Biomedical subjects

M Ptak

Publications and source records attributed to M Ptak.

At least 55 records · Page 3Linked to original sources

PubMed1992

Distortions induced in DNA by cis-platinum interstrand adducts.

A 22 base pair double-stranded oligonucleotide containing a unique interstrand adduct resulting from chelation of the two guanine residues within the central sequence d(TGCT/AGCA) by a cis-platinum residue has been studied by means of gel electrophoresis, chemical probes, and molecular mechanics. The anomalously slow electrophoretic mobility of the multimers of the platinated and ligated oligomers suggests that the platinated oligonucleotide is bent. The two cytosine residues (complementary to the platinated guanines) are hyperreactive to hydroxylamine, indicating a large exposure of the two bases to the solvent. The adduct does not induce a local denaturation within the flanking sequences since the adenine residues are not reactive with diethyl pyrocarbonate. This is confirmed by the nonreactivity of the complementary T residues with osmium tetraoxide. These results and the molecular mechanics modeling suggest that the interstrand adduct bends the double helix by approximately 55 degrees toward the major groove, that the double helix conserves its average twist angle, and that the distortion induced by the adduct is localized at the platinated sequence d(GC/CG).

Base Sequence↗

PubMed1992

Two-dimensional 1H NMR study of recombinant insect defensin A in water: resonance assignments, secondary structure and global folding.

A 500 MHz 2D 1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities, 3JNH-alpha H coupling constants as well as 1H/2H exchange rates and delta delta/delta T temperature coefficients of NH protons strongly support the existence of an alpha-helix (residues 14-24) and of an antiparallel beta-sheet (residues 27-40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from 3JNH-alpha H coupling constants, (iii) 12 hydrogen bonds mostly deduced from 1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a beta-sheet is linked to an alpha-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.

Amino Acid Sequence↗

PubMed1992

Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A.

Iturin A and surfactin are two lipopeptides extracted from a same strain of Bacillus subtilis. Iturin A possesses antibiotic and antifungal activities and surfactin is a strong surfactant. The presence of surfactin, at a concentration at which, alone, it is inactive, increases to a very large extent the haemolysis percent induced by iturin A. This synergistic effect seems to be in relation with interactions between iturin A and surfactin. Iturin A adsorbs to and penetrates into surfactin monolayers. Iturin A and surfactin are miscible and interact specifically in mixed monolayers.

Adsorption↗

PubMed1991

Two- and three-dimensional 1H NMR studies of a wheat phospholipid transfer protein: sequential resonance assignments and secondary structure.

Two- and three-dimensional 1H NMR experiments have been used to sequentially assign nearly all proton resonances of the 90 residues of wheat phospholipid transfer protein. Only a few side-chain protons were not identified because of degeneracy or overlapping. The identification of spin systems and the sequential assignment were made at the same time by combining the data of the two- and three-dimensional experiments. The classical two-dimensional COSY, HOHAHA, and NOESY experiments benefit from both good resolution and high sensitivity, allowing the detection of long-range dipolar connectivities. The three-dimensional HOHAHA-NOESY experiment offers the advantage of a faster and unambiguous assignment. As a matter of fact, homonuclear three-dimensional NMR spectroscopy proved to be a very efficient method for resonance assignments of protein 1H NMR spectra which cannot be unraveled by 2D methods. An assignment strategy which overcomes most of the ambiguities has been proposed, in which each individual assignment toward the C-terminal end is supported by another in the opposite direction originating from a completely different part of the spectrum. Location of secondary structures of the phospholipid transfer protein was determined by using the method of analysis introduced here and was confirmed by 3J alpha NH coupling and NH exchange rates. Except for the C-terminal part, the polypeptide chain appears to be organized mainly as helical fragments connected by disulfide bridges. Further modeling will display the overall folding of the protein and should provide a better understanding of its interactions with lipids.

Amino Acid Sequence↗

PubMed1991

Isolation and characterization of a new variant of surfactin, the [Val7]surfactin.

Reinvestigation of surfactin, a previously studied peptidolipid surfactant from Bacillus subtilis, by fast-atom-bombardment mass spectrometry and 1H-NMR spectroscopy, as well as by chemical methods, revealed the presence of a closely related second constituent. This new compound, [Val7]surfactin, differs from the known surfactin by the C-terminal amino acid residue which is valine instead of leucine.

Amino Acid Sequence↗

PubMed1990

Incorporation of spin-labeled fatty acids into bovine brain clathrin coated vesicles.

