TY - JOUR A1 - Schenk, Jörg A. A1 - Fettke, Jörg A1 - Lenz, Christine A1 - Albers, Katharina A1 - Mallwitz, Frank A1 - Gajovic-Eichelmann, Nenad A1 - Ehrentreich-Förster, Eva A1 - Kusch, Emely A1 - Sellrie, Frank T1 - Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G JF - Journal of biotechnology N2 - The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem. KW - Hybridoma KW - SLPI KW - Protein G KW - Progesterone KW - Serum-free Y1 - 2012 U6 - https://doi.org/10.1016/j.jbiotec.2011.12.025 SN - 0168-1656 VL - 158 IS - 1-2 SP - 34 EP - 35 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Götz, Klaus-Peter A1 - Naher, Jobadatun A1 - Fettke, Jörg A1 - Chmielewski, Frank M. T1 - Changes of proteins during dormancy and bud development of sweet cherry (Prunus avium L.) JF - Scientia horticulturae : an international journal sponsored by the International Society for Horticultural Science N2 - Trees control the flowering processes in response to both environmental and endogenous (mechanisms at cellular/tissue level) conditions. Dormancy of flower buds is characterized by the reduction of growth and the enhancement of frost and desiccation resistance. The release of endodormancy and the beginning of ontogenetic development, as two important dates for developing reliable phenological models, escape from any visible signs. Thus, we identified - to our knowledge as first - relevant proteins in sweet cherry buds occurring during these phenological phases at high time resolution in three seasons (2012/13–2014/15) under natural conditions in Northeast Germany. The protein content of buds from the first week of October to leaf fall, from leaf fall to the end of endodormancy (t1), from t1 to the beginning of ontogenetic development (t1*), and from t1* until swollen bud, was comparable in each of the seasons. The increase of the protein content began after swollen bud and markedly differences occurred at side green, green tip, tight and open cluster. SDS gel electrophoresis followed by peptide mass fingerprinting accomplished by MALDI-TOF MS was applied for protein identification. ‘Volume intensity’ has been used to demonstrate the pattern and changes of proteins. None of the analysed proteins like for cell proliferation/differentiation (Phytosulfokines 3), carbon fixation (Rubisco), and defense against pathogenes (Major allergen Pru sv 1) indicates the date of endodormancy release or the beginning of the (invisible) ontogenetic development. The stages around green tip, tight, and open cluster resulted in markedly increase of the volume intensity of the protein for cell proliferation/differentiation and the carbon fixation, whereas the volume intensity of a protein for defense against pathogens markedly decreased. The pattern and changes of the volume intensity of neoxanthin synthase (NXS) in sweet cherry buds followed the increasing demand during endo- and ecodormancy to produce neoxanthin, which is a prominent member of the group of reactive oxygen species (ROS) scavengers. KW - Dormancy phases KW - Buds KW - Prunus avium L. KW - Peptide mass fingerprinting Y1 - 2018 U6 - https://doi.org/10.1016/j.scienta.2018.05.016 SN - 0304-4238 SN - 1879-1018 VL - 239 SP - 41 EP - 49 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Brust, Henrike A1 - Orzechowski, Slawomir A1 - Fettke, Jörg T1 - Starch and Glycogen Analyses BT - Methods and Techniques JF - Biomolecules N2 - For complex carbohydrates, such as glycogen and starch, various analytical methods and techniques exist allowing the detailed characterization of these storage carbohydrates. In this article, we give a brief overview of the most frequently used methods, techniques, and results. Furthermore, we give insights in the isolation, purification, and fragmentation of both starch and glycogen. An overview of the different structural levels of the glucans is given and the corresponding analytical techniques are discussed. Moreover, future perspectives of the analytical needs and the challenges of the currently developing scientific questions are included KW - starch KW - glycogen KW - analytics Y1 - 2020 U6 - https://doi.org/10.3390/biom10071020 SN - 2218-273X VL - 10 IS - 7 PB - MDPI CY - Basel ER - TY - JOUR A1 - Staszek, Pawel A1 - Krasuska, Urszula A1 - Otulak-Koziel, Katarzyna A1 - Fettke, Jörg A1 - Gniazdowska, Agnieszka T1 - Canavanine-Induced Decrease in Nitric Oxide Synthesis Alters Activity of Antioxidant System but Does Not Impact S-Nitrosoglutathione Catabolism in Tomato Roots JF - Frontiers in plant science N2 - Canavanine (CAN) is a nonproteinogenic amino acid synthesized in legumes. In mammalians, as arginine analogue, it is an inhibitor of nitric oxide synthase (NOS) activity. The aim of this study was to investigate the impact of CAN-induced nitric oxide level limitation on the antioxidant system and S-nitrosoglutathione (GSNO) metabolism in roots of tomato seedlings. Treatment with CAN (10 or 50 mu M) for 24-72 h led to restriction in root growth. Arginine-dependent