Biomedical subjects

K Lohner

Publications and source records attributed to K Lohner.

16 recordsLinked to original sources

PubMed1997

Differential scanning microcalorimetry indicates that human defensin, HNP-2, interacts specifically with biomembrane mimetic systems.

alpha-Defensins are antimicrobial peptides with 29-35 amino acid residues and cysteine-stabilized amphiphilic, triple-stranded beta-sheet structures. We used high-precision differential scanning microcalorimetry to investigate the effects of a human neutrophil alpha-defensin, HNP-2, on the phase behavior of model membranes mimicking bacterial and erythrocyte cell membranes. In the presence of this positively charged peptide, the phase behavior of liposomes containing negatively charged phosphatidylglycerol was markedly altered even at a high lipid-to-peptide molar ratio of 500:1. Addition of HNP-2 to liposomes mimicking bacterial membranes (mixtures of dipalmitoylphosphatidylglycerol and -ethanolamine) resulted in phase separation owing to some domains being peptide-poor and others peptide-rich. The latter are characterized by an increase of the main transition temperature, most likely arising from electric shielding of the phospholipid headgroups by the peptide. On the other hand, HNP-2 did not affect the phase behavior of membranes mimicking erythrocyte membranes (equimolar mixtures of dipalmitoylphosphatidylcholine and sphingomyelin) as well as the pure single components. This is in contrast to melittin, which significantly affected the phase behavior of choline phospholipids in accordance with its unspecific lytic activity. These results support the hypothesis of preferential interaction of defensins with negatively charged membrane cell surfaces, a common feature of bacterial cell membranes, and demonstrate that HNP-2 discriminates between model membrane systems mimicking prokaryotic and eukaryotic cell membranes.

Blood Proteins↗

PubMed1996

Is the high propensity of ethanolamine plasmalogens to form non-lamellar lipid structures manifested in the properties of biomembranes?

Plasmalogens are glycerophospholipids characterized by an alk-1'-enylether bond in position sn-1 and an acyl bond in position sn-2. These ubiquitous etherlipids exhibit a different molecular structure as compared to diacyl phospholipids. The most peculiar change is a perpendicular orientation of the sn-2 acyl chain at all segments to the membrane surface. This extended conformation results in an effectively longer aliphatic chain in plasmalogen than in the diacyl analog. Moreover, the lack of the carbonyl oxygen in position sn-1 affects the hydrophilicity of the headgroup and allows stronger intermolecular hydrogen-bonding between the headgroups of the lipid. These properties favour the formation of non-lamellar structures which are expressed in the high affinity of ethanolamine plasmalogen to adopt the inverse hexagonal phase. Such structures may be involved in membrane processes, either temporarily, like in membrane fusion or locally, e.g. to affect the activity of membrane-bound proteins. The predominant distribution of ethanolamine plasmalogens in some cellular membranes like nerve tissues or plasma membranes and their distinctly different properties in model membranes as compared to diacyl phospholipids impose the question, whether these differences are also manifested in the heterogeneous environment of biological membranes. The integration of biophysical studies and biochemical findings clearly indicated that the high propensity of ethanolamine plasmalogen to form non-lamellar structures is reflected in several physiological functions. So far it seems to be evident that ethanolamine plasmalogens play an important role in maintaining the balance between bilayer and non-lamellar phases which is crucial for proper cell function. Furthermore, they are the major phospholipid component of inverse hexagonal phase inclusions in native retina and are able to mediate membrane fusion as demonstrated between neurotransmitter vesicles and presynaptic membranes.

Cell Membrane↗

PubMed1994

Effects of drug-binding on the thermal denaturation of human serum albumin.

Asymmetric thermograms of defatted albumin, alone and in the presence of two model drugs, have been obtained in phosphate buffers at three pH values. The albumin is less thermally stable in the N form, but is protected by both drugs. The nonsteroidal antiinflammatory benoxaprofen offers more protection than warfarin against thermal denaturation.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1994

Structural basis for the difference in thermodynamic properties between the two cysteine proteinase inhibitors human stefins A and B.

