Wolf D. Lehmann
University of Giessen
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Featured researches published by Wolf D. Lehmann.
Journal of Biological Chemistry | 1998
Ralf Schneider; Victor Wray; Manfred Nimtz; Wolf D. Lehmann; Ulrike Kirch; Roberto Antolovic; Wilhelm Schoner
In the search for endogenous cardiac glycosides, two different inhibitors of the sodium pump have been isolated from bovine adrenals. Inhibitor A with a molecular mass of 600 Da and a UV maximum at 250 nm was purified from 16 kg of bovine adrenals. The pure substance (<1 ng) inhibited the sodium pump of human red blood cells with an affinity similar to that of ouabain, yet it cross-reacted with antibodies against the bufadienolide proscillaridin A but not against the cardenolide ouabain. Inhibitor A was slightly more hydrophilic than ouabain on RP-C18 high pressure liquid chromatography. Hence, it showed properties similar to the proscillaridin A immunoreactivity (Sich, B., Kirch, U., Tepel, M., Zideck, W., and Schoner, W. (1996) Hypertension 27, 1073–1078) that increased in humans with systolic blood pressure and pulse pressure. Inhibitor B of the sodium pump with a molecular mass of 584 Da was purified 106-fold from 20 kg of bovine adrenals. It cross-reacted with antibodies against ouabain but not with antibodies against proscillaridin A and inhibited the sodium pump of human and rat red blood cells with the same affinity as ouabain. All other properties, such as the retention time in a C18-reversed phase chromatography, molecular mass determination by electrospray mass spectrometry and fragmentation pattern, and UV and 1H NMR spectroscopic data, were identical to ouabain. Hence, sodium pump inhibitor B from bovine adrenals is the cardenolide ouabain.
Journal of Proteome Research | 2009
Nico Zinn; Bettina Hahn; Rüdiger Pipkorn; Dominik Schwarzer; Wolf D. Lehmann
An innovative method for the production of absolutely quantified peptide standards is described. These are named phosphorus-based absolutely quantified standard (PASTA) peptides. As the first step, synthetic phosphopeptides are calibrated via a hybrid LC-(ICP+ESI)-MS system. Quantification is achieved by ICP-MS detection of 31P, and identification is performed by ESI-MS. Generation of phosphopeptide standard solutions with this system is demonstrated to provide absolute concentrations with an accuracy better than 10%. The concept was extended to the production of peptide standards by subjecting a PASTA phosphopeptide to gentle and complete dephosphorylation to obtain the cognate PASTA peptide. It is demonstrated that both enzymatic hydrolysis by alkaline or antarctic phosphatase or chemical hydrolysis by hydrofluoric acid can be employed for this purpose. Further, the introduction of one or more stable isotope-labeled amino acids (preferably labeled by 13C, 15N) results in the production of a labeled PASTA peptide, which then can be employed as an internal standard for quantitative analysis by LC-ESI-MS. Using a 1:1 mixture of a stable isotope-labeled PASTA peptide/phosphopeptide pair as dual standard, a quantification between active and inactive recombinant MAP kinase p38alpha was performed by a combination of tryptic digestion and nanoLC-MS.
Journal of Biological Chemistry | 2006
Stefan Bonn; Saturnino Herrero; Christine B. Breitenlechner; Andrea Erlbruch; Wolf D. Lehmann; Richard A. Engh; Michael Gassel; Dirk Bossemeyer
Controlling aberrant kinase-mediated cellular signaling is a major strategy in cancer therapy; successful protein kinase inhibitors such as Tarceva and Gleevec verify this approach. Specificity of inhibitors for the targeted kinase(s), however, is a crucial factor for therapeutic success. Based on homology modeling, we previously identified four amino acids in the active site of Rho-kinase that likely determine inhibitor specificities observed for Rho-kinase relative to protein kinase A (PKA) (in PKA numbering: T183A, L49I, V123M, and E127D), and a fifth (Q181K) that played a surprising role in PKA-PKB hybrid proteins. We have systematically mutated these residues in PKA to their counterparts in Rho-kinase, individually and in combination. Using four Rho-kinase-specific, one PKA-specific, and one pan-kinase-specific inhibitor, we measured the inhibitor-binding properties of the mutated proteins and identify the roles of individual residues as specificity determinants. Two combined mutant proteins, containing the combination of mutations T183A and L49I, closely mimic Rho-kinase. Kinetic results corroborate the hypothesis that side-chain identities form the major determinants of selectivity. An unexpected result of the analysis is the consistent contribution of the individual mutations by simple factors. Crystal structures of the surrogate kinase inhibitor complexes provide a detailed basis for an understanding of these selectivity determinant residues. The ability to obtain kinetic and structural data from these PKA mutants, combined with their Rho-kinase-like selectivity profiles, make them valuable for use as surrogate kinases for structure-based inhibitor design.