NOS-like activity was almost completely inhibited, demonstrating direct effect of CAN action. CAN increased total antioxidant capacity and the level of sulphydryl groups. Catalase (CAT) and superoxide dismutase (SOD) activity decreased in CAN exposed roots. CAN supplementation resulted in the decrease of transcript levels of genes coding CAT (with the exception of CAT1). Genes coding SOD (except MnSOD and CuSOD) were upregulated by CAN short treatment; prolonged exposition to 50-mu M CAN resulted in downregulation of FeSOD, CuSOD, and SODP-2. Activity of glutathione reductase dropped down after short-term (10-mu M CAN) supplementation, while glutathione peroxidase activity was not affected. Transcript levels of glutathione reductase genes declined in response to CAN. Genes coding glutathione peroxidase were upregulated by 50-mu M CAN, while 10-mu M CAN downregulated GSHPx1. Inhibition of NOS-like activity by CAN resulted in lower GSNO accumulation in root tips. Activity of GSNO reductase was decreased by short-term supplementation with CAN. In contrast, GSNO reductase protein abundance was higher, while transcript levels were slightly altered in roots exposed to CAN. This is the first report on identification of differentially nitrated proteins in response to supplementation with nonproteinogenic amino acid. Among nitrated proteins differentially modified by CAN, seed storage proteins (after short-term CAN treatment) and components of the cellular redox system (after prolonged CAN supplementation) were identified. The findings demonstrate that due to inhibition of NOS-like activity, CAN leads to modification in antioxidant system. Limitation in GSNO level is due to lower nitric oxide formation, while GSNO catabolism is less affected. We demonstrated that monodehydroascorbate reductase, activity of which is inhibited in roots of CAN-treated plants, is the protein preferentially modified by tyrosine nitration. KW - canavanine KW - cellular antioxidant system KW - GSNOR-GSNO reductase KW - nitrated proteins KW - nitric oxide-NO KW - nonproteinogenic amino acid KW - NOS-like activity KW - reactive nitrogen species (RNS) Y1 - 2019 U6 - https://doi.org/10.3389/fpls.2019.01077 SN - 1664-462X VL - 10 PB - Frontiers Research Foundation CY - Lausanne ER - TY - THES A1 - Fettke, Jörg T1 - Stärkerelevante cytosolische Heteroglykane: Identifizierung und funftionelle Analyse Y1 - 2006 CY - Potsdam ER - TY - JOUR A1 - Gietler, Marta A1 - Nykiel, Malgorzata A1 - Orzechowski, Slawomir A1 - Fettke, Jörg A1 - Zagdanska, Barbara T1 - Protein carbonylation linked to wheat seedling tolerance to water deficiency JF - Environmental and experimental botany N2 - The appearance of the first leaf from the coleoptile in wheat seedlings (Triticum aestivum L.) coincides with the development of seedling susceptibility to water deficiency on the fifth day following imbibition. In dehydrated wheat seedlings, an increase in the protein carbonyl group has been observed. The coincidence of higher protein carbonylation levels with development of dehydration intolerance drew our attention. To gain more insight into the molecular basis of wheat drought tolerance, the seedling profiles of carbonylated proteins were analysed and compared. Two-dimensional gel electrophoresis (2D-PAGE) and mass spectrometry (MALDI-TOF and LC-MS/MS) were used to indicate and identify differential carbonylated proteins. Among the protein spots with at least a two-fold change in protein abundance in dehydrated seedlings in relation to control (well-watered) plants during the tolerant phase of growth, 19 carbonylated proteins increased and 18 carbonylated proteins decreased in abundance. Among 26 differentially expressed carbonylated proteins in sensitive seedlings, the abundance of 10 protein spots increased while that of 16 proteins decreased upon dehydration. We have demonstrated a link between protein carbonylation and seedling sensitivity to dehydration. The analysis of carbonylated protein profiles clearly showed that proteins with a potential role in the maintenance of dehydration tolerance in wheat seedlings are mainly linked to energy production, anti-fungal and/or insecticidal activity, or to the regulation of both protein synthesis and degradation. KW - Protein carbonylation KW - Dehydration tolerance KW - Triticum aestivum L. KW - Seedlings KW - Proteomic Y1 - 2017 U6 - https://doi.org/10.1016/j.envexpbot.2017.02.004 SN - 0098-8472 SN - 1873-7307 VL - 137 SP - 84 EP - 95 PB - Elsevier CY - Oxford ER - TY - JOUR A1 - Fettke, Jörg A1 - Eckermann, Nora A1 - Tiessen, Axel A1 - Geigenberger, Peter Ludwig A1 - Steup, Martin T1 - Identification, subcellular localization and biochemical characterization of water-soluble heteroglycans (SHG) in leaves of Arabidopsis thaliana L. : distinct SHG reside in the cytosol and in the apoplast N2 - Water-soluble heteroglycans (SHG) were isolated from leaves of wild-type Arabidopsis thaliana L. and from two starch-deficient mutants. Major