Biomedical subjects

R A Demel

Publications and source records attributed to R A Demel.

At least 55 records · Page 3Linked to original sources

PubMed1992

SecA insertion into phospholipids is stimulated by negatively charged lipids and inhibited by ATP: a monolayer study.

SecA-lipid interactions are believed to be important for the translocation of precursor proteins across the inner membrane of Escherichia coli [Lill, R., Dowhan, W., & Wickner, W. (1990) Cell 60, 271-280]. SecA insertion into the phospholipid bilayer could a role in this process. We investigated this possibility by studying the interactions between SecA and different phospholipids using the monolayer technique. It was established that SecA is surface-active and can insert into lipid monolayers. This insertion was greatly enhanced by the negatively charged lipids DOPG and Escherichia coli cardiolipin. Insertion of SecA into these negatively charged lipids could be detected up to initial surface pressures of 34 mN/m for DOPG and 36 mN/m for Escherichia coli cardiolipin, implying a possible role for negatively charged lipids in the insertion of SecA in biological membranes. High salt concentrations did not inhibit the SecA insertion into DOPG monolayers, suggesting not only an electrostatic but also a hydrophobic interaction of SecA with the lipid monolayer. ATP decreased both the insertion (factor 2) and binding (factor 3) of SecA to DOPG monolayers. ADP and phosphate gave a decrease in the SecA insertion to the same extent as ATP, but the binding of SecA was only slightly reduced. AMP-PNP and ATP-gamma-S did not have large effects on the insertion or on the binding of SecA to DOPG monolayers. The physiological significance of these results in protein translocation is discussed.

Adenosine Triphosphatases↗

PubMed1991

Interaction of influenza virus hemagglutinin with a lipid monolayer. A comparison of the surface activities of intact virions, isolated hemagglutinins, and a synthetic fusion peptide.

In the infectious entry pathway of influenza virus, the low pH of the endosomal compartment induces an irreversible conformational change in influenza virus hemagglutinin, leading to fusion of viral and endosomal membranes. In the current report, we characterized the low-pH-induced activation of hemagglutinin of influenza strain X31 by studying its interaction with a lipid monolayer. The surface activities of virions, of isolated hemagglutinins and its proteolytic fragments, and of a synthetic peptide mimicking the amino terminus of subunit 2 of hemagglutinin are compared. The data indicate that the surface activity of both virions and isolated hemagglutinin develop as a result of the low-pH-induced conformational change in hemagglutinin. The surface activity of isolated hemagglutinin is mainly caused by penetration into the lipid monolayer of protein domains other than the amino terminus of subunit 2 of hemagglutinin; domains in subunit 1 may be involved. The surface activity of virions appears to be a secondary effect of the conformational change and is explained by assuming a net transfer of viral lipids to the lipid monolayer.

Amino Acid Sequence↗

PubMed1991

Characterization of lipid insertion into monomolecular layers mediated by lung surfactant proteins SP-B and SP-C.

Pulmonary surfactant proteins, SP-B and SP-C, if present in preformed monolayers can induce lipid insertion from lipid vesicles into the monolayer after the addition of (divalent) cations [Oosterlaken-Dijksterhuis, M. A., Haagsman, H. P., van Golde, L. M. G., & Demel, R. A. (1991) Biochemistry 30, 8276-8287]. This model system was used to study the mechanisms by which SP-B and SP-C induce monolayer formation from vesicles. Lipid insertion proceeds irrespectively of the molecular class, and PG is not required for this process. In addition to lipids that are immediately inserted from vesicles into the monolayer, large amounts of vesicles are bound to the monolayer and their lipids eventually inserted when the surface area is expanded. SP-B and SP-C are directly responsible for the binding of vesicles to the monolayer. By weight, the vesicle binding capacity of SP-B is approximately 4 times that of SP-C. For vesicle binding and insertion, the formation of close contacts between monolayer and vesicles is essential. SP-B and SP-C show very similar surface properties. Both proteins form extremely stable monolayers (collapse pressures 36-37 mN/m) of alpha-helical structures oriented parallel to the interface. In monolayers consisting of DPPC and SP-B or SP-C, an increase in mean molecular area is observed, which is mainly attributed to the phospholipid. This will greatly enhance the insertion of new lipid material into the monolayer. The results of this study suggest that the surface properties and the hydrophobic nature of SP-B and SP-C are important for the protein-mediated monolayer formation.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1991

Synthesis of acylated gramicidins and the influence of acylation on the interfacial properties and conformational behavior of gramicidin A.

