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F Paltauf

Publications and source records attributed to F Paltauf.

At least 55 records · Page 3Linked to original sources
PubMed1994

Apoprotein-phospholipid interactions in Lp(a).

Lipoprotein (a) (Lp(a)) and low-density lipoprotein (LDL) are structurally related to each other. Both exhibit identical phospholipid compositions and possess one molecule of apoprotein B-100 (apoB). Lp(a) contains, in addition, apoprotein (a) (apo(a)), which localizes to the particle surface and interacts with the apoB component by non-covalent and covalent forces. Protein-protein interaction is probably interrelated with protein-lipid interaction. Fluorescent analogs of phosphatidylcholine and sphingomyelin were inserted into the surface layer of LDL and Lp(a). The obtained fluorescence data reflecting mobility and distributional heterogeneity of the labeled lipids provided evidence that apo-proteins discriminate between choline phospholipids and preferentially associate with phosphatidylcholine. This effect is enhanced in Lp(a) because of the presence of apolipoprotein (a). Higher affinity for Lp(a) as compared with LDL was also observed with a fluorescent diether analog of phosphatidylcholine in native serum. In contrast, the time-dependent transfer of the same lipid into Lp(a) was slower compared with LDL, probably as a consequence of the more rigid surface of the former lipoprotein.

Apolipoproteins↗
PubMed1994

Isolation and characterization of a mutant of Saccharomyces cerevisiae with pleiotropic deficiencies in transcriptional activation and repression.

The isolation of the dep1 mutant of Saccharomyces cerevisiae is reported. The mutant was identified by its disability to regulate expression of structural genes involved in phospholipid biosynthesis, INO1, CHO1 and OPI3, in response to supplementation with soluble lipid precursors. Expression of the INO1, CHO1 and OPI3 genes was not fully derepressed in the absence of soluble lipid precursors, inositol and choline in the dep1 mutant, as compared to wild type. The mutant also exhibited incomplete repression of these same genes in the presence of inositol and choline. Repression by phosphate of the PHO5 gene was reduced in the mutant, as was derepression of this gene in the absence of phosphate. In addition, we show that expression of INO1 and OPI3 structural genes is strongly dependent on the growth phase both in wild-type and dep1 mutant strains. However, in the mutant, elevated basal steady-state mRNA levels were reached in the late stationary growth phase, independent of supplementation conditions. The dep1 mutation represents a new complementation group with respect to phospholipid synthesis and was mapped to a position of about 12 cM distal from the centromere on the left arm of chromosome I. Deficiencies in transcription activation and repression of metabolically unrelated genes, as well as reduced mating efficiency and lack of sporulation of homozygous diploid dep1/dep1 mutants indicate a pleiotropic regulatory function of the DEP1 gene product. Thus, Dep1p appears to be a new member of a class of transcriptional modulators, including Rpd1p/Sin3p/Ume4p/Sdi1p/Gam3p, Rpd3p, Spt10p and Spt21p.

CDPdiacylglycerol-Serine O-Phosphatidyltransferase↗
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

Acetyl-CoA carboxylase from yeast is an essential enzyme and is regulated by factors that control phospholipid metabolism.

We have isolated a 1.2-kilobase pair cDNA fragment in a screening for yeast genes regulated at the level of transcription by soluble lipid precursors, inositol and choline. Sequence analysis and comparison of the deduced amino acid sequence to protein databases unveiled 68% similarity of a 374-amino acid peptide fragment to published C termini of chicken and rat acetyl-CoA carboxylase and almost 100% identity to the product of the FAS3 gene from yeast. Several lines of evidence confirm that the cloned gene represents the yeast structural gene ACC1 encoding acetyl-CoA carboxylase. Overexpression of the ACC1 gene from a high copy number plasmid resulted in overexpression of a 250-kDa biotin-enzyme and enzymatic activity of acetyl-CoA carboxylase. Disruption of one ACC1 allele in a diploid wild-type strain resulted in 50% reduction of ACC1-specific mRNA and acetyl-CoA carboxylase specific activity and a marked decrease of biotin associated with a 250-kDa protein, compared to wild-type. After sporulation of diploid disruptants, spores containing the disrupted acc1 allele failed to enter vegetative growth, despite fatty acid supplementation, suggesting that acetyl-CoA carboxylase activity is essential for a process other than de novo fatty acid synthesis and that only a single functional copy of the ACC1 gene exists. ACC1 transcription was repressed 3-fold by lipid precursors, inositol and choline, and was also controlled by regulatory factors Ino2p, Ino4p, and Opi1p, providing evidence that the key step of fatty acid synthesis is regulated in conjunction with phospholipid synthesis at the level of gene expression. The 5'-untranslated region of the ACC1 gene contains a sequence reminiscent of an inositol/choline-responsive element identified in genes encoding phospholipid biosynthetic enzymes.

