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F Paltauf

Publications and source records attributed to F Paltauf.

At least 73 records · Page 4Linked to original sources
PubMed1991

Isolation of a phosphatidylserine transfer protein from yeast cytosol.

A phospholipid transfer protein with a broad substrate specificity was isolated from yeast cytosol. The rate of transfer catalyzed by this protein in vitro is highest for phosphatidylserine; phosphatidylethanolamine, cardiolipin, phosphatidic acid and ergosterol are transported at a lower rate. In contrast to the yeast phosphatidylinositol transfer protein (Daum, G. and Paltauf, F. (1984) Biochim. Biophys. Acta 794, 385-391) the phosphatidylserine transfer protein does not catalyze the translocation of phosphatidylinositol or phosphatidylcholine. Using chromatographic methods the phosphatidylserine transfer protein was enriched approximately 3000-fold over yeast cytosol. The protein is inactivated by heat, detergents and proteinases. Divalent cations strongly inhibit the transfer of phosphatidylserine in vitro, and EDTA at low concentrations has a stimulatory effect.

Adsorption↗
PubMed1991

Mitochondrial membrane contact sites of yeast. Characterization of lipid components and possible involvement in intramitochondrial translocation of phospholipids.

Submitochondrial membrane fractions from yeast that are enriched in inner and outer membrane contact sites were analyzed with respect to their lipid composition. Characteristic features were the significantly reduced content of phosphatidylinositol, the decreased amount of phosphatidylcholine, and the enrichment in phosphatidylethanolamine and cardiolipin. Coisolation of phosphatidylserine synthase with the outer membrane portion and enrichment of phosphatidylserine decarboxylase in the inner membrane portion of isolated contact sites provided the basis for a metabolic assay to study phosphatidylserine transfer from the outer to the inner mitochondrial membrane via contact sites. The efficient conversion to [3H]phosphatidylethanolamine of [3H]phosphatidylserine synthesized from [3H]serine in situ supports the notion that mitochondrial membrane contact sites are zones of intramitochondrial translocation of phosphatidylserine.

Biological Transport↗
PubMed1991

Protein-catalyzed transport of ether phospholipids.

The protein-catalyzed transfer of alkenylacyl-, alkylacyl-, or diacyl-glycerophosphocholines, carrying a pyrenedecanoyl residue as a fluorogenic acyl chain, was studied using unilamellar bilayer vesicles as donor and acceptor membranes in a fluorescence assay. Specific phospholipid transfer proteins, such as phosphatidylinositol transfer protein from yeast and phosphatidylcholine transfer protein from bovine liver showed higher transfer rates with ether lipid substrates. Transfer rates for alkylacyl- and alkenylacyl-glycerophosphocholine as compared to the diacyl analog were rather similar in the presence of non-specific lipid transfer proteins from maize or from bovine liver, respectively. When vesicles of fluorogenic compounds were titrated with the yeast phosphatidylinositol transfer protein, only a 15-20% higher binding affinity for alkenylacyl- and alkylacyl-glycerophosphocholine than for diacyl-glycerophosphocholine was observed. Thus the marked differences of transfer rates measured with this transfer protein cannot be attributed to different binding affinities for the respective phospholipid subclasses. A possible explanation for differences in transfer rates could be differences in the organization of the phospholipid subclasses at the hydrophobic/hydrophilic interface of bilayer membranes.

Animals↗
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

Coordinate regulation of phosphatidylserine decarboxylase in Saccharomyces cerevisiae.

Regulation of the activity of the mitochondrial enzyme phosphatidylserine decarboxylase (PSD) was measured in vitro by using membrane preparations from wild-type and mutant strains of Saccharomyces cerevisiae. PSD specific activity was not affected by carbon source, and on all carbon sources, the highest specific activity was observed in cells entering the stationary phase of growth. However, PSD activity was found to be regulated in response to soluble precursors of phospholipid biosynthesis. PSD specific activity was reduced to about 63% of the level observed in unsupplemented wild-type cells when the cells were grown in the presence of 75 microM inositol. The presence of 1 mM choline alone had no repressing effect, but the presence of 1 mM choline and 75 microM inositol together led to further repression to a level of about 28% of the derepressed activity. Regulatory mutations known to affect regulation or expression of genes encoding phospholipid-synthesizing enzymes also affected PSD specific activity. opi1 mutants, which are constitutive for a number of phospholipid-biosynthetic enzymes, were found to have high, constitutive levels of PSD. Likewise, in ino2 or ino4 regulatory mutants, PSD activity was found to be at the fully repressed level regardless of growth condition. Regulation of PSD activity was also affected in several structural-gene mutants under conditions of impaired phosphatidylcholine biosynthesis. Together, these data strongly suggest that PSD expression is controlled by the mechanism of general control of phospholipid biosynthesis that regulates many enzymes of phospholipid biosynthesis.