Five gramicidin A analogs were synthesized in which various acyl chains, differing in length and unsaturation, were covalently coupled to the C-terminal ethanolamine group. The analogs were characterized by various spectroscopic techniques and their molecular properties were investigated using monolayer techniques and circular dichroism. It is demonstrated that neither the interfacial properties nor the conformational behavior of gramicidin A at the air/water interface are seriously affected upon acylation. It is proposed that at the limiting area the gramicidin molecule is oriented with its C-terminus towards the subphase with the covalently coupled acylchain located parallel to the helical axis in between the protruding tryptophans. Circular dichroism experiments, in which gramicidin-containing vesicles were prepared from different organic solvents, indicate that the presence of a covalently coupled fatty acylchain tends to stabilize the beta 6.3 helical conformation. It is demonstrated that, like for gramicidin A, also for the acylgramicidins the single-stranded beta 6.3 helical conformation, or channel conformation, is the preferred conformation upon incorporation in bilayers.

Acylation↗

PubMed1991

Mitochondrial creatine kinase mediates contact formation between mitochondrial membranes.

Purified mitochondrial creatine kinase (Mi-CK) (EC 2.7.3.2) from chicken heart was shown to interact simultaneously with purified inner and outer mitochondrial membranes, thereby creating an intermembrane chondrial membranes, thereby creating an intermembrane were purified from rat liver and thus were fully devoid of Mi-CK. Intermembrane contact formation was demonstrated by measuring the binding of inner membrane vesicles to outer membranes spread at the air-water interface. Mi-CK also mediated intermembrane adhesion when membranes formed with total lipid extracts of both membranes were used, pointing to the role of lipids as potential membrane anchors of Mi-CK in the mitochondrial intermembrane space. Other enzymes of the intermembrane space that (like Mi-CK) are also cationic, as well as cytosolic isoenzymes of creatine kinase, failed to induce contact formation. Thus, of the proteins tested, membrane contact formation was specific for Mi-CK. The two oligomeric forms of Mi-CK (octamer and dimer) differed in their ability to mediate intermembrane adhesion, the octamer being more potent. Highly basic peptides, i.e. poly-L-lysines, were shown to strongly interact with membranes formed with lipid extracts of mitochondrial membranes: they both induced intermembrane binding and fusion. Interestingly, the extent of contact formation mediated by poly-L-lysines was lower than that of octameric Mi-CK. The implications of these findings on the function and localization of Mi-CK and on the structure of the mitochondrial intermembrane compartment are discussed.

Animals↗

PubMed1991

Lipid-peptide interactions between fragments of the transit peptide of ribulose-1,5-bisphosphate carboxylase/oxygenase and chloroplast membrane lipids.

The interactions of fragments of the transit peptide of ribulose-1,5-bisphosphate carboxylase/oxygenase with lipid monolayers was studied in order to investigate the possible involvement of the membrane lipids in the protein import process. The fragments are surface active and have a differential ability to insert in lipid monolayers. The fragments have a preference for the chloroplast galacto- and sulpholipids and phosphatidylglycerol and interact with envelope membrane lipid extracts. These results suggest that probably transit peptide-lipid interactions are involved in the chloroplast protein import process.

Amino Acid Sequence↗

PubMed1991

Interaction of lipid vesicles with monomolecular layers containing lung surfactant proteins SP-B or SP-C.

