Properties of Ornithine Carbamoyltransferase from Pisum sativum L

Abstract

Some properties of ornithine carbamoyltransferase from chloroplasts isolated from leaves of Pisum sativum L. (cv Marzia) were compared with those of the enzyme partially purified (316-fold) from shoots of seedlings after 3 weeks of cultivation.Both preparations showed a pH optimum at pH 8.3 and had the same affinity to ornithine (K,. = 1.2 millimolar) as well as to carbamoyl phosphate (K,, = 0.2 millimolar). The approximate molecular weight determined by gel sieving was 77,600.A desalted ammonium sulfate precipitate from 14-day seedlings (inclusive roots and senescing cotyledons) was applied on a column of anion exchanger. The elution pattern showed one peak of ornithine carbamoyltransferase activity. This elution pattern was the same as observed for the enzyme from chloroplasts.The results suggest the presence of one form of ornithine carbamoyltransferase in pea seedlings.In higher plants, OCT' (EC 2.1.3.3.) is involved in the synthesis of citrulline. The enzyme has been localized in chloroplasts from pea leaves (23) and plastids from soybean cell suspension cultures (21) by means of density gradient studies.Two isozymes of OCT have been found in sugarcane (8), pea seedlings (7), root nodules from black alder (13), and apple leaves (22). These isozymes show different elution patterns from an anion exchange column (7,8,13) and have different kinetic properties (7,8,13,22). A cytoplasmic and a mitochondrial enzyme with mol wt of 79,000 and 224,000, respectively, were distinguished in sugarcane (8). It has been suggested that the two isozymes from pea seedlings (7) and sugarcane (8) 3 weeks. The shoots were cut about 1 cm above the cotyledons and stored at -20°C. Freshly harvested leaves of seedlings after 3 weeks of cultivation were used for isolation of chloroplasts.Enzyme Purification. Unless stated otherwise all steps were carried out at 4°C.Protein Extraction. Proteins were extracted from 500 to 600 g (fresh weight) pea shoots along the lines described by Eid et al. (7). The dry powder obtained after acetone precipitation was kept at -20°C. Prior to use, the powder was extracted during 1 h in 1 L buffer containing 50 mm K-phosphate (pH 7.8) and 1 mM 2-mercaptoethanol. The extract was passed through a Perlon screen (mesh width 45 Am) and then centrifuged for 7 min at 20,000g. This is the filtrate referred to as step 1 in Table I.Ammonium Sulfate Precipitation. The supernatant fluid was brought to 40% saturation by solid (NH4)2SO4. The supernatant obtained after centrifugation (20 min at 22,000g) was brought to 65% (NH4)2SO4 saturation and centrifuged again. The precipitate was dissolved in about 300 ml buffer containing 50 mM Kphosphate (pH 7.8), 50 mM L-orn, and 1 mM 2-mercaptoethanol.Heat Treatment. The extract was heated under stirring to 61°C in a water bath. The solution was maintained at 61 to 62°C for 1 min, then cooled in ice and centrifuged (7 min at 20,000g) to sediment the denaturated proteins. The supernatant was filtered through filter paper and then brought to 70% saturation by addition...

Origin, structure, and regulation of argK, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase in Pseudomonas syringae pv. phaseolicola, and functional expression of argK in transgenic tobacco

“…Hybridization with single-stranded RNA probes of opposite polarity to the ROCT frame, produced by in vitro transcription from plasmid pEH10, revealed three transcripts of the size as those detected with the doublestranded probe, while a probe with the same polarity as the ROCT frame did not produce any signal. All (12,16,22) suggested that plant OCTs are present in several subcellular compartments, one of which, the chloroplast, is the most likely cellular target of phaseolotoxin (53,85). Accordingly, we constructed plasmid pEH60 (Fig.…”

Purification and Characterization of Ornithine Transcarbamylase from Pea (Pisum sativum L.)

“…It is interesting to note, however, that nontrimeric structures have been reported for other plant OTCs. For example, deRuiter and Kolloffel (10) concluded that partially purified pea OTC was a dimer (Mr = 77,600). Recently, Acaster et al (1) reported that the mean Mr value for OTCs from several plant species was 158,000 and the same value was obtained for the carrot enzyme (2), suggesting that the enzyme was most likely a tetramer.…”

“…35,000) contaminating band on SDS-PAGE gels, and it seems likely that the lower mol wt band in their preparation is the proteolytic degradation product. Pea OTC has also been purified by two other groups (10,20), but size estimates for the OTC monomer were not given.…”

Human ornithine transcarbamylase: crystallographic insights into substrate recognition and conformational changes

“…phaseolicola, Thr*" is replaced by methionine and glycine respectively [42,43]. Both of these plant OTCases exhibit elevated K m values for CP and reduced affinities for the bisubstrate analogue inhibitor, PALO, relative to other OTCases [44,45]. The relatively weak binding of CP to plant OTCases, relative to ATCases, may be important for partitioning CP between de no o pyrimidine and arginine biosynthesis, as a single glutamine-dependent CP synthetase provides the CP intermediate to both pathways [42].…”