Carboxy-Lyases↗
PubMed1991

Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae.

Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.

Cell Compartmentation↗
PubMed1991

Serum plasmalogens in ischemic cerebrovascular disease.

Plasmalogens, a subclass of glycerophospholipids are ubiquitous constituents of cellular membranes and serum lipoproteins. Comparing concentrations of plasmalogens in sera from patients suffering from ischemic cerebrovascular disease with serum levels in a normal population significantly lower values were found for patient sera.

Aged↗
PubMed1990

Dipolar solvent relaxation on a nanosecond time scale in ether phospholipid membranes as determined by multifrequency phase and modulation fluorometry.

The present study reports on the observation of dipolar solvent relaxation in phospholipid membranes using multifrequency phase and modulation fluorometry. We measured the time-resolved emission spectra of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) in artificial bilayer membranes of chemically defined acyl-, alkyl-, and alkenyl-substituted phospholipids at 15 degrees C. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 3-O-hexadecyl-2-oleoyl-sn-glycero-1-phosphocholine, or 1-O-hexadec-1'-enyl-2-oleoyl-sn-glycero-3-phosphocholine (plasmalogen) were used as matrix lipids. The chemical structures of these lipids differ only with respect to the type of linkage (carboxyl ester, ether, or enol ether bond) between glycerol and the hydrophobic chain linked to the primary hydroxyl of glycerol. At 15 degrees C, all the lipids are in the liquid crystalline state. PRODAN probably localizes at the hydrophobic-hydrophilic interface of the phospholipid bilayer [Chong, P. L. (1988) Biochemistry 27, 399-404]. We found faster solvent relaxation of PRODAN in membranes composed of the ether lipid compared to that in the ester lipid membranes. On the other hand, the fluorescence anisotropies of the label were very similar, showing that the motion of the label itself is similar in ether and carboxyl ester lipids. We conclude that the spectral differences observed for PRODAN in ether and ester lipids could be due to different dipolar relaxation of the immediate surroundings of the label, i.e., reorientation of lipid dipoles in the glycerol region and of water molecules residing therein.

Kinetics↗
PubMed1990

Stereoselectivity of lipases. I. Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique.

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol, enantiomeric 1(3)-alkyl-2-acyl-sn-glycerol, or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the same in both enantiomers, thereby excluding the possibility of effects occurring due to fatty acid specificity. We observed a porcine pancreatic lipase sn-3 stereoselectivity when using the enantiomeric 1(3)-alkyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack of stereoselectivity observed when using the enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol (triglyceride analogue). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyze the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoakyl-diacyl pair tested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of rabbit gastric lipase, whereas low rates and a clear chiral discrimination was noticed in the case of human gastric lipase during hydrolysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers. It is particularly obvious that in the case of human gastric lipase decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylamino derivatives (diglyceride analogue).

Animals↗
PubMed1990

Uptake of fluorescent plasmalogen analogs by cultured human skin fibroblasts deficient in plasmalogen.

One of the consequences of hereditary peroxisomal dysfunction in the cerebro-hepato-renal (Zellweger) syndrome (CHRS) is a dramatic decrease in the biosynthesis and cellular content of ether lipids. In the present study effects of reduced cellular plasmalogen levels on membrane-membrane interactions were investigated. Cultured CHRS fibroblasts were incubated with unilamellar phospholipid vesicles consisting of 1-O-alkenyl-2-acyl- or 1,2-diacyl-sn-glycerophosphocholines and ethanolamines, carrying either the trans-parinaroyl or the 1,6-diphenyl-1,3,5-hexatriene propionyl group in position 2. Transfer of the fluorogenic phospholipids from vesicles to cells was followed by measuring the concomitant increase in fluorescence intensity. Transfer of phospholipids from cells to vesicles was monitored by incubating cells, prelabeled with [3H]oleic acid, in the presence of phospholipid vesicles. Fibroblasts from healthy donors or CHRS fibroblasts supplemented with the plasmalogen precursor 1-O-hexadecylglycerol served as controls. Plasmalogen-deficient cells exhibited a significantly increased tendency to take up exogenous choline or ethanolamine plasmalogens. Cellular plasmalogens were transferred from control cells to vesicles at a higher rate if the acceptor vesicles consisted of plasmalogens as compared to diacylglycerophosphocholine. Thus, it appears as if mechanisms existed which preserve cellular plasmalogen levels during interaction with exogenous phospholipid pools. Preliminary experimental evidence suggests that the observed exchange of phospholipids between cultured fibroblasts and vesicles occurs by a protein-catalyzed process.