Acetyl-CoA Carboxylase↗
PubMed1993

Two yeast peroxisomal proteins crossreact with an antiserum against human sterol carrier protein 2 (SCP-2).

An antibody raised against human sterol carrier protein 2 (SCP-2) crossreacts with two yeast peroxisomal proteins. These proteins have apparent molecular weights of 35 and 58 kDa. Subfractionation of peroxisomes revealed that the 58 kDa species is a soluble matrix protein, whereas the 35 kDa protein is membrane bound. Treatment of isolated peroxisomal membranes with 0.25 M KCl released the 35 kDa crossreactive protein into the soluble supernatant. However, lipid transfer activity could be attributed neither to the 35 kDa nor to the 58 kDa protein.

Antigen-Antibody Reactions↗
PubMed1993

Interaction of the yeast phosphatidylserine transfer protein with artificial and biological membranes.

Transfer of pyrene-labeled phosphatidylserine catalyzed by the yeast phosphatidylserine transfer protein in vitro largely depends on the membrane lipid composition of artificial unilamellar acceptor vesicles. Negatively charged phospholipids markedly decrease the rate of protein-catalyzed phosphatidylserine transfer. Although biological membranes contain a significant proportion of negatively charged phospholipids they serve more effectively as acceptors than artificial membranes with a similar phospholipid composition, but without proteins. This result indicates that proteins present in biological membranes mask negative charges of phospholipids on the surface of acceptor membrane vesicles. When proteins of the membrane surface are removed by proteinase treatment this protective effect is partially lost. A correlation between the activity of the phosphatidylserine transfer protein in yeast cytosol and the extent of membrane biogenesis during growth could not be observed.

Carrier Proteins↗
PubMed1993

Import of phosphatidylserine into isolated yeast mitochondria.

A yeast phosphatidylserine transfer protein was used as a tool to transport radioactively labeled phosphatidylserine from unilamellar vesicles to isolated mitochondria of the yeast, Saccharomyces cerevisiae. Import of phosphatidylserine into mitochondria was monitored by the appearance of radioactively labeled phosphatidylethanolamine, which is produced from phosphatidylserine by the action of phosphatidylserine decarboxylase. This enzyme is located on the outer aspect of the inner mitochondrial membrane. Import of phosphatidylserine into mitochondria and formation of phosphatidylethanolamine does not require ATP or an electrochemical gradient, and is not affected by adriamycin. Evidence is presented that contact sites between the two mitochondrial membranes are zones of intramitochondrial translocation of phosphatidylserine and phosphatidylethanolamine. When phosphatidylserine decarboxylase is inhibited by hydroxylamine, transport of phosphatidylserine to the mitochondrial surface is unaffected. Under these conditions only a small amount of phosphatidylserine accumulates in the inner mitochondrial membrane indicating that the intramitochondrial transport of phosphatidylserine and its metabolic conversion to phosphatidylethanolamine are linked processes.

Carboxy-Lyases↗
PubMed1993

Functional expression of bacterial beta-glucuronidase and its use as a reporter system in the yeast Yarrowia lipolytica.

The use of beta-glucuronidase (beta-GUS) as a reporter and sensitive detection system for Yarrowia lipolytica was studied. The Escherichia coli gusA gene was expressed under control of the homologous LEU2 promoter in a transcriptional fusion. An NcoI restriction site was introduced at the translational start-ATG, conserving the most favorable context for initiation of translation. The chimeric LEU2'-gusA gene was integrated into the LEU2 locus by homologous recombination. The beta-GUS assay was very sensitive and highly reproducible, using the cytosolic fraction or a total cell extract as the source of enzyme. In a leucine-free medium, beta-GUS activity was at a high, constant level, independent of growth phase. In transformants grown on complete medium, beta-GUS activity was reduced about three-fold, but doubled during logarithmic growth. No intrinsic beta-GUS activity was detectable in untransformed Y. lipolytica and no effect of beta-GUS expression on growth was observed. beta-GUS-producing Y. lipolytica cells could be directly detected on media plates containing X-gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronide).

Base Sequence↗
PubMed1993

Molecular cloning of the yeast OPI3 gene as a high copy number suppressor of the cho2 mutation.

