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Biomedical subjects

W Stoffel

Publications and source records attributed to W Stoffel.

At least 109 records · Page 6Linked to original sources
PubMed1982

Syntheses of phosphatidylcholines, sphingomyelins and cholesterol substituted with azido fatty acids. Photocrosslinking with nearest neighbouring lipids in liposomes chemical and mass spectroscopic proof.

The chemical synthesis of azido-substituted saturated, monoenoic and dienoic fatty acids of high specific tritium radioactivity, together with improved methods for their introduction into phosphatidylcholine, sphingomyelin and cholesteryl ester molecules are outlined. Phospholipid vesicles with these photosensitive probes were irradiated and the main crosslinking products isolated and characterized by high resolution mass spectroscopy.

Azides↗
PubMed1982

Chemical studies on the structure of human serum high-density lipoprotein (HDL). Photochemical crosslinking of azido-labelled lipids in HDL.

The lipid classes of native human serum high-density lipoprotein (HDL) were exchanged against phosphatidylcholine, sphingomyelin and cholesteryl ester species substituted with photosensitive fatty acyl residues and against 25-azido-27-norcholesterol. The photosensitive fatty acyl residues were 5-and 16-azidopalmitic, 12-azidooleic and 18-azidolinoleic acid, all labelled with high tritium radioactivity. The lipid exchange method previously described was used. After UV irradiation and delipidation the apoproteins AI and AII, photocrosslinked with the radioactive lipids, were separated. The yield of covalently crosslinked lipid molecules amounted to 30% of the photosensitive lipid molecules incorporated into the HDL particle. ApoAI and apoAII were labelled by each of the photosensitive lipid classes, intergrated in the HDL particle although in very different stoichiometry. The regiospecific photochemical labelling of the lipopolypeptides AI and AII was established by cyanogen bromide cleavage and the separation of the four apoAI and two apoAII CNBr fragments. The analytical data prove the close steric relation and interaction of defined protein regions with hydrophobic regions of the lipid molecules by chemical means. These results are discussed with respect of the HDL structure.

Azides↗
PubMed1982

Analysis of the primary structure of the strongly hydrophobic brain myelin proteolipid apoprotein (lipophilin). Isolation and amino acid sequence determination of proteolytic fragments.

Proteolipid aproprotein (lipophilin) and DM-20 protein from bovine brain white matter proved to be identical in polyacrylamide gel electrophoresis and automated Edman degradation of the N-terminal end over 20 cycles. Lipophilin can be hydrolysed by trypsin, thermolysis, chymotrypsin and subtilisin. We describe here a new, effective and rapid high-performance liquid chromatographic separation method for hydrophilic polypeptides according to molecular mass on an analytical and preparative scale. Three large and several small peptides have been isolated from the tryptic and thermolysinolytic hydrolysate and purified by combined molecular sieve and high-performance chromatographic separation and purification for automated Edman degradation. 40 amino acid residues of the large tryptic fragment and sequences of 43 and 22 amino acids of two thermolysinolytic fragments have been determined. These three polypeptides are partial structures of the l4 kDa large tryptophan fragment 1 or the cyanogen bromide fragment I (18-19 kDa). Thermolysin also releases a polypeptide from incompletely reductively carboxymethylated lipophilin which is cleaved into the large thermolysin fragment mentioned, 22 residues of which were analysed, and a 14 amino acids long sequence of tryptophan fragment IV, described in the previous paper. Reductively carboxymethylated liprophilin, the lysine side chains of which were blocked with maleic anhydride, can be cleaved at arginine specific sites. Bio-Gel P-150 and high-performance chromatographic purification yielded a polypeptide, which upon performic acid oxidation was split into a 15 kDa and a 7.8 kDa polypeptide. The 15 kDa polypeptide resembles the N-terminal end as proven by 31 cycles in Edman degradation. The 7.8 kDa polypeptide corresponds to the 72 amino acid C-terminal sequence, which equals cyanogen bromide fragments II, III and IV and embraces tryptophan fragment IV.

Amino Acid Sequence↗
PubMed1982

Lipophilin (proteolipid apoprotein) of brain white matter. Purification and amino acid sequence studies of the four tryptophan fragments.