Pulmonary surfactant contains two families of hydrophobic proteins, SP-B and SP-C. Both proteins are thought to promote the formation of the phospholipid monolayer at the air-fluid interface of the lung. The Wilhelmy plate method was used to study the involvement of SP-B and SP-C in the formation of phospholipid monolayers. The proteins were either present in the phospholipid vesicles which were injected into the subphase or included in a preformed phospholipid monolayer. In agreement with earlier investigators, we found that SP-B and SP-C, present in phospholipid vesicles, were able to induce the formation of a monolayer, as became apparent by an increase in surface pressure. However, when the proteins were present in a preformed phospholipid monolayer (20 mN/m) at similar lipid to protein ratios, the rate of surface pressure increase after injection of pure phospholipid vesicles into the subphase at similar vesicle concentrations was 10 times higher. The process of phospholipid insertion from phospholipid vesicles into the protein-containing monolayers was dependent on (1) the presence of (divalent) cations, (2) the phospholipid concentration in the subphase, (3) the size of the phospholipid vesicles, (4) the protein concentration in the preformed monolayer, and (5) the initial surface pressure at which the monolayers were formed. Both in vesicles and in preformed monolayers, SP-C was less active than SP-B in promoting the formation of a phospholipid monolayer. The use of preformed monolayers containing controlled protein concentrations may allow more detailed studies on the mechanism by which the proteins enhance phospholipid monolayer formation from vesicles.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1991

Characterization of the interaction of doxorubicin with (poly)phosphoinositides in model systems. Evidence for specific interaction with phosphatidylinositol-monophosphate and -diphosphate.

The anticancer drug doxorubicin penetrates into Langmuir monolayers containing phosphoinositides. Upon binding of doxorubicin to phosphoinositide-containing SUV, its fluorescence is self-quenched due to self-association. As compared to other anionic phospholipids, as much as 2- to 3-fold larger effects were obtained with PIP and PIP2, in mixtures of these lipids with DOPC. Doxorubicin competes efficiently with the non-penetrating antibiotic neomycin for binding to PIP2. According to its penetration, specific binding of doxorubicin was half-maximal at 5-15 microM. It is likely that also in biological membranes doxorubicin binds specifically to PIP and PIP2.

Doxorubicin↗

PubMed1991

The interaction of synthetic analogs of the N-terminal fusion sequence of influenza virus with a lipid monolayer. Comparison of fusion-active and fusion-defective analogs.

The amino terminus of subunit-2 of influenza virus hemagglutinin (NHA2) plays a crucial role in the induction of fusion between viral and endosomal membranes leading to the infection of a cell. Three synthetic analogs with an amino acid sequence corresponding to NHA2 of variant hemagglutinins were studied in a monolayer set up. Comparison of the interaction of a fusion-active and two fusion-defective analogs with a lipid monolayer revealed a greater surface activity of the fusion-active analog. Pronounced differences were found if the pure peptides were spread at the air/water interface; the fusion-active analog showed a higher collapse pressure and a greater limiting molecular area. Circular dichroism measurements on collected lipid monolayers indicated a high content of alpha-helical structure for the fusion-active and one of the fusion-defective analogs. A simple relation between alpha-helical content and fusogenicity does not seem to exist. Instead, the extent of penetration, a defined tertiary structure or orientation of the alpha-helical peptide may be essential for its membrane perturbing activity.

Amino Acid Sequence↗

PubMed1990

Lipid and peptide specificities in signal peptide--lipid interactions in model membranes.

The present data show the critical importance of the anionic lipid content in monomolecular layers for the interaction with PhoE signal peptide. At 37 degrees C and 100 mM NaCl the interaction is maximal at 30-40 mol% anionic lipid. The results correlate with the reduced translocation competence of Escherichia coli strain HD3122, which has a much lower anionic lipid content as compared to the wild-type strain SD12 (De Vrije et al. (1988) Nature 334, 173-175). PhoE signal peptide analogs as N-formyl PhoE signal peptide, PhoE signal peptide +(1-7) and PhoE signal peptide Val-8----Trp-8 show the same lipid preference as PhoE signal peptide. On the other hand the affinity for an anionic lipid interface is strongly reduced for PhoE signal peptide Lys-19,-20----Asp-19,-20, which correlates with the less efficient translocation of PhoE protein carrying this signal sequence. At limiting anionic lipid concentrations there is a temperature and salt effect on the observed interaction, which is related to a conformational change of the peptide. Signal sequences show clearly conformational flexibility in responds to environmental conditions. Under the conditions used in this study FTIR spectra of PhoE signal peptide-DOPG monolayers show a high content of beta-structure and beta-turn.

