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P Laggner

Publications and source records attributed to P Laggner.

At least 37 records · Page 2Linked to original sources
PubMed1998

Structural changes of creatine kinase upon substrate binding.

Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzyme) to 48.9 A (enzyme plus Mg-ATP) and to 48.2 A (enzyme plus TSAC). M-CK showed similar changes from 28.0 A (free enzyme) to 25.6 A (enzyme plus Mg-ATP) and to 25.5 A (enzyme plus TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK also showed a change of the radius of gyration from 21.5 A (free enzyme) to 19.7 A (enzyme plus Mg-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a Mg-nucleotide-induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In CK, however, additional movements have to be involved.

Adenosine Triphosphate↗
PubMed1998

Site-specific effect of radical scavengers on the resistance of low density lipoprotein to copper-mediated oxidative stress: influence of alpha-tocopherol and temperature.

The radical scavenging capacity of active nitroxide spin label radicals located at different depths in the surface monolayer of native and alpha-tocopherol enriched low density lipoprotein (LDL) has been evaluated at early stages of copper-mediated lipid peroxidation. Spin labels induced a concentration-dependent prolongation in lag time and a pronounced decrease in the initial rate of conjugated diene (CD) formation. These effects strongly argue for a protective, antioxidative action of spin labels, which in turn become destroyed with the extent of oxidation by radical recombination reactions. The results revealed that the decrease in spectral intensity proceeds at a higher rate for nitroxide radicals located in a more hydrophobic environment. The loss in spin label activity is accompanied by simultaneous alpha-tocopherol consumption and progresses rather independently of initial alpha-tocopherol content. The data provided no evidence that spin labels either save alpha-tocopherol or compete with it for radicals. The authors, therefore, deduce that due to enhanced accessibility and mobility, spin labels located in the interior of LDL eliminate lipid-derived radicals, which otherwise would promote lipid peroxidation. Lowering of temperature clearly below the core-lipid phase transition temperature of LDL exerts a significant effect on the kinetics of copper-induced LDL oxidation, whereas the characteristics of the radical scavenging mechanisms of the spin label molecules located in the surrounding phospholipid monolayer are conserved. Taken together, the susceptibility of LDL to primary oxidative stress conditions was efficiently retarded by small amounts of radical scavengers. This effect was more pronounced for nitroxide radicals embedded deeper in the phospholipid monolayer and was rather independent of alpha-tocopherol enrichment.

Copper↗
PubMed1998

Trapping of short-lived intermediates in phospholipid phase transitions: the L* alpha phase.

Time-resolved small-angle X-ray diffraction of liquid-crystalline phospholipid-water systems under temperature or pressure jump conditions has demonstrated the existence of an ordered, intermediate L alpha phase, with a sub-second lifetime, designated as the L* alpha-phase. The lamellar repeat spacing is, universally, 0.3 nm smaller than that of the parent phase, irrespective of the lipid composition and of the jump conditions, provided that the jump leads to a net volume expansion of the phase. The presence of salts, most notably LiCl, leads to a prolongation of the lifetime. The results suggest a non-monotonic potential function for the interbilayer water thickness.

Membranes, Artificial↗
PubMed1997

Characterization of the sub-main-transition in distearoylphosphatidylcholine studied by simultaneous small- and wide-angle X-ray diffraction.

Simultaneous small- and wide-angle X-ray diffraction was used to investigate the structural conversions in the so-called sub-main-transition of fully hydrated multilamellar vesicles of distearoyl phosphatidylcholine (DSPC). The small-angle diffraction patterns show a modification in the supramolecular structure of the corrugated lipid bilayer and reveal that the sub-main-transition does not abolish the general features of the ripple phase (P(beta')). Concomitantly in the wide-angle region the diffraction patterns exhibit a rearrangement of the hydrocarbon chain packing and an onset of chain melting. The presence of KCl show an enhancement of the effects of the sub-main-transition. Moreover, in the ripple phase (P(beta')) below the sub-main-transition the hydrocarbon chains are positioned on an orthorhombic lattice in the presence of KCl.

Calorimetry, Differential Scanning↗
PubMed1996

Crystallization and preliminary X-ray analysis of a low density lipoprotein from human plasma.