The chemical cleavage of lipophilin (proteolipid apoprotein) from bovine brain white matter with HBr/dimethyl sulfoxide at the tryptophan residues, under conditions adapted to this hydrophobic protein, releases four fragments with approximate molecular masses 14 kDa (Trp I), 6.8 kDa (Trp IV), 5.2 kDa (Trp III) and 2.1 kDa (Trp II). These fragments have been separated and purified by a combination of solvent distribution, molecular sieve chromatography (Bio-Gel P-150) and high-performance liquid chromatography for automated Edman degradation and combined gas-liquid chromatography/mass spectroscopy. The complete amino acid sequences of Trp II and III and large sequences of Trp I are reported in this communication. The amino acid sequence of Trp IV and the sequences of peptides releasable from lipophilin by proteolytic enzymes (trypsin, thermolysin, subtilisin, chymotrypsin) have been described in previous reports from this laboratory. Despite two small gaps in the complete primary structure of lipophilin from myelin of central nervous system, our sequence data suggest the arrangement of four long hydrophobic sequences (30-40 apolar amino acid residues) within the hydrophobic core of the myelin lipid bilayer, linked by three hydrophilic regions at the aqueous membrane interphase. These features lend lipophilin the properties of a polytopic membrane protein.

Amino Acid Sequence↗
PubMed1982

Primary structure of the C-terminal cyanogen bromide fragments II, III and IV from bovine brain proteolipid-apoprotein.

Purified lipophilin from bovine brain white matter was reductively carboxymethylated and then cleaved by cyanogen bromide into four fragments: CNBr I 18-19 kDa, CNBr II 5 kDa, CNBr III 2.1 kDa and CNBr IV 0.7 kDa. Hydrogenbromide/dimethylsulfoxide and 3-bromo-2-(2-nitrophenylsulfenyl)skatol (BNPS-skatol) cleaved lipophilin into four fragments of molecular masses of approximately 14000 (Trp I), 2100 (Trp II), 5000 (Trp III) and 7000 Da (Trp IV). Separation and purification of the peptides for liquid phase sequenator degradation was achieved by high performance liquid chromatography. In addition proteolytic cleavage of the Trp IV fragment with trypsin facilitated the alignment of the peptides. An effective control of the sequenator data came from the partial acid hydrolysis of the Trp IV fragment, which yielded di-, tri- and tetrapeptides. The mixture was N-trifluoroacetylated, the amide (peptide) bonds and carboxyl groups were reduced with B2D6 (hexadeuterodiborane) and the polyaminoalcohols derivatized with chlorotrimethylsilane. These derivatives were separated and identified by capillary gas-liquid chromatography/mass spectrometry. Extensively overlapping sequences support the data obtained by Edman degradation in a liquid phase sequenator of the CNBr peptides II, III and IV, the 72 amino acid residues containing C-terminal sequence of lipophilin of molecular mass (7520 + x) Da, which includes the Trp IV fragment.

Amino Acid Sequence↗
PubMed1981

Application of specific extracorporeal removal of low density lipoprotein in familial hypercholesterolaemia.

A highly selective method for the removal of apolipoprotein-B-containing serum lipoproteins (low density lipoproteins [LDL] and very-low-density lipoproteins [VLDL]) from hypercholesterolaemic patients by immunoadsorption in an extracorporeal system consists of separation of plasma from the blood cells by a blood-separation centrifuge, followed by the selective adsorption of LDL from the plasma effluent, from which high-density lipoproteins (HDL) and other plasma proteins are not removed, is returned to the patient with the blood cells. Three patients with familial hypercholesterolaemia, one homozygous and two heterozygous, were treated repeatedly during a period of 9 months. No undesirable side-effects or changes in clinical, chemical, haematological, or immunological parameters have yet been observed. The new procedure has several advantages over treatments currently used; it is non-invasive, more specific, and less costly and lowers LDL to a greater degree.

Adolescent↗
PubMed1981

Selective removal of apolipoprotein B-containing serum lipoproteins from blood plasma.

