Oxidation of Proline by Plant Mitochondria '

Mitochondria isolated from etiolated shoots of com ( Zen mays), wheat (Tddk aestum, baley (HM "*are), soybean ( Glychie max L. Merr.), and mung bean ( Phasebhis awueus) exhibited a prol-pndent 02 uptake subject to respiratory controL ADP/O ratios with proline as substrate were intermediate between ratios obtained with exogenous NADH and malate + pyruvate as substrates. Isotope studies showed proline metabolism to be dependent on 02, but not NAD. The major ninhydrin-positive product formed via A1-pyrroline-5-carboxylic acid was glutamate. Mitochondria were capable of further metabolism of glutamate, as radioactive CO2, organic acids, and aspartate were recovered after 114CIprolne feeding expeiments. These results demonstrate the mitochondrial association and 02 dependence of plant proline metabolism. In mammals, insects, yeasts, and bacteria, the metabolism of proline is begun by conversion to P5C2 (5, 7, 8) or in some cases P2C (2, 8). This conversion is typically mitochondrial, and is catalyzed by proline oxidase, an 02-dependent flavoprotein (5, 7, 8, 21). Activity of this enzyme apparently has not been demonstrated in higher plants. Instead, several workers have reported the presence of proline dehydrogenase, a nonparticulate, NADlinked enzyme which has been suggested, but not proved, to catalyze P5C formation in vivo (10-12, 19). Doubt that proline dehydrogenase is the in vivo catalyst for proline oxidation has arisen from the necessity to assay it at high pH (above pH 10, refs. 10 and 12) and from the observation that it co-purifies with P5C reductase (10), the NADH-linked proline biosynthetic enzyme that is typically stable and present in relatively high activity in a variety of tissues (6, 10, 12, 16, 17). These considerations led us to look for proline oxidation by mitochondria isolated from higher plants. MATERIALS AND METHODS Plant Material. Mitochondria were isolated from 3-day etiolated shoots of corn (Zea mays cv. WF9[NJ x B37) and wheat ( Triticum aestivum cv. Abe), from 4-day etiolated shoots of barley (Hordeum vulgare cv. Prior) and from 4-day etiolated hypocotyls of soybean (Glycine max L. Merr. cv. Amsoy 71) and mung bean (Phaseolus aureus). Corn seedlings were grown at 30 C on moist paper towels saturated with 0.1 mM CaCl2. The other seedlings were grown in moist Vermiculite at room temperature (24-28 C). Mitochondrial Preparation. The procedure was the same as previously described for corn mitochondria (13). Briefly, shoots ' This work was supported by the Illinois Agricultural Experiment Station and the Iowa State University Graduate College. 2 Abbreviations: P5C: A'-pyrroline-5-carboxylic acid; P2C: A'-pyrroline-2-carboxylic acid; OAB: o-aminobenzaldehyde. were clipped, ground in a mortar and pestle, and the homogenate filtered through cheesecloth. After a low speed centrifugation to remove debris, mitochondria were peileted (28,000g, 6 min), resuspended in 0.4 M sucrose, and the suspension clarified by centrifugation at low speed. Mitochondria were then centrifuged through a 0.6 M sucrose cushion (17,500g, 18 min) with final suspension in 0.4 M sucrose. Mitochondrial protein was measured by the Lowry procedure (9). 02 Uptake Measurements. 02 concentration was monitored with a Clark 02 electrode (Yellow Springs Instrument Co.) in a stirred, temperature-controled (27 ± 0.2 C) 4-ml reaction volume. The reaction vial was fitted into the light path of a Bausch & Lomb spectronic 70 spectrophotometer, so that 02 concentration and percentage of light transmittance through the reaction suspension (520 nm) could be simultaneously recorded. The reaction medium contained 0.2 M sucrose, I mg/ml BSA, 1 mM MgSO4, 4 mM K-phosphate, and 10 mm TES (pH 7.6). Radioisotope Experiments. Radiochemicals were obtained from Amersham/Searle. L-5T-Proline was purified, dried, and redissolved before use to remove tritiated water. ['4CJProline was purified by passing through a Dowex 1-acetate column. Glutamic acid was identified by its retention on Dowex 1-acetate, elution with 0.5 M acetic acid, and subsequent TLC (4) with standards. The method of Mitra et al. (14) was used to assay the appearance of tritium in water after incubation of mitochondria with tritiated proline. When assay of "CO2 was desired, reactions were set up in vials inside a stoppered 500-ml Erlenmeyer flask containing a scintillation vial holding 1 ml of ethanolamine. The reaction was terminated by adding 0.1 ml of I N HCI to the reaction mixture. The 500-ml flask was then shaken for 1 hr to allow equilibration of CO2. The "4C02-containing ethanolamine was counted by liquid scintillation. When further fractionation of the reaction medium was required, it was diluted 5-fold and added to a Dowex 50-H+ column (4 x 0.7 cm), allowing the column effluent to pass through Dowex l-formate as described by Wang (22). The formate column was eluted with 4 N formic acid to give an organic acid fraction. The Dowex 50-H+ column was eluted with 2 N NH40H to give the amino acid fraction. After evaporating to remove ammonia, this fraction was added to a column of Dowex I-acetate. The neutral amino acid fraction (containing proline) passed through this column, while the acidic amino acid fraction (containing glutamate) was eluted from the column with 0.5 N acetic acid. Two-dimensional chromatography and autoradiography of amino acids was on thin layers of cellulose as described previously (4). Two methods were used to isolate P5C. We have observed that P5C adheres to Dowex I-acetate at pH 7.5, and can be eluted with 0.1 N acetic acid. Thus, reaction mixtures were added to Dowex 1-acetate columns, which had been washed with water to remove proline, then with 0.1 N acetic acid to elute P5C. After evaporation, the P5C-containing eluate was chromatographed in l-butanolacetic acid-water (12/3/5, v/v/v) along with standard P5C. In this 22 www.plantphysiol.org on October 15, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. PROLINE AND PLANT MITOCHONDRIA solvent P5C migrates ahead of glutamate, aspartate, P2C, and organic acids, and is recognized by its pink ninhydrin reaction. A modification of the assay of Phang et al. (18) was also used to identify P5C. This reaction was stopped by adding 2 ml of OAB (0.3%) in acidic ethanol (12 N HCI-95% ethanol, 1/7, v/v). This mixture was added to a Dowex 50-H+ column (4 x 0.7 cm) which was washed free of radioactivity with 2 N HC1. The column was then washed with water to remove excess HCI, and the P5C-OAB complex was eluted with 2 N NH40H. The eluate was evaporated and chromatographed in 1-butanol-acetic acid-water (12/3/5, v/v/v). The yellow complex was observed on the chromatogram. Proline Dehydrogenase Assay. The assay cuvettes contained 200 mm carbonate buffer (pH 10.5), 0.67 mm NAD, 10 mM Lproline, and 0.1 ml of resuspended mitochondrial pellet or 0.3 ml of supernatant. Total volume was 3 ml. Activity was measured as an increase in A at 340 nm.