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1.
J Cancer Res Clin Oncol ; 143(10): 1999-2010, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28639083

ABSTRACT

PURPOSE: Merkel cell carcinoma (MCC) is a rare but aggressive neuroendocrine tumor of the skin with an increasing incidence. The clinical course is variable and reliable prognostic factors are scarce. Tumor angiogenesis has been shown to have prognostic impact in different types of cancer. The aim of our study was to determine potential prognostic factors, including tumor vascularization, for clinical outcome of MCC. METHODS: The medical records of 46 patients with MCC diagnosed between 1997 and 2010 were analyzed retrospectively. Tissue samples were immune-stained for the lymphatic endothelial vessel marker podoplanin/D2-40 and the panvascular marker CD31. These immunostained sections were analyzed using computer-assisted morphometric image analyses. Aside from the parameters of tumor vascularization, clinicopathologic features were investigated, and progression-free survival (PFS) and tumor-specific survival (TSS) were assessed. Univariate and multivariate analyses were performed to determine prognostic factors. RESULTS: Male sex of the MCC patients and a high cross-sectional whole vessel area (WVA) in relation to the entire tumor area as determined on CD31-stained tumor sections were found to be negative prognostic factors for PFS in a univariate and multivariate regression analysis. Ulceration of the primary tumor was significantly associated with both impaired PFS and TSS. CONCLUSIONS: Our results indicate a high prognostic impact of tumor vascularization on the clinical outcome of MCC patients. Male sex and ulceration of the primary MCC were identified as independent unfavorable prognostic markers for the clinical outcome. As an outlook, MCC patients with increased angiogenesis might be identified and subjected to a targeted anti-angiogenic treatment.


Subject(s)
Carcinoma, Merkel Cell/blood supply , Carcinoma, Merkel Cell/pathology , Skin Neoplasms/blood supply , Skin Neoplasms/pathology , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Neovascularization, Pathologic/pathology , Prognosis , Sex Factors
2.
Sportverletz Sportschaden ; 26(1): 49-56, 2012 Mar.
Article in German | MEDLINE | ID: mdl-22422285

ABSTRACT

INTRODUCTION: A dance teacher teaches in various professional and amateur genres. The physical load depends on the target group and is partly submaximal or even higher. The only tool of dance teachers is their own body. There are only few other tools to facilitate such work. Therefore, keeping the body in a good health status is of great significance. Up to the present, there have been little or no data on the health status of persons teaching dance. The aim of this study is to examine the health status of persons who teach dance in order to initiate a data base for the development of further projects with a preventive medical focus. METHODS: A total of 165 (f: 154, m: 11) average aged (46.1 years) dance teachers in Germany responded to a cross-section questionnaire survey. RESULTS: Teaching dance is for 96.6 % "the job of their dreams". Of all dance teachers 89.4 % continue to work while in pain and 85.5 % cannot afford a "longer down-time". 78.2 % state that they have paused due to a work-related disease, although 56.9 % of the respondents consider their occupation to be the cause for a disease with the answer only, seldom (44.4 %) or never (12.5 %). 4.5 % consult a medical doctor only after the self-treatment has failed. The lower extremity is the most common anatomic region in acute injuries (57.6 %) as well as in chronic complaints (58.3 %). The causes are multiple and comprise physical as well as psychic factors such as a high pressure of expectation (64.8 %) or a tight schedule/time table (43 %). 29 % of all dance teachers suffer from internal and 58 % from orthopaedic complaints. 43.1 % of them state that these complaints result from their occupation. 80.5 % make an acute injury responsible for compulsary breaks. DISCUSSION: The discrepancies between the self-perception and the findings of this investigation clearly show that the psychic and physical load in this occupation should not be underestimated. Here, further research on the social, psychic and physical health status of dance teachers is required. The results of this investigation warrant further research in the field of activity of the variable and up to now unprotected dance teacher profession in Germany.