Biological Transport↗
PubMed1990

Lipid transport in microorganisms.

Microorganisms are useful model systems for the study of intracellular transport of lipids. Eukaryotic microorganisms, such as the yeast Saccharomyces cerevisiae, are similar to higher eukaryotes with respect to organelle structure and membrane assembly. Experiments in vivo showed that transport of phosphatidylcholine between yeast microsomes and mitochondria is energy independent; transfer of phosphatidylinositol to the plasma membrane and the flux of secretory vesicles take place by different mechanisms. Linkage of transfer and biosynthesis of phospholipids was demonstrated in the case of intramitochondrial phospholipid transfer. A yeast phosphatidylinositol/phosphatidylcholine transfer protein, which is essential for cell viability, was isolated and characterized. Another phospholipid transfer protein present in yeast cytosol, which has a different specificity, is currently under investigation. Transfer of phospholipids between cellular membranes was also demonstrated with prokaryotes. The cytoplasm and the periplasma of the gram-negative facultative photosynthetic bacterium Rhodopseudomonas sphaeroides contain phospholipid transfer proteins; these seem to be involved in the biosynthesis of prokaryotic membranes.

Bacteria↗
PubMed1990

Intramitochondrial transfer of phospholipids in the yeast, Saccharomyces cerevisiae.

Translocation of phosphatidylinositol, which is synthesized on the outer aspect of the outer membrane of isolated yeast mitochondria, to the inner membrane is linked to phosphatidylinositol synthesis and is therefore a vectorial process. Phosphatidylinositol once integrated into the inner mitochondrial membrane is not transferred back to the mitochondrial surface. Phosphatidylserine is also translocated from the outer to the inner mitochondrial membrane, where it is decarboxylated to phosphatidylethanolamine. We made use of this metabolic modification to characterize the intramitochondrial transfer of phosphatidylserine and phosphatidylethanolamine. Intramitochondrial phosphatidylserine transfer is insensitive to the uncoupler carbonyl cyanide m-chlorophenylhydrazone and to valinomycin and is thus independent of an electrochemical gradient across the inner membrane. Transfer of phosphatidylserine from the outer to the inner mitochondrial membrane occurs not only in intact mitochondria but also in mitoplasts which are devoid of intermembrane space proteins but have the outer membrane still adherent to the inner membrane. This result suggests that specific contact sites are involved in the intramitochondrial translocation of phospholipids. 3H-Labeled phosphatidylethanolamine synthesized from [3H]serine in isolated mitochondria is readily exported from the inner to the outer mitochondrial membrane without prior mixing with the pool of phosphatidylethanolamine of the inner membrane.

Biological Transport↗
PubMed1990

Halo lipids. 10. Synthesis and cytostatic activity of O-alkylglycerophospho-L-serine analogs.

The synthesis of O-alkylglycerophospho-L-serine analogs is described, which represent a new class of cytostatically active agents based on phospholipids. The new compounds were obtained by conversion of O-alkylglycerophosphoric ester analogs by means of phospholipase D and by condensing O-alkylglycerophosphoric acid analogs with protected L-serine followed by the removal of protective groups of the resulting intermediates. The structure of the O-alkylglycerophospho-L-serines was confirmed by fast atom bombardment mass spectrometry. The compounds were found to inhibit the growth of Ehrlich ascites tumor cells in vitro. Half maximum inhibition was observed at concentrations between 7 and 15 microM. For the 1-O-alkyl-2-methoxy glycerophosphoserine only a higher value (30 microM) was found. With most of the substances tested growth was completely inhibited at a concentration of 40 microM.

Animals↗
PubMed1990

Fluorescence lifetime distributions of parinaroyl phospholipids in choline plasmalogen and phosphatidylcholine bilayers containing different amounts of cholesterol.

