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J Salnikow

Publications and source records attributed to J Salnikow.

At least 19 recordsLinked to original sources
PubMed1999

Comparison of two-dimensional electrophoresis patterns of heat shock protein Hsp27 species in normal and cardiomyopathic hearts.

Heat shock protein Hsp27 occurs in a complex pattern in human myocardial tissue. Normal and failing explanted human heart from patients with dilated cardiomyopathy (DCM) or ischemic heart failure (IHF), respectively, were analyzed by high resolution two-dimensional electrophoresis (23x30 cm) and Hsp27 immunostaining. Twelve Hsp27 spots in DCM samples were significantly altered in intensity and ten of these were significantly changed in IHF. Four spots (h1, h2, h4, h5) in DCM samples and three spots (h2, h4, h5) in IHF at a molecular mass of 28 kDa were decreased in intensity. In this study, investigating left ventricles of human myocardium, spot h4 was only detected in normal heart samples. On the other hand, spots with a lower molecular mass of 27 kDa (h14, h15, h17, h20, h21) and 22-23 kDa (46, h47, h50) increased in intensity in failing hearts, suggesting that some form of Hsp27 degradation occurs during heart failure.

Adult↗
PubMed1999

Stability of some Cactaceae proteins based on fluorescence, circular dichroism, and differential scanning calorimetry measurements.

Characterization of three cactus proteins (native and denatured) from Machaerocereus gummosus (Pitahaya agria), Lophocereu schottii (Garambullo), and Cholla opuntia (Cholla), was based on electrophoretic, fluorescence, CD (circular dichroism), DSC (differential scanning calorimetry), and FT-IR (Fourier transform infrared) measurements. The obtained results of intrinsic fluorescence, DSC, and CD were dissimilar for the three species of cactus, providing evidence of differences in secondary and tertiary structures. Cactus proteins may be situated in the following order corresponding to their relative stability: Machaerocereus gummosus (Pitahaya agria) > Cholla opuntia (Cholla) > Lophocereu schottii (Garambullo). Thermodynamic properties of proteins and their changes upon denaturation (temperature of denaturation, enthalphy, and the number of ruptured hydrogen bonds) were correlated with the secondary structure of proteins and disappearance of alpha-helix.

Calorimetry, Differential Scanning↗
PubMed1998

Close vicinity of Lhc b1 and Lhc b4 in Photosystem II--membrane fragments as verified by chemical cross-linking.

The nearest neighbourhood of pigment-protein complexes within Photosystem II (PSII) membrane fragments has been studied by means of chemical cross-linking with o-phthalaldehyde (OPA) in conjunction with protein-chemical techniques. By means of OPA-induced cross-linking a major conjugate of about 60 kDa has been identified. This conjugate was shown to consist of two pigment-protein complexes of light-harvesting complex II (LHC II), Lhc b1 (CP27) and Lhc b4 (CP29) by means of SDS/PAGE in combination with an immunological analysis using mAbs directed against Lhc b4 and by matrix-assisted-laser-desorption-ionization mass spectrometry (MALDI-MS) and sequence analysis of peptides derived from a proteolytic digest of the conjugate. Domains of Lhc bl and Lhc b4 have been localized to a distance of not more than 5 A within LHC II. Our results are discussed in the light of recent models on the topography of the two subunits within the antenna system of Photosystem II.

Amino Acid Sequence↗
PubMed1998

Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis patterns by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS).

