title: Plasma membrane-associated malate dehydrogenase of maize (Zea mays L.) roots: native versus recombinant protein.
abstract: Malate dehydrogenase (MDH, EC 1.1.1.37) is involved in several cellular processes including plant development, nutrient uptake and oxidative stress. Evidence for a plasma membrane-associated MDH has been presented for maize (Zea mays L.) roots. In the present study isoenzymes of MDH were purified from highly enriched plasma membrane preparations of maize and compared with soluble isoenzymes (Km, pH optima, pI and molecular masses). Modified SDS-PAGE analyses revealed monomers of 41 kDa for membrane-associated MDH, whereas monomers (35 kDa) and dimers (70 kDa) were detected for soluble isoenzymes. Membrane-associated MDH of cauliflower (Brassica oleracea L.) inflorescences and spinach (Spinacia oleracea L.) leaves showed molecular masses similar to the membrane-associated MDH of maize. The specific maize MDH involved was identified by mass spectrometry (ESI-QTOF-MS/MS, MALDI-TOF-MS). The corresponding gene was cloned and the protein was characterised after heterologous expression in Escherichia coli. Enzyme kinetics and properties of the recombinant and native proteins were compared. The function of thiol groups and the presence of disulphide bonds were analysed by the effect of N-ethylmaleimide, diagonal electrophoresis and labelling. Semiquantitative reverse transcription polymerase chain reaction of maize root transcripts demonstrated a constitutive expression of the gene encoding plasma membrane-associated MDH.
authors: University of Hamburg, Biocentre Klein Flottbek and Botanical Garden, Plant Physiology, Ohnhorststraße 18, D-22609 Hamburg, Germany.
authors: Ljiljana
authors: L
authors: Menckhoff
authors: University of Hamburg, Biocentre Klein Flottbek and Botanical Garden, Plant Physiology, Ohnhorststraße 18, D-22609 Hamburg, Germany.
authors: Nicole
authors: N
authors: Mielke-Ehret
authors: University Hospital Eppendorf, Institute of Clinical Chemistry, Campus Science, Martinistrasse 52, D-20246 Hamburg, Germany.
authors: Friedrich
authors: F
authors: Buck
authors: Maize Research Institute, Zemun Polje, Slobodana Bajića 1, 11185 Belgrade, Serbia.
authors: Mirjana
authors: M
authors: Vuletić
authors: University of Hamburg, Biocentre Klein Flottbek and Botanical Garden, Plant Physiology, Ohnhorststraße 18, D-22609 Hamburg, Germany. Electronic address: sabine.luethje@uni-hamburg.de.
authors: Sabine
authors: S
authors: Lüthje
mesh_terms: D000595:Amino Acid Sequence; D001937:Brassica / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology*; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005033:Ethylmaleimide / Q000737:chemistry; D020869:Gene Expression Profiling; D008291:Malate Dehydrogenase / Q000737:chemistry*; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010940:Plant Proteins / Q000737:chemistry; D018517:Plant Roots / Q000201:enzymology; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D018724:Spinacia oleracea / Q000201:enzymology; D053719:Tandem Mass Spectrometry; D003313:Zea mays / Q000201:enzymology*
chemical_list: D004220:Disulfides; D010940:Plant Proteins; D011994:Recombinant Proteins; D008291:Malate Dehydrogenase; D005033:Ethylmaleimide