Amino Acid Sequence↗

PubMed1989

Transfer of cholesterol and oxysterol derivatives by the nonspecific lipid transfer protein (sterol carrier protein 2): a study on its mode of action.

The nonspecific lipid transfer protein (nsLTP) facilitates the transfer of both phospholipids and cholesterol between membrane interfaces. In this study, we have investigated the transport of 14C-labelled cholesterol, 7-ketocholesterol, 7 alpha-hydroxycholesterol and 25-hydroxycholesterol from a mixed lipid monolayer at the air/water interface to acceptor vesicles in the subphase. In the absence of nsLTP the transport of cholesterol was virtually nil, whereas the spontaneous transport of the oxysterol derivatives increased in the order 7-ketosterol less than 7 alpha-hydroxycholesterol less than 25-hydroxycholesterol. In the presence of nsLTP, the transport of both cholesterol and the oxysterol derivatives was greatly enhanced; the highest rate of transport was observed for 25-hydroxycholesterol. In the absence of vesicles, binding of cholesterol and of 25-hydroxycholesterol from the monolayer to nsLTP was negligible. Similarly, nsLTP did not bind cholesterol from radiolabeled bovine heart mitochondria under conditions where it stimulated the transfer of cholesterol to vesicles. In agreement with this failure to bind, nsLTP was unable to carry cholesterol between two separate monolayers. From the monolayer experiments it became apparent that nsLTP is highly surface-active. Measurement of the transport of cholesterol and of oxysterol derivatives by the monolayer-vesicles assay and of a series of pyrene-labeled phosphatidylcholine species by the fluorescent transfer assay showed a high correlation between the spontaneous and the nsLTP-mediated lipid transport. This supports the notion that nsLTP lowers the energy barrier for the lipid monomer-membrane interface equilibration process. In view of the above observations, we propose that nsLTP may facilitate the transfer of lipids by being part of a transient collisional complex between donor and acceptor membrane.

Animals↗

PubMed1989

Are fatty acid-binding proteins involved in fatty acid transfer?

The possible function of fatty acid-binding protein (FABP) to act as a fatty acid carrier protein was investigated in model systems with regard to three aspects. (1) does FABP release fatty acids from membranes? (2) does it facilitate fatty acid transport in an aqueous environment? (3) are FABP-bound fatty acids released for use by mitochondria? FABPs could bind oleic acid from liposomes and mitochondrial membranes with a ratio of 1 mol per mol protein. Oleic acid was withdrawn from negative, neutral or cholesterol-containing monolayers by FABP with rates up to 10%/min. Only about 5% of FABP penetrated into the monolayer. Spontaneous transfer of oleic acid between mitochondria and vesicles or liposomes occurred so rapidly that an effect of FABP was not detectable. When the mitochondria were separated from the vesicles in an equilibrium dialysis cell, a stimulating effect of FABP on fatty acid transfer could be demonstrated. Injected FABP increased also transfer of oleic acid between two separate monolayers. FABP-bound fatty acid was well oxidized by rat liver mitochondria. The results indicate that the FABP-fatty acid complex may function as an intermediate in the transfer of fatty acids between membranes. No functional differences were detected between heart and liver FABPs in this respect.

Animals↗

PubMed1989

Interactions of metal ions with phosphatidylserine bilayer membranes: effect of hydrocarbon chain unsaturation.