Stearic acids with a nitroxide radical at selected positions have been incorporated in the phospholipid bilayers of clathrin coated vesicles, uncoated vesicles and sonicated liposomes made from the lipids extracted from the uncoated vesicles. The extent of incorporation was found minimum for stearic acids labeled on C-12 and for bilayers of uncoated vesicles. The ESR spectra of the spin-labeled fatty acids incorporated in the bilayers showed a pronounced temperature dependence (without discontinuity) and a decrease in the hyperfine splitting as the nitroxide group was inserted deeper in the hydrophobic core of the membranes. An abrupt phospholipid phase transition or a phase separation could be excluded. The presence of the external proteins (the clathrin coat) on the membranes was not found to noticeably influence the gradient of flexibility of the fatty acid chains of the phospholipids. The influence of the internal proteins embedded in the bilayers was evidenced by a detailed analysis of the ESR spectra of (7,8)SA in terms of two components: one component arising from the labels surrounded exclusively by phospholipids, the other component arising from labels of reduced mobility perturbed by the vicinity of the proteins. These results support the persistence of lipidic domains in the endocytic vesicles despite the accumulation of receptors which follows their formation.

Animals↗

PubMed1990

Iturin lipopeptides: interactions of mycosubtilin with lipids in planar membranes and mixed monolayers.

The interactions between the antifungal lipopeptide mycosubtilin and lipids are studied. Mycosubtilin increases the ion permeability of planar lipid membranes by forming ion conducting pores. The lifetime of these pores is greatly increased when the membrane contains cholesterol. In mixed monolayers the interaction between mycosubtilin and DMPC leads to the formation of a mycosubtilin/DMPC 1:2 complex non miscible in the excess DMPC monolayer but miscible in the mycosubtilin monolayer. Mycosubtilin and cholesterol interact strongly in monolayers in all proportions and form a mycosubtilin-cholesterol (1:2) complex. These results are analyzed with reference to the overall view of the activity of iturins and the importance of the lipopeptide conformation is outlined.

Amino Acid Sequence↗

PubMed1990

Triggering of mannosyltransferase activity in inner mitochondrial membranes by dolichyl-monophosphate incorporation mediated through phospholipids or fatty acids.

The activity of GDPmannose:dolichyl monophosphate mannosyltransferase in inner mitochondrial membranes can be triggered by dolichyl-monophosphate incorporation mediated through phospholipids or fatty acids. The efficiency of this incorporation and the efficiency of the enzyme activity are not equivalent. Among a variety of amphiphiles which were tested, the highest mannosyltransferase activity was obtained with the mixture of lipids extracted from the outer mitochondrial membranes. The results presented here appear consistent only with a mechanism involving collisional contacts of the phospholipid vesicles and fusion with the membranes. ESR spectroscopy confirms that (a) the incorporation process is followed by solubilization of dolichyl monophosphate molecules in the lipid phase and (b) the general organization of the inner mitochondrial membranes is not perturbed by the addition of dolichyl monophosphate.

Animals↗

PubMed1990

A 2D NMR study of the internal flexibility of the antifungal peptide stendomycin.

A 2-D 1H NMR study (NOESY, COSY, HOHAHA and ROESY experiments) of the antifungal peptide stendomycin is presented. The variation of the NOESY cross peak intensities is measured as a function of temperature in order to discriminate between constant and fluctuating interproton distances. It is shown that among 71 NOESY cross peaks, only 12 correspond to well defined interproton distances and their correlation time is determined. The other cross peaks cannot be translated accurately in terms of distances owing to internal molecular motions. (1H)-13C nOe measurements confirm the internal mobility of the molecule. Finally a flexibility map of stendomycin can be established.

Amino Acid Sequence↗

PubMed1989

The lipopeptide antibiotic A21978C has a specific interaction with DMPC only in the presence of calcium ions.

The A21978C group are lipopeptide antibiotics which kill Gram-positive bacteria only in the presence of calcium ions. The calcium requirement of the antibacterial activity of A21978C correlates well with an in vitro calcium-dependent insertion into phospholipid vesicles. In this paper the interaction of A21978C with phosphatidylcholine is investigated in mixed monomolecular films. The spontaneity of the antibiotic-lipid mixing was determined by calculating the free energy change. On a Ca2+ containing subphase there is a specific interaction between the components at all antibiotic-lipid ratios. This is not true on K+ subphases, where specific interactions never occur. On Mg2+ subphases specific interactions occur only in monolayers containing very little lipid. By analysing the fluorescence of the kynurenine residue we have followed the effects of two factors on the penetration of the antibiotic into lipid bilayer vesicles. Firstly, the phospholipid gel to liquid crystalline phase transition which in the absence of calcium leads to an exclusion of the antibiotic from the bilayer. This trend is completely reversed in the presence of Ca2+. Secondly, the role of this lipopeptide's lipid tail was clarified by use of a series of versions of increasing fatty acyl chain length. The results indicate that the interaction promoted by calcium is not simply a hydrophobic attraction between fatty acyl chains but is more likely to be a specific interaction between polar headgroups.

