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Biomedical subjects

W Stoffel

Publications and source records attributed to W Stoffel.

At least 19 recordsLinked to original sources
PubMed1992

Proteolipid protein (PLP) of CNS myelin: positions of free, disulfide-bonded, and fatty acid thioester-linked cysteine residues and implications for the membrane topology of PLP.

Proteolipid protein (PLP), the major integral membrane protein of central nervous system myelin, contains 14 cysteine residues within its 276-residue polypeptide chain. We determined the state of all cysteine residues and localized four of them as free thiols at positions 24, 32, 34, and 168. Four cysteines are connected by disulfide bonds: Cys200-Cys219 and Cys183-Cys227. The remaining six cysteine residues at positions 5, 6, 9, 108, 138, and 140 are modified by long-chain fatty acids, mainly palmitic acid, in thioester linkage. The extreme hydrophobicity of PLP can therefore be explained by two structural features: a composition of approximately 50% apolar amino acid residues and a high degree of fatty acid acylation. A differential fluorescent-labeling technique was developed for the structural studies: the cysteine residues belonging to one of the three states were derivatized by N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-AEDANS) either directly (a), after thioester cleavage with hydroxylamine (b), or after disulfide cleavage with dithiothreitol (c). The protein was then proteolytically digested with thermolysin, and the labeled peptides were isolated by reversed-phase HPLC followed by sequence analysis. The results were further confirmed by determination of the fatty acid to protein stoichiometry. The structural data not only demand the revision of our concept of the membrane topology of PLP but will also promote more sophisticated studies on the mechanism of myelination and new functions of PLP.

Amino Acid Sequence↗
PubMed1992

Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain.

Transport systems specific for L-glutamate and L-aspartate play an important role in the termination of neurotransmitter signals at excitatory synapses. We describe here the structure and function of a 66-kDa glycoprotein that was purified from rat brain and identified as an L-glutamate/L-aspartate transporter (GLAST). A GLAST-specific cDNA clone was isolated from a rat brain cDNA library. The cDNA insert encodes a polypeptide with 543 amino acid residues (59,697 Da). The amino acid sequence of GLAST suggests a distinctive structure and membrane topology, with some conserved motifs also present in prokaryotic glutamate transporters. The transporter function has been verified by amino acid uptake studies in the Xenopus laevis oocyte system. GLAST is specific for L-glutamate and L-aspartate, shows strict dependence on Na+ ions, and is inhibited by DL-threo-3-hydroxy-aspartate. In situ hybridization reveals a strikingly high density of GLAST mRNA in the Purkinje cell layer of cerebellum, presumably in the Bergmann glia cells, and a less dense distribution throughout the cerebrum. These data suggest that GLAST may be involved in the regulation of neurotransmitter concentration in central nervous system.

Amino Acid Sequence↗
PubMed1992

PROFILEGRAPH: an interactive graphical tool for protein sequence analysis.

The computer program PROFILEGRAPH, a graphical interactive tool for the analysis of amino acid sequences, is described. The main task of the program is to integrate a variety of sliding-window methods into a single user-friendly shell. The program allows the user to combine any amino acid specific parameter with a selection of several possible types of analysis and to plot the resulting graph in one of several windows on the screen. It is also possible to calculate the moment of the amino acid specific parameter for a given secondary structure and to display both the absolute moment value and the moment angle relative to a reference residue. Also included are several utilities that facilitate visual analysis of protein primary structures like, for example, helical-wheel diagrams. It is possible to adapt the majority of published sliding-window analysis procedures for use with PROFILEGRAPH.

Algorithms↗
PubMed1992

Mouse apolipoprotein AI. cDNA-derived primary structure, gene organisation and complete nucleotide sequence.