Single crystals of human plasma low density lipoprotein (LDL), the major transport vehicle for cholesterol in blood, have been produced with a view to analysis of the three-dimensional structure by x-ray crystallography. Crystals with dimensions of approximately 200 x 100 x 50 microm have been reproducibly obtained from highly homogeneous LDL particle subspecies, isolated in the density ranges d = 1.0271-1. 0297 g/ml and d = 1.0297-1.0327 g/ml. Electron microscopic imaging of ultrathin-sectioned preparations of the crystals confirmed the existence of a regular, quasihexagonal arrangement of spherical particles of approximately 18 nm in diameter, thereby resembling the dimensions characteristic of LDL after dehydration and fixation. X-ray diffraction with synchrotron radiation under cryogenic conditions revealed the presence of well resolved diffraction spots, to a resolution of about 29 A. The diffraction patterns are indexed in terms of a triclinic lattice with unit cell dimensions of a = 16. 1 nm, b = 39.0 nm, c = 43.9 nm; alpha = 96.2 degrees, beta = 92.1 degrees, gamma = 102 degrees, and with space group P1.

Crystallography, X-Ray↗
PubMed1995

Core lipid structure is a major determinant of the oxidative resistance of low density lipoprotein.

The influence of thermally induced changes in the lipid core structure on the oxidative resistance of discrete, homogeneous low density lipoprotein (LDL) subspecies (d, 1.0297-1.0327 and 1.0327-1.0358 g/ml) has been evaluated. The thermotropic transition of the LDL lipid core at temperatures between 15 degrees C and 37 degrees C, determined by differential scanning calorimetry, exerted significant effects on the kinetics of copper-mediated LDL oxidation expressed in terms of intrinsic antioxidant efficiency (lag time) and diene production rate. Thus, the temperature coefficients of oxidative resistance and maximum oxidation rate showed break points at the core transition temperature. Temperature-induced changes in copper binding were excluded as the molecular basis of such effects, as the saturation of LDL with copper was identical below and above the core transition. At temperatures below the transition, the elevation in lag time indicated a greater resistance to oxidation, reflecting a higher degree of antioxidant protection. This effect can be explained by higher motional constraints and local antioxidant concentrations, the latter resulting from the freezing out of antioxidants from crystalline domains of cholesteryl esters and triglycerides. Below the transition temperature, the conjugated diene production rate was decreased, a finding that correlated positively with the average size of the cooperative units of neutral lipids estimated from the calorimetric transition width. The reduced accessibility and structural hindrance in the cluster organization of the core lipids therefore inhibits peroxidation. Our findings provide evidence for a distinct effect of the dynamic state of the core lipids on the oxidative susceptibility of LDL and are therefore relevant to the atherogenicity of these cholesterol-rich particles.

Calorimetry, Differential Scanning↗
PubMed1995

A comparison of structure and thermal behavior in human plasma lipoprotein(a) and low-density lipoprotein. Calorimetry and small-angle X-ray scattering.

Differential scanning calorimetry (DSC) and small-angle X-ray scattering (SAXS) studies have been performed to investigate the structural properties of lipoprotein(a) [Lp(a)] and low-density lipoprotein (LDL) obtained from the same donor. In addition, a comparison was made between autologous LDL and the remnant particle Lp(a-) obtained by removal of apo(a) through chemical reduction. With Lp(a), three distinct thermal transitions have been observed: the first one around 20 degrees C, arising from the core-located apolar lipids, similar to LDL but with a significantly lower melting temperature as compared to LDL of the same donor. The second one, at 55.7 +/- 0.25 degrees C, can be attributed to apo(a), since it was found to be absent in Lp(a-) and LDL, whereas isolated apo(a) in aqueous solution exhibited a similar transition. The third transition, at 80.4 +/- 0.9 degrees C, corresponds to apo-B100 protein unfolding. The low melting temperature of the core lipids in Lp(a) is preserved in Lp(a-); this suggests that the apolar lipid interactions are unaffected by apo(a) binding, and that the difference in the core melting behavior between Lp(a) and LDL is due to a different stabilization through interaction between the apolar core and the surface monolayer lipids. SAXS curves exhibited qualitatively the same characteristic features for LDL, Lp(a), and Lp(a-). Thus, the SAXS results showed that no major deviations from spherical particle shape occur with Lp(a), indicating that apo(a) wraps around the particle surface without major globular protrusions into the aqueous surrounding.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Squalene promotes the formation of non-bilayer structures in phospholipid model membranes.