The fluorescence decay of alkenylparinaroyl- and palmitoylparinaroyl glycerophosphocholines in vesicles of the unlabeled alkenyloleoyl and palmitoyloleoyl analogs was determined by multifrequency phase and modulation fluorometry. The measured phase angles and demodulations could be equally well fitted to a biexponential decay, as well as unimodal or bimodal continuous lifetime distributions. The latter model was applied to study the influence of cholesterol on parinaroyl phospholipid fluorescence in vesicles. The long-living component of a bimodal lifetime distribution was sensitive toward the presence of the sterol. Upon increasing cholesterol concentrations, its lifetime center increased and its distribution widths decreased. Lifetime distribution widths in vesicles of alkenyloleoyl- or palmitoyloleoyl-glycerophosphocholine (choline plasmalogen and phosphatidylcholine, respectively) were reduced by the sterol to the same extent. We interprete the sterol-induced lifetime distribution narrowing as an effect due to an increase of membrane homogeneity in cholesterol-phospholipid membranes.

Cholesterol↗
PubMed1989

Membrane properties modulate the activity of a phosphatidylinositol transfer protein from the yeast, Saccharomyces cerevisiae.

A phospholipid transfer protein from yeast (Daum, G. and Paltauf, F. (1984) Biochim. Biophys. Acta 794, 385-391) was 2800-fold enriched by an improved procedure. The specificity of this transfer protein and the influence of membrane properties of acceptor vesicles (lipid composition, charge, fluidity) on the transfer activity were determined in vitro using pyrene-labeled phospholipids. The yeast transfer protein forms a complex with phosphatidylinositol or phosphatidylcholine, respectively, and transfers these two phospholipids between biological and/or artificial membranes. The transfer rate for phosphatidylinositol is 19-fold higher than for phosphatidylcholine as determined with 1:8 mixtures of phosphatidylinositol and phosphatidylcholine in donor and acceptor membrane vesicles. If acceptor membranes consist only of non-transferable phospholipids, e.g., phosphatidylethanolamine, a moderate but significant net transfer of phosphatidylcholine occurs. Phosphatidylcholine transfer is inhibited to a variable extent by negatively charged phospholipids and by fatty acids. Differences in the accessibility of the charged groups of lipids to the transfer protein might account for the different inhibitory effects, which occur in the order phosphatidylserine which is greater than phosphatidylglycerol which is greater than phosphatidylinositol which is greater than cardiolipin which is greater than phosphatidic acid which is greater than fatty acids. Although mitochondrial membranes contain high amounts of negatively charged phospholipids, they serve effectively as acceptor membranes, whereas transfer to vesicles prepared from total mitochondrial lipids is essentially zero. Ergosterol reduces the transfer rate, probably by decreasing membrane fluidity. This notion is supported by data obtained with dipalmitoyl phosphatidylcholine as acceptor vesicle component; in this case the transfer rate is significantly reduced below the phase transition temperature of the phospholipid.

Biological Transport↗
PubMed1989

Molecular cloning, primary structure and disruption of the structural gene of aldolase from Saccharomyces cerevisiae.

A yeast cDNA genetic library in a bacteriophage expression vector was screened using an antiserum reacting with fructose 1,6-bisphosphate aldolase from Saccharomyces cerevisiae. Radio-labelled probes of selected immunopositive clones were used for screening of a yeast genomic library. From the genomic clones a yeast/Escherichia coli shuttle plasmid was constructed containing on a 1990-base-pair fragment the entire structural gene FBA1 coding for yeast aldolase. The primary structure of the FBA1 gene was determined. An open reading frame comprises 1077 base pairs coding for a protein of 359 amino acids with a predicted molecular mass of 39,608 Da. As observed for other strongly expressed yeast genes, codon usage is extremely biased. The 810 base pairs at the 5' end and the 90 base pairs at the 3' end of the coding region of the cloned FBA1 gene are sufficient for normal expression and show characteristic elements present in the noncoding sequences of other yeast genes. Aldolase is the major protein in yeast cells transformed with a high-copy-number plasmid containing the FBA1 gene. The aldolase gene was disrupted by insertion of the yeast URA3 gene into the coding region of one FBA1 allele in a homozygous diploid ura3 strain. The haploid offsprings with the defective aldolase allele fba1::URA3 lack aldolase enzymatic activity and fail to grow in media containing as a carbon source metabolites of only one side of the aldolase reaction.

Amino Acid Sequence↗