Apolipoprotein AI, the dominant protein component of serum high density lipoprotein, is intimately involved in cholesterol homeostasis. Apo AI activates the lecithin-cholesterol acyltransferase within the HDL particle and functions as ligand for a putative HDL receptor--two properties, which render this apolipoprotein a key mediator in reversed cholesterol transport. A functional analysis of the apo AI gene demands the isolation of the mouse apo AI gene for expression as transgenes in different mutant forms in the mouse. Here we describe the isolation of a full length apo AI-specific mouse liver cDNA clone with the human cDNA (892 bp) and the derived amino-acid sequence coding a polypeptide of 264 amino-acid residues. It showed a 70.7% homology to the rat and 66% to the human apo AI sequence. With this cDNA as probe the mouse apo AI gene was isolated and its organization analysed. Four exons, three of which are coding sequences, are aligned similarly to the human gene. The gene embraces 1825 bp between the transcription start, and the poly(A)+ tail attached 62 bp downstream of the stop codon. The complete nucleotide sequence of the four exons and three introns of the mouse apo AI gene was determined and its homology compared with that of the rat and human gene. Extensive deletions and a strongly reduced homology of the three introns of the two genes are obvious.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of the acid sphingomyelinase of the mouse and the organization and complete nucleotide sequence of the gene.

The cDNA and complete gene encoding mouse acid sphingomyelinase (ASM) has been isolated using a homology screening approach. Comparison with the human sequence shows 81% sequence identity at cDNA level and 82% at the protein level. Homology is markedly reduced in the N-terminal region, especially in the presumed signal peptide. The six-exon gene structure is similar to the human one, except for the position of Alu1 elements. The alternatively used splice site 40 bp downstream of the human exon 2 is not conserved in the mouse gene and accordingly no such alternative splicing was found in mouse.

Animals↗
PubMed1991

Human placental sphingomyelinase. Purification to homogeneity, antigenic properties and partial amino-acid sequences of the enzyme.

Sphingomyelinase from human placenta was purified to homogeneity in five steps: concanavalin A Sepharose, butyl agarose. Blue Sepharose, sphingosylphosphocholine Sepharose chromatography and FPLC-Mono Q. This lysosomal enzyme has a pH optimum around pH 5.0-6.0. It is a glycoprotein with an approximate molecular mass of 70 kDa which is reduced to 60 kDa by enzymatic deglycosylation. Monospecific antibodies against sphingomyelinase were isolated using sphingomyelinase covalently linked to Sepharose as affinity matrix. These antibodies effectively inhibit the sphingomyelinase activity. Peptides were released from sphingomyelinase by cyanogen bromide or proteolytically by trypsin, proteinase V8 and Lys C for gas phase sequencing. Amino-acid sequences are reported which proved to be the prerequisite for antibody and oligonucleotide screening of the respective human placenta cDNA libraries for the determination of the complete amino acid sequence of human lysosomal sphingomyelinase. In situ hybridisation with a labelled antisense RNA synthesized in vitro using cloned sphingomyelinase-specific cDNA as template, which encodes the peptide sequences described here, revealed the strong expression of sphingomyelinase in human placental villi and normal fibroblasts. Fibroblasts of a Niemann-Pick patient, however, were free of mRNA expressing the sphingomyelinase described here.

Amino Acid Sequence↗
PubMed1991

An approach to the functional analysis of lecithin-cholesterol acyltransferase. Activation by recombinant normal and mutagenized apolipoprotein AI.

Apolipoprotein AI (apo AI) of human serum high-density lipoprotein functions as an activator of lecithin-cholesterol acyltransferase (LCAT) and therefore plays an important role in reversed cholesterol transport. The mechanism of the acyltransfer, the activating polypeptide domains of apo AI and the active site of LCAT in this transesterification are not yet known. Synthetic peptides of the apo AI sequence have been designed to determine the activating structure, but did not yet lead to conclusive results. This also applies to spontaneous apo AI mutants. We therefore used the method of site-directed mutagenesis of apo AI cDNAs using the overlap extension approach by the polymerase chain reaction. These constructs were cloned into the procaryotic vector pET8c and expressed under the inducible T7 promoter. The engineered apo AI polypeptides were isolated and purified by affinity chromatography and assayed for their activator activity. The essentials of this approach to the structure and function of activators in general have successfully been exemplified for the LCAT activation by engineering apo AI mutant polypeptides a) by the deletion of two adjacent amphipathic helices (amino acid residues 146-186) and b) by introducing a point mutation (Glu111----Gln).

Amino Acid Sequence↗
PubMed1991

Transient expression of wild type and mutant human apolipoprotein AI in COS cells.