By functional complementation of the auxotrophic requirements for choline of a cdg1, cho2 double-mutant, by transformation with a genomic DNA library in a high copy number plasmid, two different types of complementing DNA inserts were identified. One type of insert was earlier shown to represent the CHO2 structural gene. In this report we describe the molecular and biochemical chemical characterization of the second type of complementing activity. The transcript encoded by the cloned gene was about 1000-nt in length and was regulated in response to the soluble phospholipid precursors, inositol and choline. A gene disruption resulted in no obvious growth phenotype at 23 degrees C or 30 degrees C, but in a lack of growth at 37 degrees C in the presence of monomethylethanolamine. Null-mutants exhibited an inositol-secretion phenotype, indicative of mutations in the lipid biosynthetic pathway. Complementation analysis, biochemical analysis of the phospholipid methylation pathway in vivo, and comparison of the restriction pattern of the cloned gene to published sequences, unequivocally identified the cloned gene as the OPI3 gene, encoding phospholipid-N-methyltransferase in yeast. When present in multiple copies the OPI3 gene efficiently suppresses the phospholipid methylation defect of a cho2 mutation. As a result of impaired synthesis of phosphatidylcholine, the INO1-deregulation phenotype is abolished in cho2 mutants transformed with the OPI3 gene on a high copy number plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, Molecular↗
PubMed1993

Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism.

Organelles of the yeast Saccharomyces cerevisiae were isolated and analyzed for sterol composition and the activity of three enzymes involved in sterol metabolism. The plasma membrane and secretory vesicles, the fractions with the highest sterol contents, contain ergosterol as the major sterol. In other subcellular membranes, which exhibit lower sterol contents, intermediates of the sterol biosynthetic pathway were found at higher percentages. Lipid particles contain, in addition to ergosterol, large amounts of zymosterol, fecosterol, and episterol. These sterols are present esterified with long-chain fatty acids in this subcellular compartment, which also harbors practically all of the triacylglycerols present in the cell but very little phospholipids and proteins. Sterol delta 24-methyltransferase, an enzyme that catalyzes one of the late steps in sterol biosynthesis, was localized almost exclusively in lipid particles. Steryl ester formation is a microsomal process, whereas steryl ester hydrolysis occurs in the plasma membrane and in secretory vesicles. The fact that synthesis, storage, and hydrolysis of steryl esters occur in different subcellular compartments gives rise to the view that ergosteryl esters of lipid particles might serve as intermediates for the supply of ergosterol from internal membranes to the plasma membrane.

Esterases↗
PubMed1993

Viscoelasticity of BLM from choline plasmalogen, alkylacyl- and diacyl-glycerophosphocholines.

Mechanical characteristics of bilayer lipid membranes (BLM) composed of alkenylacyl-, alkylacyl-, and diacyl-glycerophosphocholines were studied by measuring modulus of elasticity in the direction normal to BLM plane, E perpendicular, and coefficient of dynamic viscosity eta. Alkenylacyl-glycerophosphocholine (choline plasmalogen) BLM typically show larger values of E perpendicular and eta as compared to their analogs, suggesting a tighter packing of their hydrophobic regions. Increasing cholesterol concentrations are associated with monotonically increasing values of parameters E perpendicular and eta of plasmalogen BLM, whereas a nonmonotonic dependence of these parameters with a maximum around c = 66 mol% cholesterol is typical for the diacyl analog POPC. This may be due to the formation of cholesterol clusters at c > 66 mol%.

Cholesterol↗
PubMed1992

Organization of phosphatidylcholine and sphingomyelin in the surface monolayer of low density lipoprotein and lipoprotein(a) as determined by time-resolved fluorometry.

Fluorescent analogs of phosphatidylcholine (PC) and sphingomyelin (SM) labeled with diphenylhexatrienylpropionic acid (DPH) were prepared and incorporated into the surface layer of human low density lipoprotein (LDL) and lipoprotein(a) (Lp(a)). Fluorescence anisotropy measurements of DPH-PC and DPH-SM in both lipoprotein classes were carried out at different temperatures ranging from 20 to 37 degrees C. DPH-PC as well as DPH-SM were shown to reside in more rigid domains in Lp(a) than in LDL according to higher anisotropy values in Lp(a). In both LDL and Lp(a), DPH-PC experienced a more rigid environment than DPH-SM, suggesting different environments of PC and SM in the surface shell of the lipoproteins. Fluorescence lifetimes of the labeled lipoproteins were determined by phase and modulation fluorometry. We found bimodal Lorentzian distributions for the decay times of DPH-PC and DPH-SM in LDL and Lp(a). Lifetime distribution centers for labeled lipids were very similar except for DPH-PC in Lp(a) which was shifted to longer lifetimes, suggesting a less polar environment of PC in Lp(a) than in LDL. The distributional width of DPH-PC in Lp(a) was broader than in LDL. Accordingly, phosphatidylcholine must be localized in a more homogeneous environment in LDL as compared with Lp(a). On the other hand, no difference in distributional widths was observed for DPH-SM in both lipoproteins, showing that SM organization in Lp(a) is unaffected by apo(a). From the obtained fluorescence data we propose that apoproteins discriminate between the choline phospholipids and preferentially associate with phosphatidylcholine. This effect is enhanced in Lp(a) due to the presence of apolipoprotein(a).

