Mitochondrial malate dehydrogenase from corn. Purification of multiple forms

A method to fractionate corn (Zea mays L. B73) mitochondria into soluble proteins, high molecular weight soluble proteins, and membrane proteins was developed. These fractions were analyzed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assays of mitochondrial enzyme activitie...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 1991-12, Vol.97 (4), p.1381-1387
Main Authors: Hayes, M.K. (University of Nebraska-Lincoln, Lincoln, NE), Luethy, M.H, Elthon, T.E
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
Biological and medical sciences
Cell structures and functions
Citrates
COMPOSICION QUIMICA
COMPOSITION CHIMIQUE
Corn
Dehydrogenases
Enzyme activity
Enzymes
Fundamental and applied biological sciences. Psychology
Gels
isoenzymes
isolation
malate dehydrogenase
MALATE DESHYDROGENASE
MALATO DESHIDROGENASA
Metabolism
Mitochondria
Mitochondria and cell respiration
MITOCHONDRIE
MITOCONDRIA
Molecular and cellular biology
Mops
Phenyls
Plant physiology and development
PROTEINAS
PROTEINE
PURIFICACION
PURIFICATION
TECHNIQUE ANALYTIQUE
TECNICAS ANALITICAS
Tricarboxylic acid cycle
ZEA MAYS
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Summary:A method to fractionate corn (Zea mays L. B73) mitochondria into soluble proteins, high molecular weight soluble proteins, and membrane proteins was developed. These fractions were analyzed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assays of mitochondrial enzyme activities. The Krebs cycle enzymes were enriched in the soluble fraction. Malate dehydrogenase has been purified from the soluble fraction by a two-step fast protein liquid chromatography method. Six different malate dehydrogenase peaks were obtained from the Mono Q column. These peaks were individually purified using a Phenyl Superose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks showed that three of the isoenzymes consisted of different homodimers (I, III, VI) and three were different heterodimers (II, IV, V). Apparent molecular masses of the three different monomer subunits were 37, 38, and 39 kilodaltons. Nondenaturing gel analysis of the malate dehydrogenase peaks showed that each Mono Q peak contained a band of malate dehydrogenase activity with different mobility. These observations are consistent with three nuclear genes encoding corn mitochondrial malate dehydrogenase. Polyclonal antibodies raised against purified malate dehydrogenase were used to identify the gene products using Western blots of two-dimensional gels
ISSN:0032-0889
1532-2548
DOI:10.1104/pp.97.4.1381