A human apolipoprotein AI (apo AI) minigene and two mutants were cloned into the vector pUHD10-1 for expression studies in COS cells under the control of the strong CMV (cytomegalovirus) enhancer and the own apo AI promoter. In the mutated apo AI minigene (mutant M1) the positions of the triplets of Gln(-2)-Gln-1 at the C-terminus of the prosequence were exchanged against Gln(-8)-Ala-7, the recognition site of the signal peptidase of the wild type human apo AI. The prosequence has been deleted in mutant M2 and the presequence linked directly to the N-terminus of the mature apo AI form. We report here on expression studies in COS cells, a cell line, which does not express apo AI. They were transfected by electroporation with pUHD10-1 constructs, which contain a) the wild type apo AI minigene and b) the two mutant apo AI minigenes with mutations described above. The following results were obtained: a) the wild type and mutant apo AI constructs were efficiently transcribed and translated in COS cells, b) the expression of the wild type preproapo AI minigene in COS cells led to the secretion of proapo AI (29 kDa), that of the mutant (M2) gene, devoid of the prosequence of mature apo AI (28.4 kDa), whereas the product of mutant gene M1 (31 kDa) with the recognition site of the signal peptides transposed to the C-terminus of the prosequence remained uncleaved within the COS cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Mitochondrial 3-2trans-Enoyl-CoA isomerase. Purification, cloning, expression, and mitochondrial import of the key enzyme of unsaturated fatty acid beta-oxidation.

3-2trans-Enoyl-CoA isomerase (EC 5.3.3.8) is a key enzyme in mitochondrial beta-oxidation of unsaturated fatty acids in bacteria, plant and animal cells. The enzyme was isolated from rat liver mitochondria and purified to homogeneity by two chromatographic steps. Partial polypeptide sequences of the 29 kDa protein were derived from cyanogen bromide, tryptic, Lys-C, and protease V8 fragments by Edman degradation. Peptide-derived synthetic oligonucleotides were used for the isolation of a 990 bp long isomerase-specific cDNA from rat liver cDNA libraries. 867 bp encode the 289 amino-acid residues of the preisomerase with a molecular mass of 32,254 Da. The 1.3-kb mRNA is most strongly expressed in skeletal muscle followed by liver, heart, kidney, and weakly expressed in spleen and brain. In vitro transcription and translation yielded a 32 kDa polypeptide which was immunoprecipitated by anti rat isomerase antibodies. In the presence of mitochondria the 32 kDa precursor isomerase was processed during mitochondrial import to the 29 kDa mature form of the 3-2trans-enoyl-CoA isomerase with 264 amino-acid residues (Mr 29,706). A N-terminal signal sequence of 25 amino-acid residues directs the import into the mitochondrial matrix and is cleaved in two successive steps passing through an intermediate form of Mr 30,475. The two cysteine residues in positions 142 and 148 of the preisomerase are present as free thiol groups as shown by derivatization of the mature, native protein with the fluorescent label N-(iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid. The mitochondrial 3-2trans enoyl-CoA isomerase shows significant homology and conserved amino-acid exchanges with the mitochondrial enoyl-CoA hydratase, the N-terminal domain of the bifunctional peroxisomal enoyl-CoA-hydratase:3-hydroxyacyl-CoA dehydrogenase and to extended domains of the alpha-subunit of the procaryotic beta-oxidation complex sharing enoyl-CoA isomerase, D(-)3-hydroxyacyl-CoA epimerase, enoyl-CoA hydratase and L(+)3-hydroxyacyl-CoA dehydrogenase activity, encoded by the fad B operon of E. coli.

Amino Acid Sequence↗
PubMed1991

Evolution of the myelin integral membrane proteins of the central nervous system.