Subject(s)
Dancing/education , Health Status , Physical Education and Training , Absenteeism , Adult , Cross-Sectional Studies , Female , Germany , Health Surveys , Humans , Male , Middle Aged , Occupational Diseases/epidemiology , Occupational Diseases/etiology , Occupational Injuries/epidemiology , Occupational Injuries/etiology , Sick Leave , Surveys and Questionnaires
3.
Lett Appl Microbiol ; 48(5): 618-22, 2009 May.
Article in English | MEDLINE | ID: mdl-19416463

ABSTRACT

AIMS: To identify novel proteins secreted by the probiotic bacterium Lactobacillus rhamnosus GG after growth in de Mann-Rogosa-Sharpe broth (MRS), a complex medium often used for the culture of Lactobacillus. METHODS AND RESULTS: The proteins secreted by L. rhamnosus GG strain were precipitated using a trichloroacetic acid-based protocol, resolved by SDS-PAGE, and identified by tandem mass spectrometry (MS/MS). Among the proteins secreted by this bacterium, a leukocyte elastase inhibitor, already present in the MRS broth, was identified. Other proteins such as cell wall hydrolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, and an extracellular transcriptional regulator have been also identified. CONCLUSIONS: Lactobacillus rhamnosus GG secretes several proteins during its growth in MRS, some of them with assigned functions in the prevention of the molecular mechanisms that lead to damage in the epithelial barrier (cell wall hydrolase) and in adhesion (GAPDH). The rest of the proteins require further genetic analysis in order to establish their precise roles. None of the proteins bound to mucin or fibronectin. SIGNIFICANCE AND IMPACT OF THE STUDY: Some of these secreted proteins could be involved in the probiotic effects exerted by L. rhamnosus GG strain, their identification being the first step towards in depth functional studies.


Subject(s)
Bacterial Proteins/metabolism , Culture Media/metabolism , Lactobacillus/growth & development , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Lactobacillus/chemistry , Lactobacillus/genetics , Lactobacillus/metabolism , Molecular Sequence Data , Protein Transport
4.
Microbiology (Reading) ; 155(Pt 5): 1708-1716, 2009 May.
Article in English | MEDLINE | ID: mdl-19372165

ABSTRACT

Several Bacillus strains isolated from commercial probiotic preparations were identified at the species level, and their adhesion capabilities to three different model intestinal surfaces (mucin, Matrigel and Caco-2 cells) were assessed. In general, adhesion of spores was higher than that of vegetative cells to the three matrices, and overall strain Bacillus cereus(CH) displayed the best adhesion. Different biochemical treatments revealed that surface proteins of B. cereus(CH) were involved in the adhesion properties of the strain. Surface-associated proteins from vegetative cells and spores of B. cereus(CH) were extracted and identified, and some proteins such as S-layer components, flagellin and cell-bound proteases were found to bind to mucin or fibronectin. These facts suggest that those proteins might play important roles in the interaction of this probiotic Bacillus strain within the human gastrointestinal tract.


Subject(s)
Bacillus cereus/physiology , Bacterial Adhesion , Bacterial Proteins/metabolism , Fibronectins/metabolism , Membrane Proteins/metabolism , Mucins/metabolism , Probiotics/metabolism , Bacillus cereus/chemistry , Bacillus cereus/growth & development , Bacterial Proteins/chemistry , Caco-2 Cells , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Membrane Proteins/chemistry , Molecular Sequence Data , Protein Binding , Spores, Bacterial/growth & development , Spores, Bacterial/physiology
5.
J Microbiol Biotechnol ; 19(12): 1635-43, 2009 Dec.
Article in English | MEDLINE | ID: mdl-20075631