Identification of proteins separated by two-dimensional electrophoresis (2-DE) is a necessary task to overcome the purely descriptive character of 2-DE and a prerequisite to the construction of 2-DE databases in proteome projects. Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has a sensitivity for peptide detection in the lower fmol range, which should be sufficient for an analysis of even weakly silver-stained protein spots by peptide mass fingerprinting. Unfortunately, proteins are modified by the silver staining procedure, leading to low sequence coverage. Omission of glutaraldehyde increased the sequence coverage, but this improved sequence coverage is still clearly below the sequence coverage starting with Coomassie Brilliant Blue (CBB) R-250-stained spots. Other factors additionally seem to modify proteins during silver staining. By decreasing the protein amount, the advantage of very sensitive detection on the gel is lost during identification, because the resulting low sequence coverage is not sufficient for secure identification. Low-quantity proteins can be identified better starting with CBB G-250 or Zn-imidazol-stained proteins. In contrast, for high-quantity CBB R-250-stained spots, a sequence coverage of up to 90% can be obtained by using only one cleaving enzyme, and up to 80% was reached for medium-quantity spots after combination of tryptic digest with Asp-N- and Glu-C digest.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1997

Campylobacter jejuni major outer membrane protein and a 59-kDa protein are involved in binding to fibronectin and INT 407 cell membranes.

Campylobacter jejuni is one of the major causes of human diarrhea throughout the world. Attachment to host cells and extracellular matrix proteins is considered to be an essential primary event in the pathogenesis of enteritis. Outer membrane proteins of three C. jejuni strains, one of which was aflagellate, were investigated for their contribution to the process of adhesion to INT 407 cell membranes and the extracellular matrix protein fibronectin. Using a ligand-binding immunoblotting assay the flagellin, the major outer membrane protein and a 59-kDa protein were detected to be involved in adhesion to both substrates. The MOMP was able to inhibit the attachment of the bacteria to INT 407 cell membranes partly, when the protein was isolated under native conditions. However, it was totally lost when the protein was isolated in the presence of SDS. The 59-kDa protein of one strain was identified by N-terminal sequencing, and regarding the first 14 amino acids it was found to be identical to the 37-kDa CadF protein just recently described as fibronectin-binding protein of C. jejuni. Especially for the aflagellate strain this protein may be of special importance for adhesion of the bacteria to different substrates.

Amino Acid Sequence↗
PubMed1997

C-terminal ladder sequencing by an approach combining chemical degradation with analysis by matrix-assisted-laser-desorption ionization mass spectrometry.

A chemical procedure for the degradation of peptides and analysis by matrix-assisted-laser-desorption ionization mass spectrometry (MALDI-MS) was used for C-terminal sequence determination. The method allowed us to determine up to eight amino acid residues in the lower picomole range by mass analysis without any repetitions of the degradation nor any extraction or purification of the truncated peptide chains. The C-terminal degradation of all 20 common amino acid residues was proved by applying this method. Extended C-terminal sequence information from enzymatic digests using carboxypeptidase P was obtained by combining the enzymatic with the chemical mass spectrometric approach. Furthermore the amino acids lysine and glutamine, with the same masses, were distinguishable due to the formation of acetyl-lysine in the chemical process.

Amino Acid Sequence↗
PubMed1997

Identification and characterization of heat shock protein 27 protein species in human myocardial two-dimensional electrophoresis patterns.

Immunostaining of heat shock protein 27 (Hsp27) protein species on two-dimensional electrophoresis (2-DE) gels with enhanced sensitivity yields 59 spots reacting with anti-Hsp27 antibodies. Recombinant Hsp27 exists in 2-DE as two major protein species which comigrate in the human myocardial pattern with Hsp27 spots C754 and D899 as defined in the heart high-performance 2-DE database (http://www.mdc-berlin.de/emu/heart/). Preparative electrophoresis of human myocardial proteins and analysis of the enriched mass range 20-30 kDa by 2-DE revealed eight protein spots (C438, C582, C658, C697, C754, C595, C750) from the human myocardial database and a new spot not previously detected on silver-stained gels. These spots were identified as Hsp27 protein species by enzymatic in-gel-digestion and analysis by matrix assisted laser desorption-ionization (MALDI) peptide mass fingerprinting and, in part, MALDI-post source decay sequencing of single fragments. Possible post-translational modifications were investigated: immunostaining tests with anti-phospho-serine/-threonine/-tyrosine antibodies, although positive for other myocardial proteins, were negative for presumed Hsp27 protein species; likewise, periodate-glycostaining assays and biotinylation screening did not detect modifications in the investigated Hsp27 protein species.