Homology modelling has been used to model stefin A based on the X-ray structure of stefin B. Several models have been produced by interactive modelling or positioning of the side chains by Monte Carlo procedure with simulated annealing. The quality of models was evaluated by calculation of the free energy of hydration, 3D-1D potential or buried area of surface accessibility. Stefin A is a thermostable protein, exhibiting a two-state denaturation, while stefin B denatures at a 40 degrees C lower temperature and forms a stable molten globule intermediate under mild denaturing conditions. From the tertiary structures, thermodynamic functions were predicted, conforming closely to the experimental calorimetric results. Polar and apolar buried areas of surface accessibility were obtained by structural deconvolution of the thermograms. It is suggested that the basic difference between the stefins is the domination of hydrophobic interaction in the stabilization of stefin B, which is due to its non-specific nature leading to the formation of a molten globule intermediate. Modelling of stefin A predicts increased numbers of hydrogen bonds which stabilize it and increase the cooperativity of its denaturation.

Amino Acid Sequence↗

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

Thermal unfolding and aggregation of human complement protein C9: a differential scanning calorimetry study.

The thermotropic behavior of purified human complement protein C9 was investigated by high-sensitivity differential scanning calorimetry. When dissolved in physiological buffers (pH 7.2, 150 mM NaCl), C9 underwent three endothermic transitions with transition temperatures (Tm) centered at about 32, 48, and 53 degrees C, respectively, and one exothermic transition above 64 degrees C that correlated with protein aggregation. The associated calorimetric enthalpies of the three endothermic transitions were about 45, 60, and 161 kcal/mol with cooperative ratios (delta Hcal/delta HvH) close to unity. The total calorimetric enthalphy for the unfolding process was in the range of 260-280 kcal/mol under all conditions. The exothermic aggregation temperature was strongly pH dependent, changing from 60 degrees C at pH 6.6 to 81.4 degrees C at pH 8.0, whereas none of the three endothermic transitions was significantly affected by pH changes. They were, however, sensitive to addition of calcium ions; most affected was Tm1 which shifted from 32 to 35.8 degrees C in the presence of 3 mM calcium, i.e., the normal blood concentration. Kosmotropic ions stabilized the protein by shifting the endothermic transitions to slightly higher temperatures whereas inclusion of chaotropic ions (such as choline), removal of bound calcium by addition of EDTA, or proteolysis with thrombin lowered the transition temperatures. Previous studies had indicated the formation of at least three different forms of C9 during membrane insertion or during heat polymerization, and it is suggested that the three endothermic transitions reflect the formation of such C9 conformers. Choline, which is present at high concentrations on the surface of biological membranes, and calcium ions have the ability to shift the transition temperatures of the first two transitions to be either close to or below body temperature. Thus, it is very likely that C9 is present in vivo in a partially unfolded state when bound to a membrane surface, and we propose that this facilitates membrane insertion and refolding of the protein into an amphiphilic conformation.

Buffers↗

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

Effects of small organic molecules on phospholipid phase transitions.

Small organic molecules are known to exhibit a wide spectrum of physiological or pharmacological effects and many of them are thought to be membrane associated. Therefore a great number of studies is devoted to the interaction between these molecules and phospholipid model membranes. Results obtained for molecular species of varying hydrophobic/hydrophilic balances will be described. It will be shown that, in general, these different molecules induce similar effects on phospholipid phase transitions, although they are located differently in the membrane. Detailed studies of these interactions will help to understand these processes on a molecular level.

Alcohols↗

PubMed1988

Human complement protein C9 is a calcium binding protein. Structural and functional implications.

Human complement protein C9 is shown to be a metalloprotein that binds 1 mol of Ca2+/mol of C9 with a dissociation constant of 3 micron as measured by equilibrium dialysis. Incubation with EDTA removes the bound calcium, resulting in a apoprotein with decreased thermal stability. This loss in stability leads to aggregation and, therefore, to loss of hemolytic activity upon heating to a few degrees above the physiological temperature. Heat-induced aggregation of apoC9 can be prevented by salts that stabilize proteins according to the Hofmeister series of lyotropic ions, suggesting that the ion in native C9 may ligand with more than one structural element or domain of the protein. Ligand blotting indicates that the calcium binding site is located in the amino-terminal half of the protein. Removal of calcium by inclusion of EDTA in assay mixtures has no effect on the hemolytic activity of C9, and its capacity to bind to C8 in solution, or to small unilamellar lipid vesicles at temperatures at or below the physiological range. Although we do not know yet the precise structural and functional role of the bound calcium, it is clear that it provides thermal stability to C9 and it may have a function in regulation of membrane insertion.

