Metabolism of Citrulline and Ornithine in Mung Bean Mitochondria

D. H. Bone
1959 Plant Physiology  
The occurrence of appreciable quantities of citrulline (31) and ornithine (26) in some higher plants and recently the dliscovery of trace amounts of these amino acids in others, such as barley and wheat (8) has led to speculations about their metabolism. Little information is available on the interconversions of these amino acids in plant tissue. It is known that when ornithine-2-C"4 is administered to barley and white clover plants, the C'4 label appears in citrulline, arginine, proline and
more » ... ine, proline and glutamic acid (3). Further, Krebs and Eggleston (11) have detected citrulline phosphorylase activity in homlogenates of the beans Phaseoluis luitatus and P. vulgaris, and the pea, Pisuml sativumii. Beyond this, conjecture has had to rely mainly on data from work with bacteria and mammalian tissues. It is possible that the mechanism by which citrulline is synthesized in plants is similar to that in extracts of Streptococcus faecalis (7) and rat liver (4., 7) and involves the following reactions: ATP + C02 + ammonia = carbamyl phosphate + ADP carbamyl phosphate + ornithine = citrulline + inorganic phosphate The liver carbamyl phosphate synthetase was found to have a requirement for N-acetylglutamate which was unnecessary for S. faccalis extracts (14) . Reichard (20) purified ornithine carbamyl transferase from liver and found it identical with the citrulline phosphorylase enzyme of Krebs et al (12) . Two feasible pathways that may exist in higher plants for the biosynthesis of ornithine are suggested by studies of the synthesis of ornithine from glutamate in the microorganisms Neurospora crassa and Escherichia coli. In N. crassa, ornithine arises from the transamination of glutamic-y-semialdehyde (28), whereas in E. coli synthesis proceeds via several N-acetylated derivatives (27). It remains possible, of course, that yet another nmechanism occurs in higher plants. The present study is concerned with the enzymatic synthesis of citrulline and ornithine by mung bean (Phaseoluts auireuis) mitochondria and the role of carbamyl phosphate in the conversion of ornithine and citrulline. MATERIALS ANT) METHODS Mung bean seeds were soaked for 3 hours in tap water and then planted in moistened vermiculite con- 'Received September 8, 1958. 171 tained in wooden seed boxes. The boxes were covered with glass plates to keep the humidity maximal. The seedlings were grown in the dark at 310 C for 2 days. Mitochondria were prepared from washed seedlings by the technique of Millerd et al (17) except that the preparative medium used throughout consisted of 0.1 M phosphate buffer (KH2PO4) pH 7.4, 0.4 M sucrose and 0.01 M disodium ethylenediamine tetracetic acid (EDTA). Mitochondrial suspensions were incubated with experimental media at 300 C. Carbon dioxide was measured manometrically in the Warburg apparatus. Ammonia was determined by distillation in Conway units, followed by Nesslerization and measurement of the color produced in a Unicam spectrophotometer, Model SP 500, at 410 m,u. Citrulline was estimated by the modified ninhvdrin reaction of Vogel and Bonner (29). Glutamic acid was determined manometrically using the glutamic acid decarboxylase of Clostridium welchii (10). Glutamic-y-semialdehyde was assayed by the method of Vogel and Davis (30) using the mutant strains of E. coli, 55-25 and 55-1. To isolate citrulline, ornithine and glutamic acid, reaction mixtures were deproteinized with sufficient glacial acetic acidl to give a final concentration of 10 % acid, and then warmed gently for a few minutes. The protein was centrifuged off at 5,000 X G for 5 minutes. Aliquots of the deproteinized solution were banded onto paper strips (Whatman no. 3 MM), 5 cm x 40 cm. These strips were subjected to paper electrophoresis in a Shandon vertical electrophoresis apparatus using 0.1 M potassium bicarbonate as the electrolyte and a potential of 200 volts. A satisfactory separation of ornithine from citrulline and glutamate from aspartate was achieved in 2 hours under these conditions. Following drying at room temperature, the amino acids were located by spraying the edges of the bands with 0.05 M citric acid dissolved in acetone, then overspraying with a ninhydrin solution (0.1 % w/v in 95 % ethanol) and heating at 800 C for 10 minutes. The desired amino acids were each eluted from the paper with 5 ml of distilled water and the eluates evaporated to dryness. To free the amino acids from any radioactive contamiinants, one dimensional paper chromatograms were run using dliisopropyl ether : 90 % formic acid (3 : 2) (11) for ornithine and citrulline, and the organic laver of n-butanol : acetic acid : water (4 : 1 : 5) for glutamic acid. The solvent, 80 % aqueous phenol was also occasionally used for identification purposes. The stimulatoreffect of arsenate on citrulline www.plantphysiol.org on August 23, 2017 -Published by Downloaded from
doi:10.1104/pp.34.2.171 pmid:16655196 fatcat:safon2bme5gddlrssajikb7wce