ABSTRACT

The aim of this study was to provide new insight into the mechanism whereby the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locates to cell walls of Lactobacillus plantarum 299v. After purification, cytosolic and cell wall GAPDH (cw-GAPDH) forms were characterized and shown to be identical homotetrameric active enzymes. GAPDH concentration on cell walls was growth-time dependent. Free GAPDH was not observed on the culture supernatant at any time during growth, and provoked cell lysis was not concomitant with any reassociation of GAPDH onto the cell surface. Hence, with the possibility of cw-GAPDH resulting from autolysis being unlikely, entrapment of intracellular GAPDH on the cell wall after a passive efflux through altered plasma membrane was investigated. Flow cytometry was used to assess L. plantarum 299v membrane permeabilization after labeling with propidium iodide (PI). By combining PI uptake and cw-GAPDH activity measurements, we demonstrate here that the increase in cw-GAPDH concentration from the early exponential phase to the late stationary phase is closely related to an increase in plasma membrane permeability during growth. Moreover, we observed that increases in both plasma membrane permeability and cw-GAPDH activity were delayed when glucose was added during L. plantarum 299v growth. Using a double labeling of L. plantarum 299v cells with anti-GAPDH antibodies and propidium iodide, we established unambiguously that cells with impaired membrane manifest five times more cw-GAPDH than unaltered cells. Our results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.


Subject(s)
Cell Wall/enzymology , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Lactobacillus plantarum/metabolism , Cell Membrane Permeability , Cytoplasm/enzymology , Flow Cytometry , Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Lactobacillus plantarum/growth & development , Propidium/metabolism , Protein Multimerization , Staining and Labeling , Subcellular Fractions/metabolism
6.
J Neurol Neurosurg Psychiatry ; 74(9): 1262-6, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12933931

ABSTRACT

BACKGROUND: Peripheral neuropathies (PN) associated with monoclonal gammopathy (MG) are widely considered as autoimmune disorders, but the putative role of incriminated antigens is still not understood. OBJECTIVE: Fifty five patients with PN associated with MG were studied to investigate whether new antigens could be found, and to evaluate their relation to clinical manifestations. METHODS: An immunological study was conducted on patient sera to identify autoreactivities against nerve proteins by western blotting. Antigen proteins were purified and analysed by proteomic tools. Correlation with ultrastrucural and clinical features was then studied. RESULTS: Of the 55 patients suffering from PN associated with MG, 17 exhibited IgG autoantibodies directed against peripheral nerve proteins of 35, 58, and 60 kDa. N-terminal microsequencing and mass spectrometry analyses of the 35 kDa protein revealed perfect peptidic matching with 47% of the amino acid sequence of P0, whereas the 58 and 60 kDa proteins were identified as the reduced and non-reduced forms of a P0 dimer. Deglycosylation did not affect IgG binding to the 35 kDa P0 related protein, suggesting a peptidic epitope. In contrast, deglycosylation abolished IgG recognition of the P0 dimer protein, so that a carbohydrate moiety may be implicated in the epitope formation. This confirmed the existence of two different types of IgG, one recognising the 58 and 60 kDa proteins and one directed against the 35 kDa protein. CONCLUSIONS: This is the first report of antibody activity directed against the dimeric association of P0. Although P0 oligomerisation and adhesion properties play a crucial part in the myelin sheath compaction, the pathogenic significance of these autoantibodies needs further investigations to be elucidated.


Subject(s)
Antigens/analysis , Immunoglobulin G/analysis , Myelin P0 Protein/immunology , Paraproteinemias/immunology , Animals , Autoantibodies/analysis , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Humans , Myelin P0 Protein/analogs & derivatives , Paraproteinemias/pathology , Rats , Rats, Sprague-Dawley
7.
Mol Cell Biol ; 21(24): 8346-56, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11713271

