Purification of Multiple Forms

A method to fractionate com (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 0 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 0 peak contained a band of malate dehydrogenase activity with different mobility. These observations are consistent with three nuclear genes encoding com mitochondrial malate dehydrogenase. Polyclonal antibodies raised against purified malate dehydrogenase were used to identify the gene products using Western blots of two-dimensional gels. including many of the Krebs cycle enzymes. We are developing a purification scheme that will make it possible to separate most of the Krebs cycle enzymes on a single column. We report the purification and characterization of one Krebs cycle enzyme, MDH.2 Mitochondrial malate dehydrogenase has been previously purified from watermelon and the gene sequence obtained (9, 24, 25). However, in corn, genetic studies have indicated that there are three nuclear genes which code for the mitochondrial malate dehydrogenase (10, 11, 17, 18). The gene products and their interaction to produce the functional enzyme have not yet been characterized biochemically, and the genes have not been isolated or characterized. The purification and characterization of the different forms ofMDH provides a starting point for these investigations and a linkage to the genetic information available in the literature (10, 11, 18). MATERIALS AND METHODS All chemicals were obtained from Sigma Chemical Company. Seed corn, Zea mays inbred line B73, was obtained from the Nebraska Seed Foundation. Mitochondrial Isolations There is little information on the overall operation of the Krebs cycle in plant mitochondria, on its regulation, or its role during plant development (26). The information on most of the plant Krebs cycle enzymes is limited to the reports of activities in crude preparations with different enzymes being studied in different plants (3, 26). Little is known about their protein composition or genes. A few have been partially purified, and molecular studies have been carried out on malate dehydrogenase and citrate synthase with sequence data having been obtained (9, 23, 24). It is our intention to look at the Krebs cycle in a single plant species. To achieve this, it is first necessary to undertake structural and functional characterization ofthe enzymes. We have developed a procedure to separate the mitochondrial proteins into physiologically relevant fractions. The soluble protein fraction of our procedure contains matrix proteins, 'This work was supported by the Center for Biotechnology, University of Nebraska-Lincoln. 1381 Mitochondria were isolated from 3-d-old, dark grown corn seedling shoots by the procedure of Day and Hanson (2), modified by using the centrifugation speeds of Schwitzguebel and Siegenthaler (20). The mitochondria were stored frozen at -80°C. Fractionation of Mitochondria Mitochondria were fractionated into membrane and soluble protein fractions. Mitochondria (50 mg) were diluted in Mops buffer (30 mm Mops, pH 8.0) to give a protein concentration of 1 mg/mL and then sonicated until the suspension cleared (about 20 s). The membranes were pelleted by centrifugation for 30 min at 100,000g. The supernatant containing the soluble protein fraction was centrifuged at 200,000g for 3 h to pellet large molecular weight complexes. The resulting supernatant which contained the soluble proteins 2 Abbreviations: MDH, NAD-malate dehydrogenase; FPLC, fast protein liquid chromatography. www.plantphysiol.org on October 31, 2017 Published by Downloaded from Copyright © 1991 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 97, 1991 was then concentrated to 4 mL using a Amicon Diaflo concentrator with a YM10 membrane, and stored at -80'C. Purification of Malate Dehydrogenase The concentrated soluble protein fraction was subject to ion exchange chromatography using a Mono Q column on a Pharmacia FPLC system. The soluble protein fraction (2 mL) was loaded onto a Mono Q column (anion exchange) that had been previously equilibrated with 50 mM NaCl in Mops buffer. The column was washed with 15 mL of this buffer, and then proteins were eluted with a linear gradient of 50 mM to 250 mm NaCl in Mops buffer. The malate dehydrogenase peaks were then made up to 3 M NaCl with solid NaCl and run separately over a Phenyl Superose (hydrophobic interaction) column. The column was washed with 10 mL of 3 M NaCl in Mops buffer. Malate dehydrogenase activities were eluted with a linear gradient of 3 M to 1 M NaCl in Mops buffer. Peaks of malate dehydrogenase activity were concentrated with an Amicon Diaflo concentrator with a YM10 membrane.