On the natureof the carbamyl group donor in citrulline biosynthesis

Marshall, Richard O.; Hall, Leo M.; Cohen, Philip P.

doi:10.1016/0006-3002(55)90365-6

Cited by 35 publications

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“…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).…”

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Metabolism of Citrulline and Ornithine in Mung Bean Mitochondria.

Bone

1959

Plant Physiol.

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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 glutamic acid (3). Further, Krebs and Eggleston (11) (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...

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