A study of the lipid polymorphism of aqueous dispersions of stearoyloleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylchloline (95:5, mol/mol) in the presence of the hydrophobic molecule squalene, an intermediate in the biosynthesis of sterols, has been performed. With increasing concentration of squalene the main transition temperature was decreased from 29.8 degrees C for the pure phospholipid system to 28.1 degrees C for samples containing 5 mol% squalene without considerable changes in the phase transition enthalpy as detected by high precision differential scanning calorimetry. The structure of the phospholipid aggregates was determined by small- and wide-angle X-ray diffraction experiments showing only a minor increase of the lamellar repeat distance of the liquid-crystalline phase for the squalene containing samples. By far more pronounced was the effect of squalene on the lamellar-to-inverse-hexagonal phase transition, which was shifted from 64 degrees C to about 36 degrees C in the presence of 6 mol% squalene, thereby overlapping with the main transition. X-ray data showed that the size of the tubes of the inverse hexagonal phase are increasing linearly up to 6 mol% squalene. Experiments performed in the presence of 10 mol% squalene did not further change the phase behaviour, indicating the limiting partition of this hydrophobic molecule into the membrane. The high efficiency of squalene to promote the formation of the inverse hexagonal phase is discussed along the lines of argument based on the model of Kirk et al. (Kirk, G.L., Gruner, S.M. and Stein, D.E. (1984) Biochemistry 23, 1093-1102).

Calorimetry, Differential Scanning↗
PubMed1993

Ultrasonic study of melittin effects on phospholipid model membranes.

Low dose effects of melittin on dilute suspensions of dipalmitoylphosphatidylcholine multilamellar vesicles are investigated by studying the acoustic properties of the system. The temperature dependencies of sound velocity and absorption have been measured at 7.2 MHz in the temperature range of 20-55 degrees C, for different peptide/lipid molar ratios, R. The most pronounced effects were observed at R = 5 x 10(-3), in the vicinity of the pretransition, with a simultaneous increase in sound absorption and velocity. This indicates that melittin affects the polar head group region of the bilayer resulting in a decrease in mobility of the polar head groups. A nonmonotonic dependence of the main transition temperature, with an initial decrease followed by an increase as melittin is added, is interpreted as a consequence of a destabilizing action of the interfaces between mellitin-affected clusters and the unaffected phase.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Calorimetric measurements of thermal denaturation of stefins A and B. Comparison to predicted thermodynamics of stefin-B unfolding.

Thermal denaturation of two homologous proteins, low-M(r) cysteine-proteinase inhibitors stefins A and B, has been investigated by microcalorimetry. Calorimetric enthalpies, as well as the temperatures at maximum heat capacity, were determined as a function of pH for each protein. Transitions were found reversible at all pH values examined (5.0, 6.5, 8.1) for the thermally more stable stefin A, in contrast to stefin B. Stefin B shows a sharp irreversible transition around 65 degrees C at pH 6.5 and 8.1, probably due to unfolding of a dimeric state followed by oligomerisation. At pH 5.0, both proteins exhibit a reversible transition with temperatures of half-denaturation at 50.2 degrees C and 90.8 degrees C for stefins B and A, respectively. The calorimetric enthalpies, which equal the van't Hoff enthalpies to within 10%, are 293 kJ/mol and 490 kJ/mol for stefins B and A, respectively. Using the predictive method of Ooi and Oobatake (1991) [Proc. Natl Acad. Sci. USA 88, 2859] the thermodynamic functions of unfolding were calculated for stefin B, whose three-dimensional structure has been determined. The calculated enthalpy, heat-capacity change on unfolding and the temperature of half denaturation compare well to the microcalorimetric data.

Calorimetry, Differential Scanning↗
PubMed1992

Effect of delta-lysin on the phase transitions of lipid assemblies.

