Metabolism of Citrulline and Ornithine in Mung Bean Mitochondria.

Bone, Derek H.

doi:10.1104/pp.34.2.171

Cited by 34 publications

(11 citation statements)

References 18 publications

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“…The difference between the heat-treated control and the active enzyme reaction mixture was used as a measure of the enzymatic conversion. Step (19), and mung bean seedlings (2). DOrnithine did give a significant activity compared to L-orMithine, but this could be attributed to the possibility that there was a trace of L-ornithine in the commercial D-ornithine preparation, or that some racemase activity might exist in the enzyme preparation.…”

Section: Resultsmentioning

confidence: 95%

l-Ornithine:2-Oxoacid Aminotransferase from Squash (Cucurbita pepo, L.) Cotyledons

Lu¹,

Mazelis²

1975

Plant Physiol.

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Ornithine :2-oxoacid aminotransferase (EC 2.6.1.13) has been purified over 400-fold with a total recovery of 14% from acetone powders of cotyledons of germinating squash (Cucurbita pepo, L.) seedlings. The pH optimum of the transamination between L-ornithine and a-ketoglutarate is 8 and the Michaelis constants are 4.7 mM and 6.3 mM, respectively. The enzyme has a molecular weight of 48,000 as determined by gel filtration. The reaction is essentially specific for a-ketoglutarate as the amino group acceptor. The enzyme is inhibited very strongly by hydroxylamine, and less severely by NaCN and isonicotinylhydrazide. No inhibition is observed in the presence of 10 mM L-cysteine. The energy of activation is 7.6 kcal/mole. The stability of the enzyme preparation is enhanced by the presence of dithioerythritol and glycerol. The enzyme activity of the most purified fraction is stimulated 30% by the addition of pyridoxal phosphate; however, the evidence for the unequivocal involvement of pyridoxal phosphate was inconclusive.Proline, glutamic acid, and arginine are readily interconvertible in vivo (11). Since the reactions concerned involved either ornithine or its transamination product glutamic-y-semialdehyde, it is likely that the L-ornithine: 2-oxoacid aminotransferase (EC 2.6.1.13) plays a key role in the necessary metabolic processes.

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Section: Resultsmentioning

confidence: 95%

l-Ornithine:2-Oxoacid Aminotransferase from Squash (Cucurbita pepo, L.) Cotyledons

Lu¹,

Mazelis²

1975

Plant Physiol.

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“…Several characteristics of CPS from higher plants have been described in papers on the enzyme itself (6,7,(20)(21)(22)(23) or as part of studies on the citrulline (1), arginine (26), or pyrimidine metabolism (4,5,11,14).…”

Section: Discussionmentioning

confidence: 99%

Carbamoyl Phosphate Synthetase Activity from the Cotyledons of Developing and Germinating Pea Seeds

Kollöffel

Verkerk²

1982

Plant Physiol.

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Carbamoyl phosphate synthetase activity was measured in partially purified extracts from cotyledons of developing and germinating seeds of Piswm sativum L. Some properties of the enzyme were established. During cotyledon development, the activity initially increased sharply but de-creased during further development. The activity from germinating seeds was only one-tenth of the maximum activity at an early developmental phase. The results are discussed in relation to pea seed development and germination.Carbamoyl phosphate is utilized in the first step of two biosynthetic pathways, one leading to arginine and the other to pyrimidines. In many bacteria, a single enzyme catalyzes the synthesis of CAP' for both pathways, whereas, in most animals and fungi, there are two CPSs (13, 25), each being pathway-specific. In higher plant tissues, there appears to be only one CPS supplying CAP to both pathways (22, 23).The plant enzyme, as well as the enzyme from other sources, is a regulatory enzyme influenced by a variety of effectors, such that its activity in vivo can be strictly controlled (6, 20-23 Utrecht, The Netherlands they were allowed to germinate further in darkness at 23°C for the appropriate time. The period between the beginning ofsoaking and the moment the cotyledons were used for the experiments is referred to as 'days of germination.' Developing seeds and the seeds which were allowed to germinate were, thus, harvested from the same batches of plants.Crude Extract. All isolation and purification operations were performed at 0 to 4°C. The cotyledons (40 to 60 cotyledons, corresponding with a fresh weight of 2 to 8 g, depending on the developmental phase of the seeds) were ground with a pestle in a prechilled mortar with 5 to 8 g of sand and about 10 ml of a medium containing 400 mm mannitol, 50 mm N-morpholinopropanesulfonic acid buffer (pH 7.4), and 2.5 mg/ml BSA. The homogenate was squeezed through a Perlon screen (mesh width, 45 um), diluted to about 20 ml with grinding medium, and centrifuged at 2,500g for 5 min. The supernatant was recentrifuged at 40,000g for 10 min. The resulting pellet was washed twice and suspended in the grinding medium. This suspension was used to determine the particle-bound CPS activity. The 40,000g supernatant was used as the main source of CPS.Purification of CPS. Solid (NH4)2SO4 was added to the 40,000g supernatant to 25% saturation (at 0°C). The precipitate was removed by centrifugation (10 min at 40,000g), and additional (NH4)2SO4 was added to make a final concentration of 80%o (at 0°C). Following precipitation and centrifugation, the pellet was dissolved in 3 to 3.5 ml medium containing 50 mM Tricine-KOH buffer (pH 8.0) and 10 mm Om. An aliquot (2.5 ml) was applied to a 2.6-x 25-cm column of Sephadex G-25 equilibrated with 50 mM Tricine-KOH buffer (pH 8.0), to which was added 5 mM Orn. Fractions were collected, and their relative protein content was measured by A at 280 nm. A number of fractions were pooled; aliquots were taken and subsequently used in the enzyme as...

