Separation and properties of two ornithine carbamoyltransferases from Pisum sativum seedlings

Eid, Salwa; Waly, Yousef M.; Abdelal, Ahmed T.

doi:10.1016/s0031-9422(00)91274-3

Cited by 23 publications

(15 citation statements)

References 18 publications

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“…One peak of OCT activity was observed, which eluted from the column when a concentration of 100 mM NaCl was reached. This enzyme is probably the first enzyme (Table III) eluted in the experiment of Eid et al (7).…”

Section: Methodsmentioning

confidence: 76%

“…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.…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

Section: Introductionmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…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) have different physiological functions. An anabolic and a catabolic function have been proposed which are related to the synthesis and the degradation of citrulline, respectively.…”

mentioning

confidence: 99%

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Properties of Ornithine Carbamoyltransferase from Pisum sativum L

Ruiter¹,

Kollöffel²

1985

Plant Physiol.

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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...

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Section: Methodsmentioning

confidence: 76%

“…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.…”

Section: Methodsmentioning

confidence: 99%