A combination of surface monolayer, scanning calorimetry, 31P NMR, and spin-label ESR techniques has been used to monitor the interactions of monovalent (NH4+, Na+, and Li+) and divalent (Ca2+) cations with phosphatidylserines (PS) differing in their levels of chain unsaturation. Comparisons are made between the disaturated dimyristoyl-, dipalmitoyl-, and dihexadecyl-PS (DMPS, DPPS, and DHPS), saturated cis-monounsaturated palmitoyloleoyl-PS (POPS) (and bovine brain PS), di-trans-monounsaturated dielaidoyl-PS (DEPS), and di-cis-monounsaturated dioleoyl-PS (DOPS). Na+ and NH4+ cations interact weakly with all PS monolayers and bilayers without significant changes in molecular conformation, chain packing, or headgroup dynamics and without dependence on chain composition. In contrast, considering these structural and dynamic parameters, Li+ shows a gradation in its interaction with PS (DMPS greater than POPS approximately bovine brain PS greater than DOPS), suggesting that Li+-PS interactions depend on the interfacial properties of the PS molecules (e.g., surface area). Finally, Ca2+ interacts strongly with all PS monolayers and bilayers, without obvious chain selectivity. Thus, ion binding to PS depends not only on the properties of the cation (Na+ vs Li+ vs Ca2+) but also on the molecular details of the PS membrane surface.

Calcium↗

PubMed1989

Differential interactions of apo- and holocytochrome c with acidic membrane lipids in model systems and the implications for their import into mitochondria.

Monomolecular layers of lipid extracts of microsomal, mitochondrial outer and inner membranes, and pure lipid species have been used to measure their interaction with apo- and holocytochrome c. Large differences were observed both with respect to the nature and the lipid specificity of the interaction. The initial electrostatic interaction of the hemefree precursor apocytochrome c with anionic phospholipids is followed by penetration of the protein in between the acyl chains. Apocytochrome c shows similar interactions for all anionic lipids tested. In strong contrast the holoprotein discriminates enormously between cardiolipin for which it has a high affinity and phosphatidylserine and phosphatidylinositol for which it has a much lower affinity. For these latter lipids the interaction with cytochrome c is primarily electrostatic. The cytochrome c-cardiolipin interaction shows several unique features which suggest the formation of a specific complex between the two molecules. These properties account for the preference in interaction of the apoprotein with the lipid extract of the outer mitochondrial membrane over that of the endoplasmic reticulum and the large preference of cytochrome c for the inner over that of the outer mitochondrial membrane lipid extract. Only apocytochrome c was able to induce close contacts between monolayers of the mitochondrial outer membrane lipids and vesicles of mitochondrial inner membrane lipids. Experiments with fragments of both protein and unfolding experiments with cytochrome c revealed that the differences in interaction between the two proteins are mainly due to differences in their tertiary structure and not the presence of the heme group itself. The initial unfolded structure of apocytochrome c is responsible for the high penetrative power of the protein and its ability to induce close membrane contact, whereas the folded structure of cytochrome c is responsible for the specific interaction with cardiolipin. The results are discussed in the light of the apocytochrome c import process in mitochondria and suggest that lipid-protein interactions contribute to targeting the precursor toward mitochondria and are important for its translocation across the outer mitochondrial membrane and the final localization of cytochrome c toward the outside of the inner mitochondrial membrane.

Animals↗

PubMed1989

The importance of the amino terminus of the mitochondrial precursor protein apocytochrome c for translocation across model membranes.

The importance of the various regions of the apocytochrome c molecule and the effect of covalent coupling of the heme group to the protein for the translocation across a model-membrane was studied by using chemically and enzymatically prepared fragments of horse heart apo- and holocytochrome c and model-membranes composed of phosphatidylserine and phosphatidylcholine. Binding experiments showed that fragments of apocytochrome c with the highest net positive charge have the highest affinity for negatively charged large unilamellar phosphatidylserine vesicles. Monolayer experiments demonstrated that the amino-terminal fragments were only able to penetrate into a phosphatidylserine monolayer whereas the carboxyl-terminal fragments, in addition, penetrated into a phosphatidylcholine monolayer although with a lower efficiency. The covalent coupling of the heme group to both a small amino-terminal fragment residue numbers 1-38 and to the entire precursor protein resulted in a marked decrease in the ability to penetrate into a phosphatidylserine monolayer. Translocation experiments with trypsin enclosed in vesicles, showed that only the amino-terminal fragments of the precursor protein and not carboxyl-terminal peptides or the heme-containing fragments of the mature protein were able to cross the bilayer and become digestible by trypsin at the opposite side of the bilayer. Circular dichroism measurements with the various peptides both in an aqueous and lipidic environment were performed to investigate the conformation of apocytochrome c after interaction with model-membranes. Implications of these data for the import of apocytochrome c into mitochondria will be discussed.