ABSTRACT

In Saccharomyces cerevisiae, in vitro mRNA cleavage and polyadenylation require the poly(A) binding protein, Pab1p, and two multiprotein complexes: CFI (cleavage factor I) and CPF (cleavage and polyadenylation factor). We characterized a novel essential gene, MPE1 (YKL059c), which interacts genetically with the PCF11 gene encoding a subunit of CFI. Mpe1p is an evolutionarily conserved protein, a homolog of which is encoded by the human genome. The protein sequence contains a putative RNA-binding zinc knuckle motif. MPE1 is implicated in the choice of ACT1 mRNA polyadenylation site in vivo. Extracts from a conditional mutant, mpe1-1, or from a wild-type extract immunoneutralized for Mpe1p are defective in 3'-end processing. We used the tandem affinity purification (TAP) method on strains TAP-tagged for Mpe1p or Pfs2p to show that Mpe1p, like Pfs2p, is an integral subunit of CPF. Nevertheless a stable CPF, devoid of Mpe1p, was purified from the mpe1-1 mutant strain, showing that Mpe1p is not directly involved in the stability of this complex. Consistently, Mpe1p is also not necessary for the processive polyadenylation, nonspecific for the genuine pre-mRNA 3' end, displayed by the CPF alone. However, a reconstituted assay with purified CFI, CPF, and the recombinant Pab1p showed that Mpe1p is strictly required for the specific cleavage and polyadenylation of pre-mRNA. These results show that Mpe1p plays a crucial role in 3' end formation probably by promoting the specific link between the CFI/CPF complex and pre-mRNA.


Subject(s)
RNA-Binding Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/metabolism , Alleles , Amino Acid Sequence , Blotting, Northern , Conserved Sequence , Electrophoresis, Polyacrylamide Gel , Evolution, Molecular , Immunoblotting , Mass Spectrometry , Molecular Sequence Data , Polyadenylation , Protein Binding , RNA/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Temperature , Time Factors , Zinc/chemistry , mRNA Cleavage and Polyadenylation Factors
8.
Proteomics ; 1(8): 946-54, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11683511

ABSTRACT

In the context of proteome analysis, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) can fulfil the two tasks of primary structure verification and protein identification. As an illustration of the first of these tasks, the sequence of Eschericha coli isoleucyl-tRNA synthetase, a protein with 15 reported sequence conflicts, has been established by means of MALDI mass mapping. The identification of mitochondrial proteins participating in a yeast supramolecular complex exhibiting NADH dehydrogenase activity highlights the performances of MALDI-MS for the second task. The spectral suppression phenomenon occurring for complex peptide mixtures analysed by MALDI is discussed, as well as the role of post-source decay analysis for confident protein identification.


Subject(s)
Isoleucine-tRNA Ligase/chemistry , Methionine-tRNA Ligase/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Amino Acid Sequence , Escherichia coli/enzymology , Mitochondria/enzymology , Molecular Sequence Data , Saccharomyces cerevisiae/enzymology
9.
Biochemistry ; 40(33): 9758-69, 2001 Aug 21.
Article in English | MEDLINE | ID: mdl-11502169

ABSTRACT

Separation of yeast mitochondrial complexes by colorless native polyacrylamide gel electrophoresis led to the identification of a supramolecular structure exhibiting NADH-dehydrogenase activity. Components of this complex were identified by N-terminal Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. The complex was found to contain the five known intermembrane space-facing dehydrogenases, namely two external NADH-dehydrogenases Nde1p and Nde2p, glycerol-3-phosphate dehydrogenase Gut2p, D- and L-lactate-dehydrogenases Dld1p and Cyb2p, the matrix-facing NADH-dehydrogenase Ndi1p, two probable flavoproteins YOR356Wp and YPR004Cp, four tricarboxylic acids cycle enzymes (malate dehydrogenase Mdh1p, citrate synthase Cit1p, succinate dehydrogenase Sdh1p, and fumarate hydratase Fum1p), and the acetaldehyde dehydrogenase Ald4p. The association of these proteins is discussed in terms of NADH-channeling.


