrium with binding constants of (4.5 +/- 0.1) x 10(3) M-1 for [D-Pro2,D-Trp7,9]SP and (1.3 +/- 0.1) x 10(4) M-1 for both [D-Arg1,D-Trp7,9,Leu11]SP and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP for pure palmitoyloleoylphosphatidylcholine (POPC) membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed1991

Molecular interaction of the proteasome (multicatalytic proteinase). Evidence that the proteasome is not a constituent of the '26 S' multienzyme complex.

On the basis of recent reports that suggested that proteasomes, via an ATP-dependent process, become integral components of a '26 S' complex possessing 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity, we have investigated the molecular interaction of proteasomes in ATP-stabilized fraction II (proteins absorbed on DEAE-matrix and eluted with 0.5 M-KCl) of rabbit reticulocytes and mouse liver. Analysis of the various extracts by (NH4)2SO4 fractionation, velocity-gradient centrifugation, non-denaturing PAGE and SDS/PAGE and immunoblotting with proteasome-specific antisera failed to identify the proteasome as part of a higher-molecular-mass '26 S' multienzyme complex. In all instances proteasomes are identified in their 'free' 650 kDa '20 S' form. In addition to the proteasome and independent of the presence of MgATP, we isolated a high-molecular-mass proteinase whose electrophoretic migration behaviour and sedimentation rate correspond to that of the previously described '26 S' proteinase. This '26 S' proteinase possesses a strong 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity and is composed of several non-identical polypeptides in the molecular-mass range 20-150 kDa. Despite its similarity to proteasomal enzyme activity, protein analysis and immunoblotting experiments demonstrate that neither the intact proteasome nor subunits thereof are components of the '26 S' proteinase complex.

Amino Acid Sequence↗
PubMed1991

Subunit of the '20S' proteasome (multicatalytic proteinase) encoded by the major histocompatibility complex.

Cytotoxic T lymphocytes recognize fragments (peptides) of protein antigens presented by major histocompatibility complex (MHC) class I molecules. In general, the peptides are derived from cytosolic proteins and are then transported to the endoplasmic reticulum where they assemble with the MHC class I heavy chains and beta 2-microglobulin to form stable and functional class I molecules. The proteases involved in the generation of these peptides are unknown. One candidate is the proteasome, a nonlysosomal proteinase complex abundantly present in the cytosol. Proteasomes have several proteolytically active sites and are complexes of high relative molecular mass (Mr about 600K), consisting of about 20-30 subunits with Mrs between 15 and 30K. Here we show that at least one of these subunits is encoded by the mouse MHC in the region between the K locus and the MHC class II region, and inducible by interferon-gamma. This raises the intriguing possibility that the MHC encodes not only the MHC class I molecules themselves but also proteases involved in the formation of MHC-binding peptides.

Animals↗
PubMed1990

Substance P and antagonists. Surface activity and molecular shapes.

The molecular properties of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met amide) and three of its antagonists were derived by measuring the Gibbs adsorption isotherm, providing information on the surface activity, the molecular shape, and the pK values of the different molecules. The following three antagonists were investigated: [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, ANT I; [D-Arg1,D-Trp7,9,Leu11]SP, ANT II and [D-Pro2,D-Trp7,9]SP, ANT III. SP is only moderately surface active. The amino acid substitutions lead, however, to an increased surface activity of the antagonists. From the concentration dependence of the surface activity it was possible to quantify the packing characteristics of the individual neuropeptides. SP shows cross-sectional areas of 300 +/- 5 A2 to 240 +/- 5 A2 (pH 5 to 8, 154 mM NaCl) at concentrations below 10(-5) M, i.e., in the physiological concentration range, indicating a folded SP conformation. Upon increasing the packing density to concentrations larger than 10(-5) M the surface area was only half as large (148 +/- 5 A2 to 124 +/- 3 A2) suggesting now a relatively extended conformation of the SP molecule with its long molecular axis perpendicular to the air/water interface. In contrast, the three antagonists were characterized by surface areas of 147 +/- 3 A2 to 126 +/- 3 A2 which were almost independent of concentration. The antagonists thus adopt a relatively extended conformation in the whole concentration range measured. This is further supported by computer modelling which shows that the antagonists are motionally restricted and can adopt neither a bent nor a alpha-helical conformation. The surface activity of the neuropeptides was dependent on the pH of the solution. At low peptide concentrations (about 10(-6) M) it was possible to resolve and determine the pK values of all individual charged amino acid side chains. The pK values observed for the neuropeptides were about two pK units lower than those of the free amino acids in solution. The pK shifts of the neuropeptides at the air/water interface are explained in terms of the Gouy-Chapman theory. SP and its antagonists bind to lipid bilayers in the order of their surface activity. While the binding of SP is mainly due to electrostatic interactions, hydrophobic peptide-lipid interactions contribute to the binding of the antagonists.