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“…The first enzyme of this pathway is ornithine carbamoyltransferase (E.C.2.1.3.3). Appearance of label in glutamate (and ultimately proline) involves reversal of ornithine biosynthesis; i.e., formation of glutamic--y-semialdehyde by yornithine glutamate transaminase (2). Finally, appearance of label in proline occurs through the enzymes converting glutamate to proline (18).…”

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confidence: 99%

Mode of Action of the Toxin from Pseudomonas phaseolicola

1972

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The specificity of the Pseudomonas phaseolicola toxin for enzyme inhibition and its relationship to toxin-induced chlorosis in bean leaves (Phaseolus vulgaris L.) was examined. The toxin showed no significant inhibitory activity against glutamine synthetase, glutamine transferase, carbamyl phosphate synthetase, aspartate carbamoyltransferase, or arginase at concentrations 100-fold higher than that needed to inhibit ornithine carbamoyltransferase by 50%.Protection from and reversal of toxin-induced chlorosis in bean leaves was attempted with several amino acids. Aside from protection with L-citrulline which was previously reported, only L-arginine-HCl and to a minor extent L-leucine and L-glutamine showed protection from chlorosis. L-Citrulline and L-arginine-HCI (but not L-glutamine and L-leucine) also reversed toxininduced chlorosis.Ultrastructuraily, cells from toxin-treated chlorotic tissues showed no observable changes as compared to nontreated tissues. This, together with the ability of the two amino acids to reverse chlorosis, indicated that the toxin causes a reversible biochemical lesion in treated tissue.While tissues from bean plants inoculated with P. phaseolicola showed a large accumulation of ornithine, toxin-treated tissues showed no accumulation of ornithine. The latter finding indicated that in addition to the ornithine carbamoyltransferase inhibitor, the pathogen may produce inhibitors of other ornithine metabolizing enzymes in inoculated tissues.When bean plants (Phaseolus vulgaris L.) are infected by their pathogen Pseudomonas phaseolicola or injected with its toxic culture filtrates, they exhibit two symptoms: chlorosis and accumulation of ornithine (20,28). Chlorotic pathogen infected tissues show a 440-fold increase in ornithine content, while chlorotic toxin-treated tissues show a 106-fold increase (28). Since the ornithine pool in plants is usually small (1), this striking increase indicates a derangement in the normal ornithine metabolism of affected tissues.Ornithine is metabolized primarily through two pathways,

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Metabolism of Citrulline and Ornithine in Mung Bean Mitochondria.

Cited by 34 publications

References 18 publications

l-Ornithine:2-Oxoacid Aminotransferase from Squash (Cucurbita pepo, L.) Cotyledons

l-Ornithine:2-Oxoacid Aminotransferase from Squash (Cucurbita pepo, L.) Cotyledons

Carbamoyl Phosphate Synthetase Activity from the Cotyledons of Developing and Germinating Pea Seeds

Mode of Action of the Toxin from Pseudomonas phaseolicola

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