Fluorescence Polarization↗
PubMed1992

Transport of phospholipids between subcellular membranes of wild-type yeast cells and of the phosphatidylinositol transfer protein-deficient strain Saccharomyces cerevisiae sec 14.

The transfer of glycerophospholipids between microsomes and mitochondria, and from internal membranes to the plasma membrane of Saccharomyces cerevisiae was characterized. Cellular energy production was found to be essential for intracellular translocation of phospholipids, but neither a membrane potential nor an intact cytoskeleton are required for this process. Using the temperature-sensitive mutant strain Saccharomyces cerevisiae sec 14, which is defective in the phosphatidylinositol transfer protein, it could be demonstrated that this protein is not involved in the transport of phosphatidylinositol and phosphatidylcholine from internal membranes to the plasma membrane. Our results also confirm earlier findings that phosphatidylinositol and phosphatidylcholine can be delivered to the plasma membrane in a process independent of the flux of vesicles competent for protein secretion.

Biological Transport↗
PubMed1992

Altered positional specificity of human plasma lecithin-cholesterol acyltransferase in the presence of sn-2 arachidonoyl phosphatidyl cholines. Mechanism of formation of saturated cholesteryl esters.

The positional specificity of purified human lecithin-cholesterol acyltransferase (LCAT) was studied by analyzing the labeled cholesteryl ester (CE) species formed in the presence of proteoliposome substrates containing mixed chain phosphatidylcholine (PC) species, labeled cholesterol and apoprotein A-I. Whereas over 90% of the acyl groups used for CE synthesis were derived from the sn-2 position of most of the naturally occurring PC substrates, about 75% of the CE species formed in the presence of sn-1-myristoyl 2-arachidonoyl PC, sn-1-palmitoyl-2-arachidonoyl (PAPC) and sn-1-palmitoyl 2-docosahexaenoyl PC were derived from the sn-1-position. On the other hand, rat LCAT utilized mostly sn-2-acyl group from either PAPC or from sn-1-palmitoyl 2-linoleoyl PC. The positional specificity of the human enzyme was not affected by the alteration in the matrix fluidity, type of the apoprotein activator used, or by the free cholesterol/PC ratio in the substrate. These results show that the positional specificity of human plasma LCAT is altered in the presence of sn-2-arachidonoyl PC, or sn-2-docosahexaenoyl PC, probably due to steric restrictions at the active site, and this may account for the formation of disproportionately high concentrations of saturated CE, and low concentrations of long-chain polyunsaturated CE in human plasma, relative to the composition of sn-2-acyl groups in plasma PC.

Animals↗
PubMed1991

Sulfhydryl-selective fluorescence labeling of lipoprotein(a) reveals evidence for one single disulfide linkage between apoproteins(a) and B-100.

Human lipoprotein(a) and low-density lipoprotein were labeled with two different sulfhydryl-selective fluorescence markers. The hydrophilic fluorophore lucifer yellow iodoacetamide and the apolar compound 6-acryloyl-2-(dimethylamino)naphthalene were used to derivatize free -SH groups in the lipoproteins. Three sulfhydryls could be detected in low-density lipoprotein, whereas only two cysteines were available in lipoprotein(a). One of the three -SH groups in low-density lipoprotein was shown to be located in close proximity to the particle surface. We suggest that this surface-exposed cysteine of apoprotein B-100 serves as a component for the disulfide linkage to apoprotein(a) in lipoprotein(a).

2-Naphthylamine↗
PubMed1991

The effect of monoacylglycerol on the phase behavior of egg phosphatidylcholine.

Phosphatidylcholine bilayers can accommodate large quantities of monoacylglycerol. Incorporating up to 40% monoacylglycerol has little effect on the orientation and motion of the phosphatidylcholine polar group. Briefly heating mixed dispersions of 1-monooleoylglycerol/egg phosphatidylcholine (1:1, weight ratio; 2.1:1, mole ratio) to 50-60 degrees C induced spontaneous vesiculation: unilamellar and some oligolamellar vesicles bud off the large multilamellar particles. The size of the resulting vesicles ranges from 100 to 1000 nm, with the bulk of the vesicles having diameters between 100 and 500 nm. The spontaneous vesiculation process is reflected in the visual clearance of the mixed lipid dispersion and in the collapse of the 31P powder NMR spectrum to a sharp, asymmetric peak. The narrowing of the 31P-NMR spectrum is explained in terms of additional molecular and/or segmental motion of the lipid polar groups. In mixed dispersions of 1-monooleoylglycerol/egg phosphatidylcholine containing an excess of 1-monooleoylglycerol (greater than or equal to 50%) domain formation takes place, i.e., the formation of local clusters enriched in either of the two lipids. As a result the mechanical properties of these mixed lipid bilayers seem to be quite different from those of pure egg phosphatidylcholine.

Chromatography, Gel↗