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Biomedical subjects

W Stoffel

Publications and source records attributed to W Stoffel.

At least 127 records · Page 7Linked to original sources
PubMed1979

Covalent binding of photosensitive 1-(12-azido-[9,10(-3)H2]oleoyl)glycero-3-phosphocholine (lysolecithin) to human serum high density apolipoproteins.

Human serum high density apoproteins were complexed with increasing concentrations of 1-(12-azido-[9,10(-3)H2]oleoyl)glycero-3-phosphocholine up to the saturation concentration (72 mol lysolecithin per mol apo HDL). Ultraviolet irradiation generated the nitrene which led to crosslinking with the two main apolipoproteins AI and AII. Methods are described for the removal of excess, unbound lipid and the column chromatographic separation of the lipopolypeptides AI and AII.

Apolipoproteins↗
PubMed1978

Inhibition of cholesterol synthesis in cultured cells by 25-azidonorcholesterol.

25-Azidonorcholesterol, a side chain analogue of cholesterol, inhibits cholesterol biosynthesis in BHK 21 cells in tissue culture; but 3-azido-5-cholestene, the azido analogue of cholesterol has no inhibitory effect. Cell growth was unimpaired by the azido analogue, which showed only a minimal uptake by the cell, compared with that of cholesterol or 25-azidonorcholesterol. 25-Azidonorcholesterol is incorporated into the membranes of BHK 21 cells in place of cholesterol as demonstrated by isotope experiments. The incorporation of radioactive acetate is strongly inhibited by the side chain azido substituted cholesterol derivative; the incorporation of radioactive mevalonate is, however, unimpaired. We conclude that the inhibition occurs at the hydroxymethylglutaryl-CoA-reductase step. Cell growth is not significantly altered.

Animals↗
PubMed1978

Purification and properties of 3-cis-2-trans-enoyl-CoA isomerase (dodecenoyl-CoA delta-isomerase) from rat liver mitochondria.

The mitochondrial enzyme 3-cis-2-trans-enoyl-CoA isomerase, responsible for the positional and geometric isomerization of beta,gamma-unsaturated fatty acyl-CoA intermediates arising during beta-oxidation of unsaturated long chain fatty acids, has been isolated from rat liver, purified to homogeneity by a heat step and a combination of gel filtration and ion-exchange chromatographic procedures. The enzyme has a molecular weight of 30,000, as determined by dodecylsulfate polyacrylamide gel electrophoresis. The isomerase has a strong tendency to form a dimer. It elutes from a calibrated Sephadex G-200 column with an apparent Mr = 60000. The basic isoelectric point, pI 9.0-9.2, is due to its high content in basic amino acids. The amino acid composition determined by the ninhydrin and o-phthalaldehyde detection method is presented.

Acyl Coenzyme A↗
PubMed1977

Lipid-protein interactions between human apolipoprotein A-I and defined sphingomyelin species. A 13C-NMR spectroscopic study.

Chromatographyically and immunologically homogeneous apolipoprotein A-I (apoLp A-I) from human serum has been recombined in separate experiments with three species of sphingomyelin. The differed in the degree of saturation of their fatty acyl residues, stearoyl (18:0), oleoyl (18:1) and linoleoyl (18:2). The lipoprotein complexes formed were purified by CsCl density gradient centrifugation between 1.07 - 1.09 g/cm3 and by gel filtration. Stearoylsphingomyelin does not recombine with the apoprotein A-I below its phase transition temperature (tc = 41.5 degrees C). The lipoproteins eluted with the following apparent molecular weights: 18:0-sphingomyelin apoLp A-I, 8.0 X 10(5); 18:1-sphingomyelin apoLp A-I, 4.0 X 10(5); and 18:2-sphingomyelin apoLp A-I, 4.0 X 10(5). In electron microscopy the particles appear as discs of 160 - 170 A diameter and 50 - 60 A thickness. Their tendency to form stacked aggregates of discs decreases with the degree of their unsaturation. CD measurements underline the considerable increase in alpha-helicity of the secondary structure of apo A-I after recombination with the phospholipids. This increase in order is equal for the three sphingomyelin species (alpha-helicity of apoLP A-I = 0.46, after recombination 0.89). If the three sphingomyelin species are used in equal molar amounts in the recombination experiment, no preference for any one sphingomyelin species is observed. Recombination of apoLp A-I with sphingomyelin, labelled with the isotope 13C in the choline group, C-14 of stearic or linoleic, or C-11 of oleic acid, were performed for spin lattice relaxation time (T1) experiments. Compared with sphingomyelin liposomes, the polar head groups of these lipids in the lipoprotein particles possess a considerably higher mobility, whereas the changes in T-1-times of the C-atoms in the centre of the fatty acid chains of the lipids refer to their interactions with the polypeptide side chains. A model of the lipoprotein complexes formed is proposed on the basis of the experimental data.

Apolipoproteins↗
PubMed1977

Chemical proof of lipid-protein interactions by crosslinking photosensitive lipids to apoproteins. Intermolecular cross-linkage between high-density apolipoprotein A-I and lecithins and sphingomyelins.

