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Marc Baumann

Publications and source records attributed to Marc Baumann.

9 recordsLinked to original sources
PubMed2007

Toward an integrated microchip sized 2-D polyacrylamide slab gel electrophoresis device for proteomic analysis.

We describe a miniaturized instrument capable of performing 2-DE. Our miniaturized device is able to perform IEF and polyacrylamide slab gel electrophoresis (PASGE) in the same unit. It consists of a compartment for a first-dimensional IEF gel, which is connected to a second-dimensional PASGE gel. The focused samples are automatically transferred from the IEF gel to the PASGE gel by electromigration. Our preliminary experiments show that the device is able to focus and separate a mixture of proteins in approximately 1 h, excluding the time required for the staining procedure. On average, the gel-to-gel retardation factor (Rf) variation was 6.2% (+/-0.9%) and pI variation was 2.5% (+/-0.6%). Separated protein spots were excised from stained gels, digested with trypsin, and further identified by MS, thus enabling direct proteomic analysis of the separated proteins.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

Identification of a histidine-tyrosine cross-link in the active site of the cbb3-type cytochrome c oxidase from Rhodobacter sphaeroides.

The heme-copper oxidases constitute a superfamily of terminal dioxygen-reducing enzymes located in the inner mitochondrial or in the bacterial cell membrane. The presence of a mechanistically important covalent bond between a histidine ligand of the copper ion (Cu(B)) in the active site and a generally conserved tyrosine residue nearby has been shown to exist in the canonical cytochrome c oxidases. However, according to sequence alignment studies, this critical tyrosine is missing from the subfamily of cbb(3)-type oxidases found in certain bacteria. Recently, homology modeling has suggested that a tyrosine residue located in a different helix might fulfill this role in these enzymes. Here, we show directly by methods of protein chemistry and mass spectrometry that there is indeed a covalent link between this tyrosine and the copper-ligating histidine. The identity of the cross-linked tyrosine was determined by showing that the cross-link is not formed when this residue is replaced by phenylalanine, even though structural integrity is maintained. These results suggest a universal functional importance of the histidine-tyrosine cross-link in the mechanism of O(2) reduction by all heme-copper oxidases.

Amino Acid Sequence↗
PubMed2004

Techniques for studying protein heterogeneity and post-translational modifications.

Proteins often undergo several post-translational modification steps in parallel to protein folding. These modifications can be transient or of a more permanent nature. Most modifications are, however, susceptible to alteration during the lifespan of proteins. Post-translational modifications thus generate variability in proteins that are far beyond that provided by the genetic code. Co- and post-translational modifications can convert the 20 specific codon-encoded amino acids into more than 100 variant amino acids with new properties. These, and a number of other modifications, can considerably increase the information content and functional repertoire of proteins, thus making their analysis of paramount importance for diagnostic and basic research purposes. Various methods used in proteomics, such as 2D gel electrophoresis, 2D liquid chromatography, mass spectrometry, affinity-based analytical methods, interaction analyses, ligand blotting techniques, protein crystallography and structure-function predictions, are all applicable for the analysis of these numerous secondary modifications. In this review, examples of some of these techniques in studying the heterogeneity of proteins are highlighted. In the future, these methods will become increasingly useful in biomarker searches and in clinical diagnostics.

Humans↗
PubMed2002

Expression and characterization of recombinant human UDP-glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than other UGTs and was purified as an active dimeric enzyme.

Eight human liver UDP-glucuronosyltransferases (UGTs) were expressed in baculovirus-infected insect cells as fusion proteins carrying a short C-terminal extension that ends with 6 histidine residues (His tag). The activity of recombinant UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B15 was almost fully inhibited by 0.2% Triton X-100. In the case of UGT1A9, however, glucuronidation of alpha-naphthol and scopoletin was resistant to such inhibition, whereas glucuronidation of entacapone and several other aglycones was sensitive. His-tagged UGT1A9 was purified by immobilized metal-chelating chromatography (IMAC). Purified UGT1A9 glucuronidated scopoletin at a high rate, whereas its glucuronidation activity toward entacapone was low and largely dependent on phospholipid addition. Recombinant UGT1A9 in which the His tag was replaced by hemagglutinin antigenic peptide (HA tag) was also prepared. Insect cells were co-infected with baculoviruses encoding both HA-tagged and His-tagged UGT1A9. Membranes from the co-infected cells, or a mixture of membranes from separately infected cells, were subjected to detergent extraction and IMAC, and the resulting fractions were analyzed for the presence of each type of UGT1A9 using tag-specific antibodies. In the case of separate infection, the HA-tagged UGT1A9 did not bind to the column. When co-infected with His-tagged UGT1A9, however, part of the HA-tagged enzyme was bound to the column and was eluted by imidazole concentration gradient together with the His-tagged UGT1A9, suggesting the formation of stable dimers that contain one His-tagged and one HA-tagged UGT1A9 monomers.