constituents of the SHG are arabinose, galactose, rhamnose, and glucose. SHG was separated into low (< 10 kDa; SHG(S)) and high (> 10 kDa; SHG(L)) molecular weight compounds. SHG(S) was resolved into approximately 25 distinct oligoglycans by ion exchange chromatography. SHG(L) was further separated into two subfractions, designated as subfraction I and II, by field flow fractionation. For the intracellular localization of the various SHG compounds several approaches were chosen: first, leaf material was subjected to non-aqueous fractionation. The apolar gradient fractions were characterized by monitoring markers and were used as starting material for the SHG isolation. Subfraction I and SHG(S) exhibited a distribution similar to that of cytosolic markers whereas subfraction II cofractionated with crystalline cellulose. Secondly, intact organelles were isolated and used for SHG isolation. Preparations of intact organelles (mitochondria plus peroxisomes) contained no significant amount of any heteroglycan. In isolated intact microsomes a series of oligoglycans was recovered but neither subfraction I nor II. In in vitro assays using glucose 1-phosphate and recombinant cytosolic (Pho 2) phosphorylase both SHG(S) and subfraction I acted as glucosyl acceptor whereas subfraction II was essentially inactive. Rabbit muscle phosphorylase a did not utilize any of the plant glycans indicating a specific Pho 2-glycan interaction. As revealed by in vivo labeling experiments using (CO2)-C-14 carbon fluxes into subfraction I and II differed. Furthermore, in leaves the pool size of subfraction I varied during the light-dark regime Y1 - 2005 SN - 0960-7412 ER - TY - JOUR A1 - Eckermann, Nora A1 - Fettke, Jörg A1 - Steup, Martin T1 - Identification of polysaccharide binding proteins by affinity electrophoresis in inhomogeneous polyacrylamide gels and subsequent SDS-PAGE/MALDI-TOF analysis Y1 - 2002 ER - TY - JOUR A1 - Martins, Marina Camara Mattos A1 - Hejazi, Mahdi A1 - Fettke, Jörg A1 - Steup, Martin A1 - Feil, Regina A1 - Krause, Ursula A1 - Arrivault, Stephanie A1 - Vosloh, Daniel A1 - Figueroa, Carlos Maria A1 - Ivakov, Alexander A1 - Yadav, Umesh Prasad A1 - Piques, Maria A1 - Metzner, Daniela A1 - Stitt, Mark A1 - Lunn, John Edward T1 - Feedback inhibition of starch degradation in arabidopsis leaves mediated by trehalose 6-phosphate JF - Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants N2 - Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2- to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by beta-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 mu M in the cytosol, 0.2 to 0.5 mu M in the chloroplasts, and 0.05 mu M in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night. Y1 - 2013 U6 - https://doi.org/10.1104/pp.113.226787 SN - 0032-0889 SN - 1532-2548 VL - 163 IS - 3 SP - 1142 EP - 1163 PB - American Society of Plant Physiologists CY - Rockville ER - TY - JOUR A1 - Hejazi, Mahdi A1 - Fettke, Jörg A1 - Koetting, Oliver A1 - Zeeman, Samuel C. A1 - Steup, Martin T1 - The Laforin-like dual-specificity phosphatase SEX4 from Arabidopsis hydrolyzes both C6-and C3-phosphate esters introduced by starch-related dikinases and thereby affects phase transition of alpha-glucans N2 - The biochemical function of the Laforin-like dual-specific phosphatase AtSEX4 (EC 3.1.3.48) has been studied. Crystalline maltodextrins representing the A- or the B-type allomorph were prephosphorylated using recombinant glucan, water dikinase (StGWD) or the successive action of both plastidial dikinases (StGWD and AtPWD). AtSEX4 hydrolyzed carbon 6-phosphate esters from both the prephosphorylated A- and B-type allomorphs and the kinetic constants are similar. The phosphatase also acted on prelabeled carbon-3 esters from both crystalline maltodextrins. Similarly, native starch granules prelabeled in either the carbon-6 or carbon-3 position were also dephosphorylated by AtSEX4. The phosphatase did also hydrolyze phosphate esters of both prephosphorylated maltodextrins when the (phospho)glucans had been solubilized by heat treatment. Submillimolar concentrations of nonphosphorylated maltodextrins inhibited AtSEX4 provided they possessed a minimum of length and had been solubilized. As opposed to the soluble phosphomaltodextrins, the AtSEX4- mediated dephosphorylation of the insoluble substrates was incomplete and at least 50% of the phosphate esters were retained in the pelletable (phospho) glucans. The partial dephosphorylation of the insoluble glucans also strongly reduced the release of nonphosphorylated chains into solution. Presumably, this effect reflects fast structural changes that following dephosphorylation occur near the surface of the maltodextrin particles. A model is proposed defining distinct stages within the phosphorylation/dephosphorylation-dependent transition of alpha-glucans from the insoluble to the soluble state. Y1 - 2010 UR - http://www.plantphysiol.org/ U6 - https://doi.org/10.1104/pp.109.149914 SN - 0032-0889 ER -