Amino Acid Sequence↗
PubMed1993

Crosslinking of components of the cytochrome b6f complex from spinach thylakoids by o-phthalaldehyde.

Chemical modification of a purified spinach cytochrome (cyt) b6f complex was carried out using micromolar concentrations of o-phthalaldehyde (OPA). The o-phthalaldehyde-modified complex exhibited a strong and stable fluorescence characteristic of the thioisoindole derivative. o-Phthalaldehyde also caused crosslinking of the polypeptides and on polyacrylamide gel electrophoresis a new band with a molecular mass of 38-40 kDa exhibiting this characteristic fluorescence was observed as the major crosslinked component; in addition, minor protein bands in the mass range 50-60 kDa appeared. Immunostaining with specific antibodies identified the 38-kDa crosslink as a conjugate of cyt b6 with subunit IV and an additional conjugate of 54 kDa involving cyt f and subunit IV. Crosslinking of the Rieske protein with these components was not observed. In OPA-treated cyt b6f complex, the electron transfer from plastoquinol to cyt f is inhibited as well as proton translocation after incorporation of the complex into liposomes. The EPR spectral analysis of the complex modified at various o-phthalaldehyde concentrations showed that the characteristic EPR signal of the Rieske Fe-S center was lost.

Chloroplasts↗
PubMed1989

Proteinchemical and kinetic features of gramicidin S synthetase.

The amino-acid compositions of both enzymes of gramicidin S synthetase were determined. These proteins contain a high number of acidic amino-acid residues. Phenylalanine racemase, the light enzyme, was sequenced from the N-terminus until position 10. The kinetics of the thioester formation reactions were studied. The half-life times of these processes under substrate saturation conditions were found in the range between seconds and a few minutes. The valine activation at the heavy enzyme was detected as one of the rate-limiting steps of the biosynthesis of gramicidin S.

Amino Acid Isomerases↗
PubMed1988

Studies on the biosynthesis of surfactin, a lipopeptide antibiotic from Bacillus subtilis ATCC 21332.

The biosynthesis of the lipopeptide antibiotic surfactin was studied in whole cells of Bacillus subtilis ATCC 21332 which incorporate 14C-labeled precursor amino acids directly into the product. [14C]Acetate appeared in the fatty acid portion of surfactin and was also partially converted into leucine. An enzyme was isolated and partially purified from a cell-free extract of the bacillus which catalyzes ATP-Pi-exchange reactions which are mediated by the amino acid components of surfactin. This activation pattern is consistent with a peptide synthesizing multienzyme which activates its substrate amino acids simultaneously as reactive aminoacyl phosphates.

Acetates↗
PubMed1988

Evidence for a cytochrome f-Rieske protein subcomplex in the cytochrome b6f system from spinach chloroplasts.

The cytochrome b6f complex of spinach chloroplasts was prepared with minor modification according to the method of E. Hurt and G. Hauska (1981) Eur. J. Biochem. 117, 591-599) replacing, however, the final ultracentrifugation step by hydroxyapatite chromatography as suggested by M. F. Doyle and C.-A Yu (1985) Biochem. Biophys. Res. Commun. 131, 700-706). The purified complex was partially dissociated by treatment with 4 M urea or 0.1% sodium dodecyl sulfate (SDS) in the absence of reducing agents. A binary subcomplex consisting of cytochrome f and the Rieske iron-sulfur protein was observed under these conditions by three different methods: (a) hydroxyapatite chromatography; (b) extraction with an isopropanol/water/trifluoroacetic acid mixture; and (c) gel filtration in the presence of low SDS concentrations. The subcomplex dissociated into its components by treatment with mercaptoethanol. These results suggest a close interaction of the cytochrome f with the Rieske protein involving SH groups which under reducing conditions leads to complete dissociation of the subcomplex.