The molecular interactions and spatial arrangements of phospholipids and apoproteins of human high-density lipoprotein were studied by a chemical approach. Phosphatidylcholines and sphingomyelins substituted with fatty acyl residues of high specific radioactivity and labelled with the photosensitive azido group in specific positions were prepared by chemical synthesis. They were recombined with apolipoprotein A-I of human serum high density lipoprotein. The lipoprotein complexes containing either azido lecithins or azidosphingo-myelins were purified by agarose chromatography from excess lipids. The irradiation was performed under conditions which prohibit the interference with the apoprotein structure as proven by circular dichroism, fluorescence spectroscopy, immunodiffusion test and disc electrophoresis. Non-covalently bound lipid molecules were removed by Sephadex LH 20 chromatography. Mild alkaline treatment liberated radioactive fatty acids which were not directly linked to the polypeptide chain, but rather via neighbouring phospholipid molecules. The lipoprotein appeared as a single radioactive band in dodecylsulfate polyacrylamide gel electrophoresis as seen by radioscanning, which further proved the covalent linkage of the fatty acyl residues to the polypeptide chain. In the immunodiffusion test, there is no difference between covalently crosslinked phospholipid-apoLp A-I complex and the non-photolyticall treated complex. This is the first chemical proof of the spatial relationship of the hydrophobic side chains of the lipid and polypeptide chains in a lipoprotein complex.

Apolipoproteins↗
PubMed1976

Asymmetry of the lipid-bilayer of Sindbis virus.

The organization of the lipid bilayer of the enveloped Sindbis virus has been studied. In the model membrane which consists only of two virus specific glycoproteins and host derived lipids the latter were radioactively labelled with 14C-palmitic acid by prelabelling their BHK 21 host cell lipids. The purified virus particles were submitted to neuramidase, bromelain and combromelain-neuraminidase treatment. It could be demonstrated that N-acetyl neuraminic acid residue of the total hematoside present in the virion is hydrolyzed by neuraminidase leaving the particles fully intact. Proteolysis of the spikes leads to particle aggregation yet an unchanged hematoside content. This was fully transformed into ceramidelactoside by subsequent neuraminidase treatment. The analyses of the ceramide species present in hematoside of the control particles and ceramidelactoside derived thereof by neuraminidase hydrolysis are in very close agreement. From these experiments it is concluded that all hematoside molecules are organized in the outer half of the bilayer of the envelope.

Bromelains↗
PubMed1976

Human high denisty apolipoprotein A-I-lysolecithin-lecithin and sphingomyelin complexes. A method for high yield recombinations to lipoprotein complexes of reproducible stoichiometry.

High denisty apolipoprotein A-1 (apoLp A-I) has been prepared in a chromatographically and immunochemically homogeneous form. This apoprotein forms trimeric and tetrameric aggregates in aqueous solutions at higher concentrations. ApoLp A-I has been recombined in almost quantitative yield in the presence of lysolecithin with phosphatidylcholine and sphingomyelin to particles of reproducible stoichiometry. Lysolecithin is not required for the interactions of lecithin and sphingomyelin with the apoprotein A-I or for the stability of these complexes. Dialysis removes most of the lysolecithin without the loss of lecithin and sphingomyelin. ApoLp A-I-lecithin particles have a molecular weight of 200 000 and contain 50 molecules lecithin and 25 of lysolecithin. ApoLp A-I-sphingomyelin complexes contain 50 sphingomyelin and 13 lysolecithin molecules. The former particles show up as discs of 100 A diameter, and the latter particles are 250 A in diameter. Their thickness was estimated as 25 A in the apoLp A-I lecithin and 60 A in the apoLp A-I-sphingomyelin particles. ApoLp A-I and lysolecithin form complexes whose densities depend on the lysolecithin concentration. Lysolecithin enhances the binding of phosphatidylcholine to apoLP A-I, yielding lipoprotein complexes with decreasing density. The yield of apoLp A-I-sphingomyelin-lysolecithin complexes is proportional to the lysolecithin concentration. The ratio of apoLp A-I to sphingomyelin in all these complexes remains constant.

Apoproteins↗
PubMed1976

Lipid-lipid and lipid-protein interactions as studied with a novel type of fluorescent fatty acid and phospholipid probes.

A novel fluorescent-labelled group of fatty acids and phospholipids has been applied to determine phase transitions in liposomes by fluorescence intensity and polarisation measurements. The chromophore of these amphiphilic lipids proved to be very suitable to demonstrate temperature-dependent lipid-lipid interactions. Liposomes from 1,2-dipalmitoyl-3-sn-glycero-phosphoethanolamine and from lipids isolated from membranes of E. coli K 1062 mutant grown on elaidic acid were used in these studies. These probes also made it possible to observe conformational changes in membrane proteins in isolated plasma membranes from this mutant. The changes in protein conformation were dependent on structural changes in the lipid phase.

Binding Sites↗
PubMed1976

Chemical syntheses of novel fluorescent-labelled fatty acids, phosphatidylcholines and cholesterol esters.