X-ray small-angle diffraction, differential scanning calorimetry (DSC), and temperature scanning densitometry (TSD) were used to study the effect of delta-lysin on the phase transitions of lipid assemblies from 1,2-0-dixehadecyl-sn-glycero-3-phosphocholine (DHPC). The experiments were carried out in excess of water in a temperature range of 0-55 degrees C, and at low peptide concentrations between 10(-4) and 10(-2) moles peptide per mole phospholipid. The incorporation of delta-lysin into lipid assemblies alters the lipid structure without significant changes on the temperatures of phase transition from gel to liquid crystalline phase. The temperature of the main transition was nearly unaffected. A reduction in the transition volume of the lipids with increasing concentrations of delta-lysin was observed. The minor changes in these parameters were interpreted as long-range structural changes caused by the peptide incorporation. The results are discussed in terms of the concept of cooperative phase transition of entire clusters occurring within a membrane implying that relative stable domains of gel phase, and liquid crystalline phase co-exist.

Bacterial Proteins↗
PubMed1991

Stabilization of non-bilayer structures by the etherlipid ethanolamine plasmalogen.

The thermotropic phase behavior of mixtures between diradylphosphatidylethanolamines and diacylphosphatidylcholine was studied using polarized light microscopy, 31P-NMR spectroscopy and synchrotron X-ray diffraction. Multilamellar liposomes composed of alkenylacylphosphatidylethanolamine (ethanolamine plasmalogen) undergo a phase transition from a lamellar to an inverse hexagonal lipid structure at 30 degrees C, which is about 20 degrees C and 30 degrees C lower as compared to its alkylacyl- and diacyl-analog, respectively. These results indicate a higher affinity to non-bilayer structures for the ether lipids. In the presence of the bilayer stabilizing phospholipid, palmitoyloleoylphosphatidylcholine, the transition is shifted to higher temperature without any significant changes in the overall structural parameters as revealed by X-ray diffraction experiments. Again, ethanolamine plasmalogen stabilizes the inverted hexagonal phase to the highest extent, i.e. even in the presence of 40 mol% palmitoyloleoylphosphatidylcholine a pure inverse hexagonal phase is formed at 60 degrees C. Such a result was not reported so far for a diacylphosphatidylethanolamine. This property of ethanolamine plasmalogen might be predominantly explained by an optimized packing of the hydrocarbon chains in the corners and interface region of the hexagonal tubes, owing to a different conformation of the sn-2 chain, which was deduced from 2H-NMR experiments (Malthaner, M., Hermetter, A., Paltauf, F. and Seelig, J. (1987) Biochim. Biophys. Acta 900, 191-197). Data obtained by time resolved X-ray diffraction show a coexistence of lamellar and inverse hexagonal structures in the phase transition region, but do not indicate the existence of non-lamellar intermediates or disorder within the sensitivity limits of the method.

Kinetics↗
PubMed1991

The influence of low amounts of cholesterol on the interdigitated gel phase of hydrated dihexadecylphosphatidylcholine.

Dihexadecylphosphatidylcholine (DHPC)/cholesterol binary mixtures in excess of water have been characterized by small-angle X-ray diffraction and differential scanning calorimetry and a temperature-composition phase diagram for this binary has been constructed. The property of cholesterol to perturb the hydrocarbon chain interdigitation in the lamellar gel phase of DHPC and to convert it into a non-interdigitated state has been observed by small- angle X-ray diffraction at cholesterol concentrations as low as 0.1 mol%. The interdigitated and non-interdigitated lamellar gel phases coexist in the range up to 5 mol% cholesterol. At this and higher cholesterol concentrations only non-interdigitated phases have been found in the phase diagram of the mixture. It is suggested that the ability of cholesterol in low concentration to eliminate the hydrocarbon chain interdigitation is related to the free energy increase due to unfavourable line boundaries between the interdigitated and non-interdigitated lipid domains.

Calorimetry, Differential Scanning↗
PubMed1991

Phospholipid phase transitions: kinetics and structural mechanisms.

A brief review is given on the principles and methods used to investigate structural phase transitions in phospholipid supramolecular structures. The conceptual differences of approaches close to and far from equilibrium are addressed, and the consequences in terms of the limits of interpretation for different types of methods, in particular referring to jump-relaxation and steady-state techniques, are surveyed. With the emphasis on connecting dynamic and structural information, the results obtained so far from different techniques are reviewed, and the open questions addressed. The more recent advances by millisecond time-resolved X-ray diffraction with synchrotron radiation and their main results obtained for transitions triggered by IR-laser temperature jumps are summarized. As a major novel aspect in the field, the necessity of considering martensitic, diffusionless transformation mechanisms and the occurrence of intermediate structures is highlighted.