Calcium↗

PubMed1987

Structure and thermodynamics of the dihexadecylphosphatidylcholine-water system.

X-ray small- and wide-angle diffraction, differential scanning calorimetry (DSC), temperature scanning densitometry (TSD) and electron microscopy were used to study the lyotropic and thermotropic properties of the system 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine-water over a wide range of compositions from the dry lipids to a large excess of water, and in the temperature range between 0 degrees C and 150 degrees C. The results were used to construct a temperature-composition phase diagram. The phases have been characterized with respect to their molecular arrangements and hydrocarbon chain packing subcells. In the fully hydrated state (greater than 45 wt% H2O) four thermotropic phases were found, with readily reversible transitions at 5 degrees C, 32.5 degrees C and 43.6 degrees C, respectively. The two lower temperature phases deviate from all others in consisting of bilayers with fully interdigitated hydrocarbon chains, while above 32.5 degrees C the structures resemble closely those of the analog diester lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). At hydrations between 30 and 45 wt% H2O, and below 32 degrees C, interdigitated and non-interdigitated multilayers coexist in one coherent phase. A bilayer tilting mechanism is proposed for the formation of this coexistence of two regular structures. Below 30 wt% H2O, hydrated 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) exists in lamellar, non-interdigitated bilayers, showing very weak interbilayer swelling. There, the water molecules appear to occupy voids between the polar headgroups.

Calorimetry, Differential Scanning↗

PubMed1987

Thermal phase behaviour and structure of hydrated mixtures between dipalmitoyl- and dihexadecylphosphatidylcholine.

Mixtures of 1,2-dipalmitoyl- and 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DPPC and DHPC) in dispersion with excess water were studied by differential scanning calorimetry (DSC) and X-ray diffraction techniques. The transition parameters of the main gel-to-liquid crystalline transition show a monotonous dependence on the composition, indicating ideal miscibility of the two lipids, in keeping with the closely similar structures of the pure, hydrated lipids in the P beta' and L alpha states. The pre-transition shows a depression to a minimum temperature of 23 degrees C occurring around equimolar mixtures. Below the pre-transition temperatures, the L beta' gel phase of DPPC maintains bimolecular structure up to DHPC admixtures of 50 mol%, with adaptations in hydrocarbon chain packing and multilayer periodicity. On the side of DHPC, the interdigitated gel structure shows full solubility for DPPC up to equimolarity without major structural changes. The crystalline Lc-phase of DPPC exhibits immiscibility with DHPC, demonstrated by the fact that the subtransition is abolished already at less than 15 mol% DHPC. DHPC, below its subtransition, can accommodate up to 50 mol% DPPC within an interdigitated layer structure with unperturbed, crystalline hydrocarbon chain packing.

1,2-Dipalmitoylphosphatidylcholine↗

PubMedDate not supplied

Effect of cholesterol on vesicle bilayer geometry of choline plasmalogen and comparison with dialkyl-, alkylacyl- and diacyl-glycerophosphocholines.

Small unilamellar vesicles containing alkenylacyl-, alkylacyl-, dialkyl- or diacyl-glycerophosphocholine were prepared by sonication. Their size was determined from the average internal volume after chromatography on Sepharose 2B and from 31P-NMR linewidths. Alkenylacyl glycerophosphocholine (choline plasmalogen) was found to form the largest vesicles. By addition of 30 mol% cholesterol, the size of plasmalogen vesicles, but not of those containing the alkyl and acyl analogue lipids, was significantly increased. The presence of 50 mol% sterol led to highly increased vesicle sizes of alkylacyl, dialkyl and diacyl-glycerophosphocholine. Mixtures of plasmalogens with 50 mol% cholesterol did not form unilamellar vesicles upon sonication. Bilayer thickness and surface area per phospholipid molecule were determined by small angle X-ray scattering and measurement of partial specific volumes. There is little difference between alkenylacyl glycerophosphocholine and the corresponding diacyl-analog, whereas bilayers consisting of dioleoyl glycerophosphocholine are significantly thinner. Correspondingly their molecular surface area is by about 8% larger than that of the mixed-chain diradyl glycerophosphocholine, since the partial molar volumes are similar for all vesicles tested.

Cholesterol↗