Amino Acid Sequence↗
PubMed1990

The use of monolayers for simple and quantitative analysis of lipid-drug interactions exemplified with dibucaine and substance P.

The interaction between lipids and water soluble amphiphiles was investigated by means of a monolayer technique, monitoring the area increase at constant surface pressure. The area increase could be quantitated and binding isotherms at different surface pressures were measured. A comparison of dibucaine binding to monolayers and bilayers showed that a surface pressure of 32 mN/m best represents the packing density in a lipid bilayer (Seelig, 1987). Binding isotherms measured for charged dibucaine and substance P (SP) were analyzed by means of two different models. If electrostatic effects were ignored the binding of dibucaine and SP showed biphasic Scatchard plots. If, however, electrostatic effects were taken into account by means of the Gouy-Chapman theory, the insertion of both amphiphiles was best described in terms of a partitioning into the monolayer lipids. The hydrophobic binding constant was Kp = 660 +/- 80 M-1 for charged dibucaine inserting into coarse liposomes or monolayers at 32 mN/m (Seelig et al., 1986) and 1-1.8 M-1 for SP inserting into monolayers at 32 mN/m (Seelig and Macdonald, 1989).

Dibucaine↗
PubMed1989

Binding of a neuropeptide, substance P, to neutral and negatively charged lipids.

The binding of substance P (SP), a positively charged neurotransmitter peptide, to neutral and to negatively charged phospholipids has been investigated by means of a monolayer technique. Monolayers formed at room temperature from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or mixtures of the two, were maintained throughout the course of a binding experiment at a constant surface pressure while the monolayer surface area was monitored. Injection of SP into the aqueous subphase (154 mM NaCl, 10 mM Tris adjusted to pH 7.4) led to an expansion of the monolayer surface area that was attributed to a spontaneous insertion of SP between the lipid molecules. A quantitative evaluation of the area increase at constant pressure yielded SP insertion isotherms that showed that levels of SP insertion increased directly with the monolayer POPG content and decreased to negligible levels at surface pressures above 35 +/- 1 mN/m. If electrostatic effects were ignored, these data showed biphasic behavior in Scatchard plots. The apparent binding constants ranged, at 20 mN/m, from (3.2 +/- 0.3) X 10(4) M-1 for 100% POPG monolayers to (2.0 +/- 0.05) X 10(3) M-1 for 25% POPG/75% POPC monolayers. At 32 mN/m, a monolayer surface pressure that mimics bilayer conditions, the apparent binding constant for a 100% POPG monolayer was measured to be (1.1 +/- 0.05) X 10(3) M-1. However, for a monolayer containing only 25% charged lipids, corresponding to a natural membrane composition, K app at 32 mN/m was estimated to be at most 41 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycerophosphates↗
PubMed1988

Partitioning of local anesthetics into membranes: surface charge effects monitored by the phospholipid head-group.

The binding of the charged form of two local anesthetics, dibucaine and etidocaine, to bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was measured simultaneously with ultraviolet spectroscopy and deuterium magnetic resonance. Because of their amphiphilic molecular structure, both drugs intercalate between the lipid molecules, increasing the surface area and imparting a positive electric charge onto the membrane. The ultraviolet (UV) binding isotherms were therefore analyzed in terms of a model which specifically took into account the bilayer expansion as well as the charge-induced concentration variations near the membrane surface. By formulating a quantitative expression for the change in surface area upon drug intercalation and combining it with the Gouy-Chapman theory, the binding of charged dibucaine and etidocaine to the lipid membrane was best described by a partition equilibrium, with surface partition coefficients of 660 +/- 80 M-1 and 11 +/- 2 M-1 for dibucaine and etidocaine, respectively (pH 5.5, 0.1 M NaCl/50 mM buffer). Deuterium magnetic resonance demonstrated further that the binding of drug changed the head-group conformation of the lipid molecules. Invoking the intercalation model, a linear variation of the deuterium quadrupole splittings of the choline segments with the surface charge density was observed, suggesting that the phosphocholine head-group may act as a 'molecular electrometer' with respect to surface charges.

Acetanilides↗
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↗
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↗