Kinetics↗
PubMed1990

Conformational changes in bovine heart myosin as studied by EPR and DSC techniques.

Thermal behavior of intact and LC-2 deficient myosin obtained from bovine heart was studied using EPR and DSC techniques. The reactive thiol sites (Cys 704) of myosin was labelled with 4-maleimidopiperidine-nitroxyl, and the measurements were taken in X-band in the conventional and saturation transfer EPR time domains. DSC scans were made from 5 degrees up to 60 degrees C with 0.25 degree C/min scan rate. Bovine heart myosin was isolated by standard methods. The LC-2 deficient myosin was prepared by cleaving myosin with alpha-chymotrypsin (400:1 molar ratio) for 1.5 min at 25 degrees. Our basic finding was a conformational change in LC-2 deficient myosin detected at 18 degrees C. It was not observed in intact myosin suggesting that the dissociation of the regulatory light chain resulted in a local structural change in the neighbourhood of the attached label in the 20 kD domain. The rotational correlation time of the label and the microwave saturation behavior of myosin at 25 degrees C exhibited no significant differences after removal of the LC-2 light chain. However, the mobility of the same label was significantly diminished in skeletal muscle. Studying the melting behavior of myosin, six endothermic peaks were detected at 19; 41.3; 43.3; 45.5; 48.5; and 54.3 degrees C (enthalpies: 708.4; 399; 773.8; 1089; 1612.8; and 3304.8 kJ/mol). They were assigned to the segment containing the essential thiols: HMM S-2, HMM S-1 (50kD and 20kD plus 27kD) and LMM. Removal of the LC-2 light chain was associated with the disappearance of the 18 degrees transition showing again a structural change in LC-2 deficient myosin which extended to a larger region.

Adenosine Diphosphate↗
PubMed1988

Structure and phase behavior of hydrated mixtures of L-dipalmitoylphosphatidylcholine and palmitic acid. Correlations between structural rearrangements, specific volume changes and endothermic events.

Several new features of the phase diagram of L-dipalmitoylphosphatidylcholine (DPPC)/palmitic acid mixtures in excess water were established by means of static and time-resolved X-ray diffraction, densitometry and differential scanning calorimetry (DSC). At low temperatures, palmitic acid has a biphasic effect on the lamellar subgel phases: at concentrations below 5-6 mol%, it prevents formation of the DPPC subgel phase (Lc), while at higher contents (between about 40 and 90 mol%) another subgel phase (Lccom) is formed as a result of lipid co-crystallization at 1 DPPC: 2 palmitic acid stoichiometry. A crystalline palmitic acid phase separates from Lccom above 70-80 mol% of fatty acid. The Lccomphase transforms into a lamellar gel phase (L beta) in an endothermic transition centered at 38 degrees C. At high temperatures, the mixtures form hexagonal liquid-crystalline phase (HII) in the region of 60-70 mol% and an isotropic phase (I) at 90-100 mol% of palmitic acid. No coexistence of HII phase with the fluid lamellar phase of DPPC was observed at intermediate compositions (20 and 50 mol% of palmitic acid) but rather formation of a complex phase with non-periodic geometry characterized by molten chains and a broad, continuous small-angle scattering band. No evidence for fluid phase coexistence was found also at compositions between HII and I phases. The L beta--HII transition at 60-70 mol% of palmitic acids is readily reversible and two-state in both heating and cooling modes. It is characterized by the coexistence of initial and final phases with no detectable intermediates by time-resolved and static X-ray diffraction. The crystalline-isotropic transition in palmitic acid is two-state only in heating direction. On cooling, it is characterized by strong undercooling and gradually relaxing lamellar crystalline structures. The slowly reversible Lccom--L beta transition proceeds continuously through intermediate states. Although clearly discernible by both DSC and X-ray diffraction, it is not accompanied by specific volume changes.

1,2-Dipalmitoylphosphatidylcholine↗