Subject(s)
Mitochondria/enzymology , Oxidoreductases/chemistry , Saccharomyces cerevisiae/enzymology , 1-Pyrroline-5-Carboxylate Dehydrogenase , Aldehyde Oxidoreductases/chemistry , Cell Membrane/enzymology , Chromatography, High Pressure Liquid , Citrate (si)-Synthase/chemistry , Electrophoresis, Polyacrylamide Gel , Flavoproteins/chemistry , Fumarate Hydratase/chemistry , Glycerolphosphate Dehydrogenase/chemistry , L-Lactate Dehydrogenase/chemistry , Malate Dehydrogenase/chemistry , Models, Biological , NADH Dehydrogenase/chemistry , NADH Dehydrogenase/metabolism , Oxygen/metabolism , Phosphorylation , Protein Binding , Saccharomyces cerevisiae Proteins , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Succinate Dehydrogenase/chemistry , Time Factors
10.
J Pept Sci ; 7(11): 588-97, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11763363

ABSTRACT

The HTLV-I integrase N-terminal domain [50-residue peptide (IN50)], and a 35-residue truncated peptide formed by residues 9-43 (IN35) have been synthesized by solid-phase peptide synthesis. Formation of the 50-residue zinc finger type structure through a HHCC motif has been proved by UV-visible absorption spectroscopy. Its stability was demonstrated by an original method using RP-HPLC. Similar experiments performed on the 35-residue peptide showed that the truncation does not prevent zinc complex formation but rather that it significantly influences its stability. As evidenced by CD spectroscopy, the 50-residue zinc finger is unordered in aqueous solution but adopts a partially helical conformation when trifluoroethanol is added. These results are in agreement with our secondary structure predictions and demonstrate that the HTLV-I integrase N-terminal domain is likely to be composed of an helical region (residues 28-42) and a beta-strand (residues 20-23), associated with a HHCC zinc-binding motif. Size-exclusion chromatography showed that the structured zinc finger dimerizes through the helical region.


Subject(s)
Human T-lymphotropic virus 1/enzymology , Integrases/chemistry , Peptide Fragments/chemistry , Zinc Fingers , Amino Acid Sequence , Enzyme Stability , Integrases/metabolism , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary/physiology , Sequence Alignment
12.
Eur J Biochem ; 267(15): 4789-98, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10903513

ABSTRACT

Bromomethyl ketone derivatives of L-valine (VBMK), L-isoleucine (IBMK), L-norleucine (NleBMK) and L-phenylalanine (FBMK) were synthesized. These reagents were used for qualitative comparative labeling of Escherichia coli valyl-tRNA synthetase (ValRS), an enzyme with Val/Ile editing activity, in order to identify the binding sites for L-valine or noncognate amino acids. Labeling of E. coli ValRS with the substrate analog valyl-bromomethyl ketone (VBMK) resulted in a complete loss of valine-dependent isotopic [32P]PPi-ATP exchange activity. L-Valine protected the enzyme against inactivation. Noncognate amino acids analogs isoleucyl-, norleucyl- and phenylalanyl-bromomethyl ketones (IBMK, NleBMK and FBMK) were also capable of abolishing the activity of ValRS, FBMK being less efficient in inactivating the synthetase. Matrix-assisted laser desorption-ionization mass spectrometry designated cysteines 424 and 829 as the target residues of the substrate analog VBMK on E. coli ValRS, whereas, altogether, IBMK, NleBMK and FBMK labeled His266, Cys275, His282, His433 and Cys829, of which Cys275, His282 and His433 were labeled in common by all three noncognate amino-acid-derived bromomethyl ketones. With the exception of Cys829, which was most likely unspecifically labeled, the amino-acid residues labeled by the reagents derived from noncognate amino acids were distributed between two fragments 259-291 and 419-434 in the primary structure of E. coli ValRS. In fragment 419-434, Cys424 was specifically labeled by the substrate analog VBMK, while His433 was labeled in common by all the used bromomethyl ketone derivatives of noncognate amino acids, suggesting that the synthetic site where aminoacyl adenylate formation takes place on E. coli ValRS is built up of two subsites. One subsite containing Cys424 might represent the catalytic locus of the active center where specific L-valine activation takes place. The second subsite containing His433 might represent the binding site for noncognate amino acids. The fact that Cys275 and His282, fragment 259-291, were labeled by IBMK, NleBMK and FBMK, but not by the substrate analog VBMK, suggests that these residues might be located at or near the editing site of E. coli ValRS. Comparison of fragment 259-291 with all the available ValRS amino-acid sequences revealed that His282 is strictly conserved, with the exception of its replacement by a glycine in a subgroup corresponding to the archaebacteria. Because a nucleophile is needed in the editing site to achieve hydrolysis of an undesired product at the level of the carbonyl group thereof, it is proposed that the conserved His282 of E. coli ValRS is involved in editing.