Studies were undertaken to determine the applicability and effectiveness of immunoadsorption chromatography on anti-low density lipoprotein (LDL) columns as a plasma-cholesterol-lowering procedure. Mass production and isolation of monospecific antibodies against swine LDL (rho = 1.006-1.063 g/ml) was carried out by immunization of sheep with swine LDL and selective antibody adsorption from their antisera by chromatography on LDL-Sepharose. The isolated LDL antibodies were then covalently linked to Sepharose CL-4B. Pig plasma LDL was effectively removed in vitro by the anti-LDL-Sepharose beads. In vivo studies were performed in pigs with (i) a plasma-separator membrane permeable to solutes below Mr 2,000,000 or (ii) a blood centrifuge interposed into an arteriovenous shunt to separate the corpuscular elements of blood from the plasma. In either case, the plasma was passed through the anti-LDL-Sepharose column and recirculated into the venous part of the shunt. Plasma cholesterol levels were reduced 70-80% by this procedure and rebounded 3-4 days after the extracorporeal immunoadsorption procedure. This continuous plasma separation-immunoadsorption procedure may have broad applicability for the elimination of any plasma component with antigenic properties.

Animals↗
PubMed1981

Membrane attack complex of complement: distribution of subunits between the hydrocarbon phase of target membranes and water.

Membrane destruction by complement is effected by the membrane attack complex (MAC) which is the dimer of a fusion product of the complement proteins C5b, C6, C7, C8, and C9. Phospholipid bilayer vesicles were used as target membranes for the MAC and its intermediate complexes. The subunits of these membrane-bound complexes were explored as to their relative exposure to the hydrocarbon phase of the lipid bilayer and to water surrounding the lipid vesicles. Protein exposed to the aqueous phase was labeled with 125I; protein exposed to the hydrocarbon phase was labeled by using tritiated azido phospholipids and irradiation. Analysis of the membrane-bound MAC showed that subunits C5b, C8 beta, and C9 were exposed to the aqueous phase. The subunits C8 alpha-gamma and C9 were primarily in contact with the hydrocarbon phase. C6 and C7 were little exposed to either phase, suggesting that these proteins are inaccessible within the MAC. Analysis of the intermediate complexes showed that C5b was the subunit most exposed to water in membrane-bound C5b-7, and C5b and C8 beta were the water-exposed subunits in C5b-8. Subunit exposure to the hydrocarbon phase of the lipid bilayer changed during MAC assembly. Whereas all three subunits of C5b-7 carried the phospholipid photolabel; most of the label was bound to the C8 subunit in C5b-8 and to C9 in the MAC. It is proposed that contact with the hydrocarbon core of membranes is established by C5b-7 through each of its subunits, by C5b-8 through C8, and by the MAC through C8 and, particularly, C9.

Affinity Labels↗
PubMed1980

Carbon-13 nuclear magnetic resonance studies on the interaction of glycophorin with lecithin in reconstituted vesicles.

Glycophorin, the MN blood group substance, is a major intrinsic glycoprotein in erythrocyte membranes. The interaction of glycophorin with phosphatidylcholine, 13C-labeled in specific positions in reconstituted unilamellar vesicles, was investigated by using the 13C NMR technique. 1-Palmitoyl-2-([14-13C]linoleoyl)-sn-glycero-3-phosphocholine was synthesized and used as a probe. At 37 degrees C the spin-lattice relaxation time (T1) of vesicle bilayers consisting of this phospholipid was 0.74 s in the absence of glycophorin. The incorporation of glycophorin decreased the T1 to 0.63 s, indicating that the bulk lipid molecules are somewhat immobilized by glycophorin. In addition to the reduction in time, a broad component (delta H1/2 = approximately 40 Hz) superimposing the sharp resonance was observed in the 13C NMR spectrum of reconstituted vesicles. The T1 of the broad component was 0.32 s, suggesting that the lipid molecules contributing to the broad component may be more restricted than that of the sharp component. In order to quantify the broad component, a computer simulation was performed. The intensity of the broad component estimated from the simulation depended linearly on the concentration of glycophorin. Therefore, the broad component is considered to be contributed by a phospholipid domain surrounding the glycophorin molecules, a so-called "boundary lipid". The relationship between the broad component and the stoichiometry of the reconstituted vesicles allows the conclusion that about 30 lipid molecules are immobilized by one glycophorin monomer.