Amino Acid Sequence↗

PubMed1988

Penetration of the signal sequence of Escherichia coli PhoE protein into phospholipid model membranes leads to lipid-specific changes in signal peptide structure and alterations of lipid organization.

In order to obtain more insight in the initial steps of the process of protein translocation across membranes, biophysical investigations were undertaken on the lipid specificity and structural consequences of penetration of the PhoE signal peptide into lipid model membranes and on the conformation of the signal peptide adopted upon interaction with the lipids. When the monolayer technique and differential scanning calorimetry are used, a stronger penetration is observed for negatively charged lipids, significantly influenced by the physical state of the lipid but not by temperature or acyl chain unsaturation as such. Although the interaction is principally electrostatic, as indicated also by the strong penetration of N-terminal fragments into negatively charged lipid monolayers, the effect of ionic strength suggests an additional hydrophobic component. Most interestingly with regard to the mechanism of protein translocation, the molecular area of the peptide in the monolayer also shows lipid specificity: the area in the presence of PC is consistent with a looped helical orientation, whereas in the presence of cardiolipin a time-dependent conformational change is observed, most likely leading from a looped to a stretched orientation with the N-terminus directed toward the water. This is in line also with the determined peptide-lipid stoichiometry. Preliminary 31P NMR and electron microscopy data on the interaction with lipid bilayer systems indicate loss of bilayer structure.

Bacterial Outer Membrane Proteins↗

PubMed1988

Characterization of the interfacial behavior and structure of the signal sequence of Escherichia coli outer membrane pore protein PhoE.

The behavior of the chemically synthesized PhoE signal peptide and signal peptide fragments on hydrophilic-hydrophobic interfaces was studied with circular dichroism and monolayer techniques. The experimental results were compared with computer-calculated predictions of peptide structure, orientation, and molecular area. The complete signal sequence was found to aggregate in a beta-sheet structure when introduced in an aqueous environment; on the other hand, in sodium dodecyl sulfate micelles approximately 75% alpha-helical structure was observed. Assuming this to reflect the actual structure in a peptide monolayer and taking into account the orientations predicted for the fragments, the measured molecular areas suggest a looped orientation of the signal sequence with both N and C terminus in the water phase.

Bacterial Outer Membrane Proteins↗

PubMed1988

Phase behaviour of lipid X.

The phase behaviour of aqueous dispersions of lipid X, a precursor of bacterial lipopolysaccharides has been investigated by a variety of physico-chemical techniques. The results are consistent with the presence of disk-shaped micelles with an average diameter of 13 +/- 1.8 nm. The critical micellar concentration in water and physiological saline is 4 x 10(-5) M. Consistent with the formation of micelles in water and physiological saline is the finding that lipid X is in the liquid-crystalline state at temperatures higher than 0 degrees C. The packing and the dynamics of lipid X are characteristic of micelles. Close to the polar group the hydrocarbon chains are significantly more mobile and disordered than in the corresponding region of lipid bilayers. From monolayer studies an estimate of the molecular area of lipid X is derived; under physiological conditions the area/molecule is about 0.50 nm2 at 30 mN/m indicating that lipid X has a wedge-like shape. The two pK values of the primary phosphate group of lipid X are pK1 approximately 1.3 and pK2 = 8.2. At pH values less than 7, the area/molecule decreases, i.e. the packing of the lipid X molecules becomes tighter, and there is also a decrease in the solubility of lipid X. As is characteristic of charged lipids, the state of aggregation (phase behaviour) of lipid X depends on pH, the ionic strength and the nature of the counterion.

Calorimetry, Differential Scanning↗