Subject(s)
Escherichia coli/enzymology , Valine-tRNA Ligase/chemistry , Valine/metabolism , Amino Acid Sequence , Binding Sites , Diethyl Pyrocarbonate/chemistry , Escherichia coli/chemistry , Escherichia coli/metabolism , Ethylmaleimide/chemistry , Isoleucine/metabolism , Ketones/metabolism , Kinetics , Molecular Sequence Data , Norleucine/metabolism , Peptides/chemistry , Phenylalanine/metabolism , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Thermus thermophilus/chemistry , Thermus thermophilus/metabolism , Time Factors
13.
J Chromatogr B Biomed Sci Appl ; 737(1-2): 55-61, 2000 Jan 14.
Article in English | MEDLINE | ID: mdl-10681041

ABSTRACT

High quality purification of membrane-spanning peptides and proteins remains a challenging problem. In this work we describe a tailored chromatographic purification of a synthetic 35-residue peptide corresponding to the transmembrane region of the tyrosine kinase receptor c-erb2/neu. Composed to over 70% by the amino acids alanine, isoleucine, leucine, phenylalanine and valine, this peptide presents a very hydrophobic character. Product isolation from the complex peptide mixture, obtained after acid cleavage of the resin matrix used during the solid-phase synthesis, represents a difficult task. We propose a three step strategy based on gel permeation and reversed-phase high-performance liquid chromatography, using aprotic polar solvent mixtures. The challenge consisted in obtaining a sufficient amount of an extremely pure sample, in order to allow structural analysis by NMR spectroscopy. Keeping trace of the synthetic peptide throughout the different purification steps was assured by MALDI TOF mass spectrometry, and the final product purity was checked by coupled liquid chromatography-ESI TOF mass spectrometry.


Subject(s)
Membrane Proteins/chemistry , Peptide Fragments/isolation & purification , Receptor, ErbB-2/chemistry , Amino Acid Sequence , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
14.
Biochemistry ; 38(45): 15017-24, 1999 Nov 09.
Article in English | MEDLINE | ID: mdl-10555984

ABSTRACT

A component of the stator of the yeast ATP synthase (subunit 4 or b) showed many cross-linked products with the homobifunctional reagent dithiobis[succinimidyl propionate], which reacts with the amino group of lysine residues. The positions in subunit 4 that were involved in the cross-linkings were determined by using cysteine-generated mutants constructed by site-directed mutagenesis of ATP4. Cross-linking experiments with the heterobifunctional reagent p-azidophenacyl bromide, which has a spacer arm of 9 A, were performed with mitochondria and crude Triton X-100 extracts containing the solubilized enzyme. Substitution of lysine residues by cysteine residues in the hydrophilic C-terminal part of subunit 4 allowed cross-links with subunit h from C98 and with subunit d from C141, C143, and C151. OSCP was cross-linked from C174 and C209. A cross-linked product, 4+beta, was also obtained from C174. It is concluded that the C-terminus of subunit 4 is distant from the membrane surface and close to F(1) and OSCP. The N-terminal part of subunit 4 is close to subunit g, as demonstrated by the identification of a cross-linked product involving subunit g and the cysteine residues 7 or 14 of subunit 4.


Subject(s)
Multienzyme Complexes/chemistry , Phosphotransferases (Phosphate Group Acceptor)/chemistry , Saccharomyces cerevisiae/enzymology , ATP Synthetase Complexes , Amino Acid Sequence , Cross-Linking Reagents/pharmacology , Dimerization , Indicators and Reagents/pharmacology , Molecular Sequence Data , Protein Conformation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Succinimides/pharmacology
15.
J Pept Sci ; 5(6): 245-50, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10463779

ABSTRACT

Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem. Strong interchain associations within the peptide-resin matrix have to be overcome. A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures. A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis. In this work, the total chemical synthesis of the membrane-embedded yeast mitochondrial ATP synthase subunit 8 is described. The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt alpha-helical secondary structure is evidenced by circular dichroism spectroscopy.