Amino Acid Sequence↗
PubMed2002

Interaction of purified NDH-1 from Escherichia coli with ubiquinone analogues.

The NADH:ubiquinone oxidoreductase (NDH-1 or Complex I) of Escherichia coli is a smaller version of the mitochondrial enzyme, being composed of 13 protein subunits in comparison to the 43 of bovine heart complex I. The bacterial NDH-1 from an NDH-2-deficient strain was purified using a combination of anion exchange chromatography and sucrose gradient centrifugation. All 13 different subunits were detected in the purified enzyme by either N-terminal sequencing or matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis. In addition, some minor contaminants were observed and identified. The activity of the enzyme was studied and the effects of phospholipid and dodecyl maltoside were characterized. Kinetic analyses were performed for the enzyme in the native membrane as well as for the purified NDH-1, using ubiquinone-1, ubiquinone-2 or decylubiquinone as the electron acceptors. The purified enzyme exhibited between 1.5- and 4-fold increase in the apparent K(m) for these acceptors. Both ubiquinone-2 and decylubiquinone are good acceptors for this enzyme, while affinity of NDH-1 for ubiquinone-1 is clearly lower than for the other two, particularly in the purified state.

Bacterial Proteins↗
PubMed2002

Activated mast cells increase the level of endothelin-1 mRNA in cocultured endothelial cells and degrade the secreted Peptide.

Subendothelial mast cells have been implicated in the pathogenesis of allergic inflammation, in atherosclerosis, and in the regulation of vascular tone. Because endothelin-1 (ET-1) is an important regulator of vascular tone and has also been implicated in the pathogenesis of atherosclerosis, we studied the role of mast cells in the metabolism of endothelial cell-derived ET-1. In mast cell-endothelial cell cocultures, activation of the mast cells with ensuing degranulation was accompanied by the increased expression of ET-1 mRNA in the endothelial cells, yet the immunoreactive ET-1 protein in the coculture medium disappeared almost completely during the 24-hour coculture. Activation of the mast cells with the ensuing degranulation resulted in proteolytic degradation of ET-1 by the 2 neutral proteases, chymase and carboxypeptidase A, of the exocytosed mast cell granules. With synthetic ET-1 and purified mast cell granule enzymes, efficient degradation of ET-1 by chymase and carboxypeptidase A was verified. These in vitro results imply a novel role for mast cell-derived neutral proteases in ET-1 metabolism and suggest that activated subendothelial mast cells are important local regulators of ET-1 metabolism.

Animals↗
PubMed2002

Phosphorylation of a peptide related to subunit c of the F0F1-ATPase/ATP synthase and relationship to permeability transition pore opening in mitochondria.

A phosphorylated polypeptide (ScIRP) from the inner membrane of rat liver mitochondria with an apparent molecular mass of 3.5 kDa was found to be immunoreactive with specific antibodies against subunit c of F0F1-ATPase/ATP synthase (Azarashvily, T. S., Tyynelä, J., Baumann, M., Evtodienko, Yu. V., and Saris, N.-E. L. (2000). Biochem. Biophys. Res. Commun. 270, 741-744. In the present paper we show that the dephosphorylation of ScIRP was promoted by the Ca2+-induced mitochondrial permeability transition (MPT) and prevented by cyclosporin A. Preincubation of ScIRP isolated in its dephosphorylated form with the mitochondrial suspension decreased the membrane potential (delta psiM) and the Ca2+-uptake capacity by promoting MPT. Incorporation of ScIRP into black-lipid membranes increased the membrane conductivity by inducing channel formation that was also suppressed by antibodies to subunit c. These data indicate that the phosphorylation level of ScIRP is influenced by the MPT pore state, presumably by stimulation of calcineurin phosphatase by the Ca2+ used to induce MPT. The possibility of ScIRP being part of the MPT pore assembly is discussed in view of its capability to induced channel activity.

Animals↗
PubMed2002

Peptides corresponding to gelsolin derived amyloid of the finnish type (AGelFIN) adopt two distinct forms in solution of which only one can polymerize into amyloid fibrils and form complexes with apoE.

Familial amyloidosis of the Finnish type is caused by a point mutation at position 187 of gelsolin. The mutation generates a proteolytic fragment, which forms amyloid fibrils in vivo. In the present study we have elucidated the aggregation properties of synthetic peptides corresponding to the wild type and mutated gelsolin fragments. We show by high performance size exclusion chromatography and mass spectrometry that a part of these peptides polymerize with an increase in incubation time, while a portion of the same peptide can adopt a soluble, non-polymerizing structure. Peptides that remain soluble are in their monomeric stage, while the polymerizing peptides prefer a dimerized intermediate stage. We also show that the initialform of these peptides in the solution dramatically influence their capability to interact with amlyloid associated proteins, such as apolipoprotein E. Our results indicate that the amyloidogenic fragment of gelsolin can adopt two distinct forms, of which only one can form amyloid fibrils in vitro.

Amyloid↗