Chlorophyll↗
PubMed1988

Ribulose-1,5-bisphosphate carboxylase/oxygenase from Zea mays: amino-acid sequence of the small subunit.

The amino-acid sequence of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Zea mays has been determined by alignment of peptides generated by digestion with trypsin, chymotrypsin, staphylococcal protease and thermolysin. The protein-chemically determined structure is in complete agreement with the nucleotide-derived sequence as published recently (Matsuoka et al. (1987) J. Biochem. 102, 673-676), but in addition possesses, however, sixteen experimentally verified dimorphies at various positions and possibly nine more for which evidence is tentatively pointing to two (or more) different expressed nuclear genes for the small subunit. These protein dimorphies represent a protein family corresponding to a gene family, the components of which have not been separated. The closest homologous polypeptide is the small subunit from wheat.

Amino Acid Sequence↗
PubMed1987

Formation of D-Phe-Pro-Val-cyclo-Orn by gramicidin S synthetase in the absence of L-leucine.

The preparation of both enzymes of gramicidin S synthetase was efficiently improved by introduction of the fast protein liquid chromatography technique. High-resolution anion-exchange chromatography on Pharmacia Mono Q HR 5/5 was used as the final purification step. D-Phe-Pro-Val-cyclo-Orn was obtained as a product of the multienzyme by omission of L-leucine from the complete bioassay mixture. This tetrapeptide was formed by cyclization of the C-terminal ornithine to 3-amino-2-piperidone. It was identified and characterized by chromatographic and spectroscopic procedures using chemically synthesized reference compounds.

Amino Acid Isomerases↗
PubMed1985

Chromosomal and plasmid locations for phosphoribulokinase genes in Alcaligenes eutrophus.

Genes coding for phosphoribulokinase (PRK), a key enzyme of the Calvin cycle, were localized in the genome of the chemoautotroph Alcaligenes eutrophus. The NH2-terminal sequence of the PRK subunit was determined. With a synthetic oligodeoxynucleotide probe complementary to a portion of this sequence, hybridization analysis revealed PRK genes to be located on both the chromosome and the megaplasmid pHG1 of A. eutrophus H16.

Alcaligenes↗
PubMed1984

Hydroxyproline-Rich Protein Material in Wood and Lignin of Fagus sylvatica.

The nitrogen content, distribution, and amino acid composition of protein material were determined in wood and lignin of Fagus sylvatica. The data indicated that the nitrogen originated from hydroxyproline-rich cell wall glycoprotein, about half of which may be bound to the lignin polymer. The implications for lignocellulose biodegradation are discussed.

Journal Article↗
PubMed1983

Gramicidin S synthetase. Stability of reactive thioester intermediates and formation of 3-amino-2-piperidone.

The reactive thioester complexes of gramicidin S synthetase with substrate amino acids and intermediate peptides are slowly hydrolyzed in neutral buffer solutions under mild conditions. Fully active enzyme is recovered. These processes are strongly accelerated by certain thiol protective agents. In the presence of 1 mM dithioerythritol the half-life times of these hydrolysis reactions are in the range of 1-90 h at 3 degrees C. The thioester complex of gramicidin S synthetase 2 (GS2, the heavy enzyme) with the tripeptide DPhe-Pro-Val is distinguished by the highest stability of all these intermediates. A different decomposition pattern is observed for the thioester complex of GS2 with LOrn. Here 3-amino-2-piperidone (cyclo-LOrn) is formed in a rapid cyclization reaction. This product specifically blocks the activation center of GS2 for LOrn at the thioester binding site. All other activation reactions of gramicidin S synthetase are unaffected. A procedure for a specific labelling of the reaction centers of the multienzyme is outlined.

Amino Acid Isomerases↗