Plasma membrane-associated malate dehydrogenase of maize (Zea mays L.) roots: Native versus recombinant protein

• Published in: Journal of proteomics Vol. 80; pp. 66 - 77
• Main Authors: Menckhoff, Ljiljana, Mielke-Ehret, Nicole, Buck, Friedrich, Vuletić, Mirjana, Lüthje, Sabine
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 27.03.2013
• Subjects: Amino Acid Sequence; Brassica - enzymology; Brassica oleracea; cauliflower; Cell Membrane - enzymology; corn; disulfide bonds; Disulfides - chemistry; enzyme kinetics; Escherichia coli; Escherichia coli - enzymology; Ethylmaleimide - chemistry; gene expression; Gene expression profile; Gene Expression Profiling; genes; inflorescences; isozymes; leaves; Malate dehydrogenase; Malate Dehydrogenase - chemistry; mass spectrometry; Molecular Sequence Data; nutrient uptake; Oxidative Stress; plant development; Plant Proteins - chemistry; Plant Roots - enzymology; Plasma membrane; polyacrylamide gel electrophoresis; Protein Interaction Mapping; Protein Processing, Post-Translational; Proteomics - methods; Recombinant MDH; recombinant proteins; Recombinant Proteins - chemistry; reverse transcriptase polymerase chain reaction; roots; Sequence Homology, Amino Acid; Spectrometry, Mass, Electrospray Ionization; spinach; Spinacia oleracea; Spinacia oleracea - enzymology; Tandem Mass Spectrometry; Thiol-groups; thiols; Zea mays; Zea mays - enzymology; MALDI-TOF-MS; ESI-QTOF-MS/MS; Zea mays; HRP; Malate dehydrogenase; IEF; PVDF; Gene expression profile; MDH; Recombinant MDH; ICM; NEM; Plasma membrane; Thiol-groups; GAPDH; PM; mPEG-Mal
• ISSN: 1874-3919; 1876-7737
• DOI: 10.1016/j.jprot.2012.12.015

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 41kDa for membrane-associated MDH, whereas monomers (35kDa) and dimers (70kDa) 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. [Display omitted] ► MDH was purified from the PM of distinct plant species, developmental states and tissues. ► Full length amino acid sequence was identified for ZmMDH by mass spectrometry. ► PTM, structure and orthologs of ZmMDH were predicted by in silico analysis. ► Enzyme kinetics of native and recombinant proteins were compared. ► Function of SH-groups and putative disulphide bonds was investigated.