Erythrocytes↗
PubMed1980

Studies in vitro on the biosynthesis of ceramide and sphingomyelin. A reevaluation of proposed pathways.

The postulated biosynthetic-pathways of ceramide and sphingomyelin were reinvestigated in extensive investigations by means of synthetic stereo- and radio-chemically pure substrates of high specific radioactivity. As a result, the synthesis of ceramides requires the acyl-CoA-mediated acyltransfer to the long chain bases sphingenine and sphinganine. During the biosynthesis of sphingomyelins, phosphocholine is being transferred from the donor CDP-choline to the primary alcohol group of ceramides. Neither can the free long chain sphingosine bases act as acceptor molecule for the phosphocholine group from CDP-choline, nor has a transfer of [N-14CH3]phosphocholine from [N-14CH3]phosphatidyl choline to ceramide by rat liver enzyme preparations been observed. In agreement with previous studies in vivo, the acylation of sphingenylphosphocholine by acyl-CoA or free fatty acid, ATP and CoASH as an alternative pathway in sphingomyelin biosynthesis has been excluded. Other parameters of the CDP-choline:ceramide cholinephosphotransferase reaction (pH-optimum, ion requirement, competitive inhibition by diacyl glycerols, chain length of fatty acids) are reported. Sphingenine-containing ceramide species are preferred as acceptor molecules. Ceramide species with the L-threo (2S,3S)-enantiomeric long-chain bases are better acceptors than the corresponding D-erythro (2S,3R)-isomeric compounds. The meaning of the steric arrangement for the reaction is discussed.

Acyl Coenzyme A↗
PubMed1980

Studies on the biosynthesis of ceramide. Does the reversed ceramidase reaction yield ceramides?

During studies on the biosynthetic pathways of sphingomyelin, the condensation reaction between free fatty acids and sphingenine to ceramide catalysed in the reversed ceramidase reaction as proposed by Gatt (J. Biol. Chem. 238, PC 3131-3133 (1963) was reinvestigated. Radioactive long-chain fatty acids (palmitate and oleate) condense with ethanolamine, one of the buffer components of the original studies and yield N-acylethanolamine. N-Palmitoylethanolamine was isolated on a preparative scale from incubation mixtures and identified directly and also after derivatization by mass spectroscopy and comparison with the authentic synthetic compound. It is concluded that only the acyl-CoA-dependent ceramide synthesis is of biological significance.

Amidohydrolases↗
PubMed1979

Interactions of a photosensitive analog of cholesterol with hydroxymethyglutaryl-CoA reductase (NADPH) and acyl-CoA:cholesterol acyltransferase.

25-Azido[25-(3)H]norcholesten-3beta-ol, a radioactive photosensitive analog of cholesterol, was synthesized as a probe to study lipid-protein interactions [Stoffel, W. & Klotzbücher, R. (1978) Hoppe-Seyler's Z. Physiol Chem. 359, 199-209]. Upon UV irradiation a reactive nitrene is generated which can insert into carbon-hydrogen bonds of proteins to form covalent linkages. The compound has properties similar to those of cholesterol. When administered intravenously to rats it was transported to the liver, and within 4 hr it was found in all of the subcellular fractions, accounting for 8% of the microsomal sterol content. Concomitantly, it altered the activities of two membrane-bound enzymes. 3-Hydroxy-3-methylglutaryl (HMG)-CoA (NADPH) reductase [mevalonate:NADP(+) oxidoreductase (CoA-acylating), EC 1.1.1.34] activity was reduced to 20% of control values, and acyl-CoA:cholesterol acyltransferase (ACAT; acyl-CoA:cholesterol O-acyltransferase, EC 2.3.1.26) was stimulated at least 2-fold. Esterified products of the analog were found in both blood and liver, and no evidence was obtained for production of any hydroxylated or polar derivatives. UV irradiation of microsomes increased the radioactivity bound to the protein fraction and decreased it in the total lipid extract; in parallel a sharp decline in ACAT activity but unaltered HMG-CoA reductase activity was observed. These results are interpreted as a possible consequence of crosslinking of the sterol to the enzyme proteins and discussed in view of the evidence that HMG-CoA reductase is an extrinsic and ACAT an intrinsic membrane protein.