Anti-Bacterial Agents↗

PubMed1989

Interactions of the lipopeptide antifungal iturin A with lipids in mixed monolayers.

The miscibility and the interactions of the antifungal lipopeptide iturin A with lipids, DMPC and cholesterol, are studied in monolayers at the air/water interface and a comparison of the respective behaviour of iturin A and the biologically inactive methylated derivative MeTyr-iturin A is made. Each lipopeptide is miscible with anyone of the lipids. This behaviour is revealed by the dependence of the transition pressure upon composition and by deviations from the additivity rule of the mean molecular area. The thermodynamic properties of the mixed systems are studied by the method of Goodrich. The mixed monolayers are always more stable than the two separate components, subsequently there are interactions between the components. However, the excess free energy of mixing delta Gexm is positive for the iturin A/DMPC system which is an indication that the interactions between lipopeptide and lipid molecules are weaker than the interactions between the pure components themselves. This is compatible with the presence of self-associated lipopeptide molecules. However, delta Gexm is highly negative for the iturin A/cholesterol system giving evidence of the formation of a specific complex between iturin A and cholesterol which is not the case with the methylated derivative. These data are analysed in connection with previous results concerning the pore-forming properties of these lipopeptides and the lack of biological activity of MeTyr-iturin A.

Anti-Bacterial Agents↗

PubMed1989

Interaction of clathrin coat proteins with unilamellar and multilamellar vesicles of phosphatidylcholine.

The binding of clathrin and accessory coat proteins to small unilamellar vesicles and to liposomes of uncharged phospholipids has been followed by chromatography, 31P-NMR, ESR and fluorescence anisotropy. At pH 6.5 and at an ionic strength value (0.1 M Mes) close to that used during the purification of clathrin-coated vesicles, the proteins do not restore the characteristic network found around the natural vesicles. Instead, a limited fusion leads to enlarged structures in which the perturbation of the dynamics of the phospholipids decreases gradually with the depth in the membrane. While the rate of motion of the outer polar heads is lowered, the order parameter of doxyl groups located either under or in the vicinity of the glycerol backbone is not affected by the proteins. In the inner core of the membrane, the main thermotropic transition of the hydrocarbon chains is unchanged. All the effects are the results of interactions limited to the membrane surface. The electrostatic nature of these interactions is evidenced when the embedded spin labels have a charge protruding at the membrane surface. An 'anchoring' effect appears which is due to the charged groups of the proteins. The lateral diffusion of the probes is reduced and, at low ionic strength, a cationic derivative no longer detects the thermotropic transition of the hydrocarbon chains. These results indicate that, although it is known that clathrin and accessory proteins bind to membranes by a series of protein-protein interactions, this system is not devoid of lipid-protein interactions, at least when it is not organized as in the natural system.

Adaptor Proteins, Vesicular Transport↗

PubMed1989

Methylation of the antifungal lipopeptide iturin A modifies its interaction with lipids.

Iturin A, extracted from the culture media of Bacillus subtilis, is an antifungal lipopeptide, the peptide cycle of which includes a D-Tyr residue in position 2. The antibiotic strength of iturin A is related to a change in the permeability of the membrane cells which leads to a leakage of K+ from the intracellular medium. Methylation of the D-Tyr residue dramatically decreases the biological activity of iturin A. Using the intrinsic fluorescence of D-Tyr we have shown that both iturin A and O-methyl-tyrosine iturin A enter the lipid membranes. When dimyristoylphosphatidylcholine vesicles contain iturin A we observe a change in the order degree of the lipid phase and an increase in the transition temperature. The methylated derivative has no effect. Two model membranes have been used to study the permeability changes induced by iturin A and O-methyltyrosine iturin A. Studying ionic permeability we have found that the conductance of a planar lipid membrane increases very much less when the lipopeptide is methylated. On the other hand, the release of carboxyfluorescein trapped in lipid vesicles is less upon addition of O-methyltyrosine-iturin A. We conclude that the Tyr residue of the peptide cycle plays a role in determining the interactions of iturin A with lipid membrane.

Anti-Bacterial Agents↗

PubMed1989

Structure of the intrastrand cis-[Pt(NH3)2(d(GpCpG))] adduct in a dodecanucleotide duplex: I. A 1H and 31P n.m.r. study.

The structure of an intrastrand cis-[Pt(NH3)2(d(GpCpG))] adduct in a dodecanucleotide duplex has been investigated by using ultraviolet absorption, circular dichroism, 1H and 31P n.m.r. The binding of cis-DDP does not inhibit the formation of a duplex but it induces a lowering of congruent to 26 degrees C of its melting temperature. A broadening of the 1H spectrum prevents an accurate analysis of the platination site. Nevertheless, by considering its thermal behavior and the number of imino protons a model of structure of the platinated duplex is proposed in which the central C.G. pair is disrupted and a neighboring C.G pair is very accessible or distorted. The environment of two phosphate groups is disturbed by the cis-DDP binding.