Subject(s)
Intracellular Membranes/enzymology , Mitochondria/ultrastructure , Proton-Translocating ATPases/chemical synthesis , Amino Acid Sequence , Chromatography, High Pressure Liquid , Circular Dichroism , Membrane Proteins/chemical synthesis , Protein Structure, Secondary , Saccharomyces cerevisiae/ultrastructure , Yeasts/ultrastructure
16.
Biochimie ; 80(4): 313-9, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9672751

ABSTRACT

The transglutaminase (TGase; EC 2.3.2.13) from Streptoverticillium cinnamoneum CBS 683.68 has been purified, characterised and its gene cloned. The purified enzyme had a relative molecular mass of 37,660 determined by mass spectrometry and contained a single Cys residue that was essential for the catalytic activity. Contrary to eukaryotic TGases, this enzyme was calcium-independent. The fact that TGase was capable to incorporate a wide variety of aliphatic and aromatic non-polar compounds suggested that the amine fixation site could be an hydrophobic pocket. S cinnamoneum CBS 683.68 TGase was synthesised as a protein precursor of 411 amino acid residues corresponding to a signal peptide of 81 amino acid residues and a mature TGase of 330 amino acid residues. Amino acid sequence analysis revealed that the S cinnamoneum CBS 683.68 TGase had little sequence homology with eukaryotic TGases, but shared high identity with the sequence of Streptoverticillium strain S-8112. In accordance with kinetics data, hydropathy analysis showed that the active site of the enzyme was in an hydrophobic environment as for eukaryotic TGases.


Subject(s)
Genes, Fungal , Streptomycetaceae/enzymology , Streptomycetaceae/genetics , Transglutaminases/genetics , Transglutaminases/metabolism , Amino Acid Sequence , Base Sequence , Chromatography, Affinity , Chromatography, Gel , Chromatography, Ion Exchange , Cloning, Molecular , Kinetics , Molecular Sequence Data , Peptide Fragments/chemistry , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity , Transglutaminases/isolation & purification
17.
J Chromatogr B Biomed Sci Appl ; 697(1-2): 123-33, 1997 Sep 12.
Article in English | MEDLINE | ID: mdl-9342662

ABSTRACT

Characterization of a human anti-Rhesus(D) monoclonal antibody, developed for the treatment of Rh(D) haemolytic disease of the newborn, was performed. Capillary electrophoresis (CE) has been employed for peptide mapping of the IgG heavy chain and glycopeptide identification. The combination of the high resolution and low solvent consumption of CE and the ultrasensitive detection and precise identification properties of mass spectrometry led to a complete glycosylation analysis of the protein. Glycopeptides were easily isolated from a single injection in a 100 microns i.d. capillary of the preparative CE system and collected for molecular mass determination using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The off-line CE-MS characterization revealed the presence of different oligosaccharides linked to the unique N297-S-T glycosylation site of the IgG heavy chain. The differences between calculated and experimental masses of the glycopeptides suggested the presence of a fucosylated biantennary structure containing one or two galactose units as major oligosaccharide, together with similar species bearing a bisecting N-acetylglucosamine. CE conditions were optimized to allow the MS identification of sialylated forms.