Journal of Biological Chemistry | 2017
Andreas Kulawik; Raphael Engesser; Christian Ehlting; Andreas Raue; Ute Albrecht; Bettina Hahn; Wolf D. Lehmann; Matthias Gaestel; Ursula Klingmüller; Dieter Häussinger; Jens Timmer; Johannes G. Bode
The IL-1β induced activation of the p38MAPK/MAPK-activated protein kinase 2 (MK2) pathway in hepatocytes is important for control of the acute phase response and regulation of liver regeneration. Many aspects of the regulatory relevance of this pathway have been investigated in immune cells in the context of inflammation. However, very little is known about concentration-dependent activation kinetics and signal propagation in hepatocytes and the role of MK2. We established a mathematical model for IL-1β-induced activation of the p38MAPK/MK2 pathway in hepatocytes that was calibrated to quantitative data on time- and IL-1β concentration-dependent phosphorylation of p38MAPK and MK2 in primary mouse hepatocytes. This analysis showed that, in hepatocytes, signal transduction from IL-1β via p38MAPK to MK2 is characterized by strong signal amplification. Quantification of p38MAPK and MK2 revealed that, in hepatocytes, at maximum, 11.3% of p38MAPK molecules and 36.5% of MK2 molecules are activated in response to IL-1β. The mathematical model was experimentally validated by employing phosphatase inhibitors and the p38MAPK inhibitor SB203580. Model simulations predicted an IC50 of 1–1.2 μm for SB203580 in hepatocytes. In silico analyses and experimental validation demonstrated that the kinase activity of p38MAPK determines signal amplitude, whereas phosphatase activity affects both signal amplitude and duration. p38MAPK and MK2 concentrations and responsiveness toward IL-1β were quantitatively compared between hepatocytes and macrophages. In macrophages, the absolute p38MAPK and MK2 concentration was significantly higher. Finally, in line with experimental observations, the mathematical model predicted a significantly higher half-maximal effective concentration for IL-1β-induced pathway activation in macrophages compared with hepatocytes, underscoring the importance of cell type-specific differences in pathway regulation.
Archive | 2003
Andreas Schlosser; Wolf D. Lehmann
The advent of new analyzer systems based on Fourier-transform ion cyclotron resonance (FT-ICR) and quadropole time-of-flight (Q-TOF) technologies allows protein digests to be analyzed under high-resolution conditions, resulting in mass spectometry (MS) and tandem MS (MS/MS) peptide mass spectra characterized by a resolution of 10,000 [full width half-maximum (FWHM) definition] or better. From mass spectra recorded under these conditions, the m/z values of peptides can be measured with mass errors between somewhat below 1 ppm and 50 ppm, depending mainly on the type of instrument used, the sample purity, the signal intensity, and the mode of calibration employed.
Analytical Chemistry | 2001
Andreas Schlosser; Ruediger Pipkorn; Dirk Bossemeyer; Wolf D. Lehmann
Analytical Chemistry | 2006
Ralf Krüger; Dieter Kübler; Roser Pallisse; Andreas Burkovski; Wolf D. Lehmann
Journal of Proteome Research | 2007
Wolf D. Lehmann; Ralf Krüger; Mogjiborahman Salek; Chien-Wen Hung; Florian Wolschin; Wolfram Weckwerth
Analytical Chemistry | 2005
Mathias Wind; Ansgar Wegener; Roland Kellner; Wolf D. Lehmann
Analytical Chemistry | 2003
Mogjiborahman Salek; Angel Alonso; Rüdiger Pipkorn; Wolf D. Lehmann