Acyltransferases↗
PubMed1979

Covalent cross-linking of photosensitive phospholipids to human serum high density apolipoproteins (apoHDL).

Human serum high density apolipoproteins were reassociated with three different lecithin species substituted with radioactively labelled photosensitive azido fatty acids, bis([3H]-16-azidopalmitoyl)-, bis([3H]12-azidooleoyl)- and bis([3H]18-azidolinoleoyl)glycerophosphocholine. The lipoprotein particles were reconstituted from a mixture of azido-labelled phosphatidylcholine and non-labelled dioleoylglycerophosphocholine (1:9). Excess lipid was separated from the homogeneous particles by Bio-Gel A-5m. The molecular weight, stoichiometry, fluorescence and circular dichroism of the reconstituted particles were determined before and after photoactivation with covalent cross-linking of the phospholipids with the apoproteins. The physical parameters of the reconstituted lipoproteins remained unperturbed by the cross-linking reaction between the generated nitrenes and apolipoprotein A-I and A-II. Thus the hydrophobic interactions of the phospholipid molecules with the apoproteins have been proved for the first time by a chemical method.

Apolipoproteins↗
PubMed1979

13C-NMR spectroscopy of human serum high density lipoprotein enriched with labelled phospholipids.

Native human serum high density lipoprotein (HDL) (d = 1.063--1.21g x cm-3) was enriched with phosphatidylcholines labelled with 13C in the polar head group ([N-13CH3]choline) and in the fatty acyl chains ([26-13C]cholesterol) and its linoleic acid ester using the previously described exchange method (Stoffel et al. 1978). The properties of the HDL particles with the exchanged lipid classes were the same as those of the native particles (Mr, CD, fluorescence, lipid and apoprotein stoichiometry, electrophoretic mobility). The T1-times were very similar to those obtained previously with recombined apolipoprotein-[13C]lipid complexes and further support our proposals concerning lipid and apoprotein interactions in the HDL particle.

Carbon Isotopes↗
PubMed1979

Surface localization of apolipoprotein AII in lipoprotein-complexes.

Lipoprotein particles reconstituted from the apolipoprotein AII (apo AII) component of human serum high density lipoprotein, phosphatidylcholine and lysophosphatidylcholine were covalently linked to the imidoester groups of a polystyrene residue. Apo AII was proteolytically digested with thermolysin after delipidation. The covalently bound peptides remaining at the resin were cleaved and separated by combined two-dimensional electrophoresis/chromatography. The peptides were isolated, hydrolyzed and their amino acid composition determined. They were assigned to the apo AII sequence. Since the imidoester groups on the surface of the resin carrier cannot react with buried lysine residues, this method gives strong chemical evidence for the spreading of the apo AII polypeptide chain over the surface of the lipoprotein particle, as far as the sequence carrying lysine residues between residue 22 and 55 of each symmetrical half is concerned.

Amino Acid Sequence↗
PubMed1979

Conformational analysis of serum apolipoprotein AII in lipoprotein complexes with bifunctional crosslinking reagents.

Apolipoprotein AII isolated from human serum high density lipoproteins was recombined with phosphatidylcholine to yield homogeneous particles of 80-120 A diameter. The radioactive bifunctional crosslinkers dimethyl [1,1'-14C]suberimidate and dimethyl 4,4'-dithiobis([1-14C]butyrimidate) were reacted with these particles. The kinetics of the reactions and the localisation of the crosslinked lysines of the polypeptide chains were determined. Thermolysin hydrolysis followed by two-dimensional separation of the peptides and isolation of the mono- and bifunctionally modified peptides allowed the assignment of the crosslinked peptides of the apoprotein AII seqeunce. The crosslinking pattern indicates a close-neighbour relationship (13-15 A) of the peptide chains between amino acid residues 3, 23, 46 and 55 of the symmetrical halves of the apo AII molecule. A reconstruction of the secondary structure of Apo AII in the lipoprotein complex on the basis of theoretical calculations is given and correlated with the chemical data.

Amino Acid Sequence↗