The synthesis of a novel class of fluorescent-labelled fatty acids of different chain lengths and unsaturation, phospholipids and cholesterol esters has been developed. The following omega-anthracene-labelled cis-unsaturated fatty acids have been synthesized: omega-(9-anthryl)-6c-octenoic, -7c-nonenoic, -10c-dodecenoic, -6c,9c-undecadienoic, -10c,13c-pentadecadienoic acid. They have been introduced into the 2-position of 1-stearoyl- and 1-linoleoyl-3-sn-glycerophosphocholine and cholesterol. Mass spectroscopy, 1H-NMR, IR and fluorescence spectroscopy and different chromatographic procedures have been applied to confirm and characterize their structures. The properties of the different fluorescent-labelled phosphatidylcholines in monomolecular films have been determined by the Langmuir technique.

Cholesterol↗
PubMed1976

13C-NMR studies of the membrane structure of enveloped virions (vesicular stomatitis virus).

The mobility of the lipids in the bilayer of the envelope of vesicular stomatitis virus has been probed over its complete space by the biosynthetic incorporation of [N-13CH3]- choline as a probe for the polar head groups and [3-13C]- and [11-13C] oleic acid and [16-13C]- palmitic acid for the hydrophobic region of the bilayer. These precursors were effectively incorporated as established by the concomitant administration of the same precursors in radioactive form. Spin lattice relaxation time measurements (T1) of the 13C enriched segments in complete virus envelope allowed estimation of their mobility. The mobility of the polar head groups is restricted, probably due to ionic interactions with neighbouring acidic phospholipids (phosphatidylserine) and/or acidic side chains of the glycoprotein (G-protein). The rigidity of the hydrophobic part of the bilayer is due to the high cholesterol content and interaction with the immersing polypeptide chains of the G- and possibly M-protein. The rigidity is limited to a depth of about 15 A ranging from the inner and outer surface, whereas the inner core of the bilayer is fluid. Tryptic cleavage of the hydrophilic part of the G-protein allows the lipophilic immersing polypeptide fragment to enter further the bilayer which then reduces the fluidity of the hydrocarbon chains in the core region by lipid-protein interactions.

Animals↗
PubMed1976

Biosynthetic incorporation of fatty acids with photosensitive groups into membrane lipids of cells in tissue culture.

The physical methods (13C-NMR-spectroscopy and fluorescence spectroscopy) hitherto used for the elucidation of lipid-lipid and lipid-protein interactions in artificial and simple natural membranes were extended to the application of fatty acids, phospholipids and sphingolipids with photochemical labels (azide group) in defined positions, which on photolysis generate nitrenes. These highly reactive groups react with neighbouring molecules, either lipids or polypeptide chains, with insertion or addition. Highly radioactive 12-azido[9,10-3H2]stearic acid, 12-azido[12-3H]oleic acid and 18-azido-[9,10,12,13-3H4]linoleic acid were added to the growth medium of eukaryotic cell lines in tissue culture (BHK 21 cells and Chang liver cells). They were incorporated into neutral, phosphoand sphingolipids in amounts comparable with the unsubstituted parent fatty acids. The distribution of the azido fatty acids in the phospholipids has been determined by enzymatic hydrolysis (phospholipase A2) on the basis of the distribution of their radioactivity. Radio gas chromatography and combined gas chromatography and mass spectroscopy revealed that the azide group of the radioactive fatty acids remained unaltered.

Animals↗
PubMed1976

Biosynthetic labelling of membrane lipids of eukaryotic cells in tissue culture by a novel type of fluorescent fatty acids.

W-Anthryl labelled fatty acids with hydrocarbon chains of different lengths (C8, C11, C15) and different degrees of unsaturation have been incorporated into the membrane lipids of three different cell lines in tissue culture by addition of these 3H-labelled precursor fatty acids to the growth medium. The cell lines were baby hamster kidney cells (BHK 21), Chang liver cells and the RN6 cell line derived from a chemically induced Schwannoma tumor cell clone. Cell growth was normal. The quantitative analysis on the basis of radioactivity determinations demonstrated that the fluorescent-labelled fatty acids were introduced into the neutral lipid fraction (triglycerides, diglycerides, and cholesterol esters, all present in small amounts), but mainly into the phospholipid classes phosphatidylcholine, -ethanolamine and -serine, and to a lesser extent, as N-acyl component of sphingolipids (sphingomyelins, ceramides, mono- and diglycosylceramides). Cell fractionation studies indicated that the membranes of all subcellular particles were labelled with the fluorescent probes in their lipid moieties. These w-anthryl fatty acids are the first type of fluorescent lipid precursors which can be incorporated biosynthetically in vivo into membrane lipids of eukaryotic cells. The effective incorporation of the bulky fluorescent anthryl group in the terminal position of fatty acids of different chain lengths into the complex membrane lipids of the cell gives proff of 1) their uninhibited membrane transport, 2) their activation by the acyl-CoA synthetase and 3) their substrate properties for the O- acyl and N-acyl transferases in phospho- and sphingolipid biosynthesis.

Animals↗