The predominant integral membrane protein of the CNS myelin of amphibia, reptiles, birds and mammals is proteolipid protein (PLP) and P0, the main glycoprotein in PNS myelin. Alternative splicing of the transcripts of the single genes of PLP and myelin basic protein (MBP) is the underlying mechanism by which the isoforms of the two main proteins of the myelin membrane arise. DM20 is an isoform of PLP in mammalian, avian and reptilian myelin. It does not occur in the CNS myelin of amphibia. DM20 lacks an extended hydrophilic sequence exposed on the extracytoplasmic surface of the lipid bilayer as a result of the usage of a cryptic donor splice site within exon III. We report about comparative studies on PLP and its DM20 isoform on the protein and DNA level of frog, chicken, rat CNS and the P0-related IP proteins of the CNS of trout. Chemical cleavage at tryptophan residues with N-chlorosuccinimide yields identical patterns of PLP peptides which refers to a high conservation between amphibia, birds and mammals and is totally different from the cleavage pattern of hydrophobic myelin proteins IP-1 and IP-2 of trout CNS and that of P0 of rat PNS. The N-terminal 19 amino-acid residues of IP-1 of trout CNS- and P0 of frog PNS myelin were sequenced and proved to be homologous on one hand with the P0 analogue of CNS of the shark, a cartilage fish, and on the other hand with P0 protein of PNS of birds and mammals. The complete amino-acid sequence of chicken CNS PLP was derived from its cDNA. Coding and noncoding segments of the PLP gene of frog were sequenced: there is a high degree of conservation between amphibian and mammalian PLP within the hydrophobic domains. Numerous mutations were found within the part of exon III encoding the hydrophilic domain. Base exchanges within the putative splice site in exon III explain the absence of DM20 in the protein pattern of amphibia CNS myelin. This result is being discussed in view of the membrane organization and the function of PLP.

Amino Acid Sequence↗
PubMed1990

In vivo 13C nuclear magnetic resonance investigations of choline metabolism in rabbit brain.

The metabolism of choline in rabbit brain was studied by the noninvasive approach of in vivo 13C NMR spectroscopy. 13C-Enriched precursors were introduced into the brain. Surgery of the head skin was avoided through controlled localization of the surface coil. For long-term accumulation studies in brain, repeated subcutaneous injections proved to be advantageous over other forms of application. The resorption kinetics was calculated to be zero order which suggests slow delivery from the subcutaneous depots. Choline metabolism was studied by two approaches: [N-13CH3]choline and S-[13CH3]methionine were administered separately to adult and myelinating rabbits (Days 5 to 32), respectively, over 4 weeks. [N-13CH3]Choline and the 13CH3 group of methionine were incorporated into lecithin and sphingomyelin of brain myelin. In vivo kinetic studies of the turnover of these labeled structures were carried out. Choline and methionine are readily transported through the blood-brain barrier and utilized by the myelinating brain for the biosynthesis of its phospholipids.

Animals↗
PubMed1990

31P NMR spectroscopy for in vitro viability testing of porcine pancreatic islets.

The quality of pancreatic islets prepared by an intraductal pancreas collagenase perfusion technique was tested using three independent methods: 31P NMR spectroscopy, an insulin secretion test, and a staining method. The viability of pancreatic islet tissue was evaluated using the ratio of phosphate diester to phosphate monoester (PDE/PME) as a new criterion obtained by 31P NMR spectroscopy. According to this criterion, three types of tissue fragments could be characterized: vital (PDE/PME 0.5-0.9), damaged (PDE/PME less than 0.2), and necrotic (no PDE, no PME). The findings in the three different groups could be correlated to three trends of insulin secretion of the preparations following glucose challenge: good response to the glucose challenge, continuous decrease of insulin production, and no insulin secretion. We feel that 31P NMR spectroscopy offers a rapid and suitable method for classifying the viability of isolated pancreatic islets.

Animals↗
PubMed1990

The 3'-flanking region shared by the human apolipoprotein AI and CIII gene regulates gene expression in cooperation with 5'-flanking elements.

The genes of the apolipoproteins AI and CIII are localized in opposite orientation on chromosome 11 in close vicinity, separated from each other by a non-coding intergenic region of 2.1 kb. The interdependence of their expression has been studied in cotransfection experiments in Hep G2 cells. An expression vector harboring the intergenic region with CAT as reporter gene and likewise pUC 19 derivatives with the putative cis-elements were constructed. Inhibition of gene expression by 5'-flanking elements of the apo AI and CIII gene was observed. Four proteins have been identified in nuclear extracts of Hep G2 cells that bind to several sequences of the intergenic domain but not to the 5'-flanking regions of apo AI or CIII gene. These proteins might be responsible for the silencer effect. The pathway of the regulation of apo CIII and AI expression derived from the experiments described here is supported by mutations in the intergenic region, leading to the phenotype of hypertriglyceridemia, and the stimulatory effect of cholesterol on apo AI transcription in Hep G2 cells.

