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

TY - JOUR

T1 - Plasma membrane-associated malate dehydrogenase of maize (Zea mays L.) roots: Native versus recombinant protein

AU - Menckhoff, Ljiljana

AU - Mielke-Ehret, Nicole

AU - Buck, Friedrich

AU - Vuletić, Mirjana

AU - Lüthje, Sabine

N1 - Copyright © 2013 Elsevier B.V. All rights reserved.

PY - 2013/1/10

Y1 - 2013/1/10

N2 - 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.

AB - 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.

U2 - 10.1016/j.jprot.2012.12.015

DO - 10.1016/j.jprot.2012.12.015

M3 - SCORING: Journal article

C2 - 23313174

VL - 80C

SP - 66

EP - 77

JO - J PROTEOMICS

JF - J PROTEOMICS

SN - 1874-3919

ER -