Subject(s)
Antibodies, Monoclonal/analysis , Rh-Hr Blood-Group System/immunology , Antibodies, Monoclonal/immunology , Chromatography, High Pressure Liquid , Electrophoresis, Capillary , Glycopeptides/analysis , Glycoside Hydrolases , Glycosylation , Humans , Immunoglobulin Heavy Chains/analysis , Immunoglobulin Heavy Chains/isolation & purification , Immunoglobulin Light Chains/analysis , Peptide Mapping , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
18.
Biochemistry ; 36(36): 11021-6, 1997 Sep 09.
Article in English | MEDLINE | ID: mdl-9283094

ABSTRACT

Chicken gizzard telokin was purified to apparent homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This preparation yielded upon mass spectrometry analysis seven mass peaks spanning from 15 858 to 17 100 Da. Anion exchange-high performance liquid chromatography of the purified telokin revealed a high diversity of telokin molecules. By combining protein chemistry to chromatography and mass spectrometry, the telokin heterogeneity was analyzed. Three acetylated N-termini were found, AMI, MIS, and SGR. Cyanogen bromide cleavage of telokin yielded six different C-terminal peptides corresponding to the removal of one to six C-terminal glutamyl residues from the protein sequence deduced from the cDNA. Phosphorylation of telokin was detected, thus increasing the heterogeneity of the telokin preparation. In addition, peptide sequencing has shown that telokin contained either an aspartyl or a glutamyl residue at position 27, probably resulting from chicken polymorphism.


Subject(s)
Muscle Proteins/chemistry , Amino Acid Sequence , Animals , Chickens , Chromatography, High Pressure Liquid , Mass Spectrometry , Molecular Sequence Data , Myosin-Light-Chain Kinase , Peptide Fragments , Peptides
19.
J Protein Chem ; 16(5): 403-7, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9246620

ABSTRACT

Axonemal tubulin exhibits a high degree of heterogeneity mostly due to several posttranslational modifications (PTM). The aim of this work was to chemically characterize the different PTM occurring in the C-terminal tail of axonemal tubulin purified from sea urchin, Paracentrotus lividus, spermatozoa. After its purification, tubulin was enzymatically cleaved. The C-terminal peptides were chromatographically isolated, first by anion exchange and then by reverse-phase HPLC. Peptides were characterized by their sequence, determined by Edman degradation, and by their mass, determined by MALDI-TOF/MS. The two major conclusions are that the majority of the isolated C-terminal peptides were unmodified and that polyglycylation and polyglutamylation can occur simultaneously on one molecule of alpha-tubulin.


Subject(s)
Protein Processing, Post-Translational , Sperm Tail/metabolism , Tubulin/metabolism , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid/methods , Isoelectric Focusing , Male , Molecular Sequence Data , Peptide Fragments/analysis , Peptide Fragments/isolation & purification , Sea Urchins , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Sperm Tail/chemistry , Spermatozoa/metabolism , Tubulin/isolation & purification
20.
Protein Sci ; 6(11): 2426-35, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9385645

ABSTRACT

Methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by methionyl-tRNA synthetase (MetRS) is capable of reacting with this synthetase or other proteins, by forming an isopeptide bond with the epsilon-NH2 group of lysyl residues. It is proposed that the mechanism for the in vitro methionylation of MetRS might be accounted for by the in situ covalent reaction of methionyl-adenylate with lysine side chains surrounding the active center of the enzyme, as well as by exchange of the label between donor and acceptor proteins. Following the incorporation of 7.0 +/- 0.5 mol of methionine per mol of a monomeric truncated methionyl-tRNA synthetase species, the enzymic activities of [32P]PPi-ATP isotopic exchange and tRNA(Met) aminoacylation were lowered by 75% and more than 90%, respectively. The addition of tRNA(Met) protected the enzyme against inactivation and methionine incorporation. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-114, -132, -142 (or -147), -270, -282, -335, -362, -402, -439, -465, and -547 of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. These lysyl residues are distributed at the surface of the enzyme between three regions [114-150], [270-362], and [402-465], all of which were previously shown to be involved in catalysis or to be located in the binding sites of the three substrates, methionine, ATP, and tRNA.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Escherichia coli/enzymology , Methionine-tRNA Ligase/metabolism , Methionine/analogs & derivatives , Methionine/metabolism , Adenosine Monophosphate/metabolism , Amino Acid Sequence , Lysine/metabolism , Models, Molecular , Molecular Sequence Data , Peptide Fragments/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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