Circular Dichroism↗

PubMed1989

Structure of the intrastrand cis-[Pt(NH3)2(d(GpCpG))] adduct in a dodecanucleotide duplex: II. A molecular mechanics modeling study.

Molecular mechanics modeling has been carried out for the intrastrand cis-[Pt(NH3)2(d(pGpCpG))] adduct in a dodecanucleotide duplex. In a first step, an analysis of the conformation of a platinated trinucleotide shows the tendency of the two chelating guanines to take respectively anti-syn or syn-anti positions and the role of phosphate-platinum amino group interactions. In a second step, duplex structures are examined. The distortions induced by platination are especially analysed for kinked and unkinked forms of the dodecanucleotide. In all models the central C.G pair in the platination site is disrupted and the double helix is more disturbed on the 5' side of this site than on the 3' side. Most of the structural features arising from the modeling are in agreement with the conclusions of an n.m.r. study reported in the preceding paper.

Binding Sites↗

PubMed1988

A comparative monomolecular film study of antibiotic A21978C homologues of various lipid chain length.

A21978C is a calcium-dependent lipopeptide antibiotic whose biological properties are modulated by changes in its lipid chain length. This article reports on the monolayer characteristics of this cyclic lipopeptide and of LY146032 a semi synthetic homologue. The equilibrium spreading pressure pi e increases linearly with the ionic concentration of the subphase and is higher with divalent cations. The nature of the divalent cation plays a crucial role in the spreading as indicated by the variation in the molecular free energy delta Gs.delta Gs decreases in the order K+ greater than Mg2+ greater than Ca2+, which indicates privileged interactions with Ca2+. Also, the larger the lipid chain, the easier the spreading of antibiotic molecules. The compression isotherm curves are shown. The mean area of the uncompressed molecules is around 220-240 A 2 which is compatible with the size of the peptide cycle lying at the interface. The isotherm curves of the natural compounds show a transition region where the molecules are more compressible. At a given area/molecule, the surface pressures increase with the acyl chain length. When the molecules are spread on various salt solutions, the surface pressures increase in the order K+ less than Mg2+ less than Ca2+. The isotherm curves are not reversible upon a compression-expansion cycle and a wide amplitude hysteresis is observed. If a second compression is done, the curve shape is that of a liquid-expanded state and the transition region is no longer observed. This implies a conformational change of the molecules during the first compression process.

Anti-Bacterial Agents↗

PubMed1988

Fluorescence indicates a calcium-dependent interaction between the lipopeptide antibiotic LY146032 and phospholipid membranes.

LY146032 is one of the A21978C family of calcium-dependent antibiotics. This paper reports on its interactions with membranes as studied by its intrinsic fluorescence. The Trp residue was found to have a low fluorescence yield because of Förster-type energy transfer to the kynurenine residue (Kyn) (epsilon = 5000 at 364 nm). However, the Kyn fluorescence (lambda max = 465 nm in H2O) was a sensitive probe of the membrane interactions, and it was used in steady-state fluorescence measurements including fluorescence polarization anisotropy. Initial binding of the peptide to phospholipid vesicles occurs in calcium-free solutions. When calcium is added, the resulting 10-fold fluorescent enhancement and 15-nm blue shift show that it causes the antibiotic to penetrate further into the lipid bilayer. Calcium is bound with an association constant of 151 M-1, while a phospholipid titration in the presence of calcium gave an association constant of 5 x 10(3) M-1 for egg phosphatidylcholine. Magnesium and cadmium cause very slight fluorescence enhancements, but a more significant effect is caused by the trivalent lanthanide ions. Analysis of these data indicates that the calcium-selective site is on the peptide and that ion binding to the phospholipid headgroups has a secondary role. Comparison with the divalent cation dependent antibiotics bacitracin and amphomycin shows that LY146032 has a quite different activity and that a calcium-dependent membrane interaction could account for results obtained in vivo.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1988

Conformational change on calcium binding by the lipopeptide antibiotic amphomycin. A C.D. and monolayer study.

The acidic linear lipopeptide amphomycin is a calcium dependent antibiotic which is thought to bind to carrier lipids such as dolichol monophosphate. The actual role of Ca++ is not definitely established and in this article we have examined the peptides interactions with a range of divalent cations. By CD we have shown that a conformational change is induced by Ca++, Sr++ and Ba++ but not by Mg++, Zn++, Cd++ or Gd+++. Monolayer studies show a decrease in molecular area and an increase in film stability when the subphase contains Ca++. The ensemble of results provides preliminary evidence for the formation of a beta hairpin structure on ion binding (Ka (Ca++) = 2.4 x 10(3)M-1) which could enhance amphomycin's bilayer solubility.

Anti-Bacterial Agents↗