Apolipoprotein A-I↗
PubMed1990

A point mutation at the X-chromosomal proteolipid protein locus in Pelizaeus-Merzbacher disease leads to disruption of myelinogenesis.

A group of inherited neurological disorders are the X-chromosome linked dysmyelinoses, in which myelin membranes of the CNS are missing or perturbed due to a strongly reduced number of differentiated oligodendrocytes. In animal dysmyelinoses (jimpy mouse, msd-mouse, md rat, shaking pup) mutations of the main integral myelin membrane protein, proteolipid protein, have been identified. Pelizaeus-Merzbacher disease (PMD) or sudanophilic leucodystrophy is an X-linked dysmyelinosis in humans. We report here on the molecular basis of the defect of affected males of a PMD kindred. Rearrangements of the PLP gene were excluded by Southern blot hybridisation analysis and PCR amplification of overlapping domains of the PLP gene. Sequence analysis revealed one single C----T transition in exon IV, which leads to a threonine----isoleucine substitution within a hydrophobic intramembrane domain. The impact of this amino-acid exchange on the structure of PLP in the affected cis membrane domain is discussed. A space filling model of this domain suggests a tight packing of the alpha-helices of the loop which is perturbed by the amino-acid substitution in this PMD exon IV mutant. The C----T transition in exon IV abolishes a Hph I restriction site. This mutation at the recognition site for Hph I (RFLP) and allele-specific primers have been used for mutation screening the PMD kindred.

Base Sequence↗
PubMed1990

Purification and characterization of bovine liver 3-cis-2-trans-enoyl-CoA isomerase.

The purification to homogeneity of 3-cis-2-trans-enoyl-CoA isomerase from bovine liver is described. This procedure has also been successfully applied to the isolation of the enzyme from rat liver mitochondria. Its molecular mass was determined to be 30 kDa by several methods. Km of the isomerase for 3-cis-dodecanoyl-CoA, the physiological intermediate in the beta-oxidation of oleic acid and of the 3-trans-isomer was determined to be 3.2 x 10(-5) M. The velocity of isomerization of the 3-trans-substrate is reduced 10-15 times. Inhibition experiments and pH-dependency of the reaction kinetics suggest the participation of a histidine residue in the isomerization. Protein analytical studies revealed that the N-terminus is blocked (acetylated). Cyanogen bromide cleavage of the purified enzyme resulted in five fragments. The N-terminus of one fragment is blocked. Partial amino-terminal sequences of two of the fragments were obtained by Edman degradation, a prerequisite for further studies on the structure of this isomerase on the DNA level.

Amino Acid Sequence↗
PubMed1990

A facile method for the isolation and preparation of proteins and peptides for sequence analysis in the picomolar range.

We report a new and facile extraction method of proteins and polypeptides in the range of 100 to 1 kDa previously separated by high-resolution SDS/polyacrylamide-gel electrophoresis. Proteins and polypeptides obtained by chemical or proteolytic cleavage of proteins can directly be applied to high-sensitivity N-terminal amino-acid sequence analysis by gas-phase sequencing. The Coomassie Blue-stained protein bands are eluted from the gel slices with 0.1 M sodium acetate buffer, pH 8.5, 0.1% SDS in high yield and directly applied to the filter disc of the gas-phase sequencer. The superior efficiency for the isolation of proteins and polypeptides from polyacrylamide gels for microsequencing has been documented by a quantitative comparison of the procedure described here and the favoured electroblot-transfer method using 14C-labeled marker proteins. This highly efficient isolation has been successfully reproduced and applied to the analysis of a variety of proteins and peptides with rather divergent physical properties, particularly to hydrophobic peptides isolated from SDS/polyacrylamide gels. The electrophoretic transfer onto activated glass filters. Immobilon membranes (polyvinylidene-difluoride membranes), siliconized or chemically activated glass fiber supports can be omitted. The method considerably simplifies and speeds up the isolation, and improves the sensitivity as compared to the electroblotting procedures due to the reproducibly high recoveries.

Amino Acid Sequence↗