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

Abstract

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...

Activity of Enzymes of Arginine Metabolism in the Cotyledons of Developing and Germinating Pea Seeds

“…In higher plants, synthesis of carbamoyl phosphate (10) and argininosuccinate (13) are considered to be possible regulating steps of the arginine synthesis. This supposition is now supported by the observation that ASA synthetase activity is relatively low and by our earlier results on carbamoyl phosphate synthetase (6).…”

“…Incubation of broad bean cotyledons of 10-d germinating seeds with labeled arginine gave rise to all urea cycle intermediates (3). Our results indicate that the activity of the arginine-synthesizing enzymes, including carbamoyl phosphate synthetase (6), is low compared with their activity in developing seeds. The presence of these enzymes in germinating seeds might be considered perhaps as being a less meaningful remnant of an enzymic machinery necessary for the synthesis of arginine in developing seeds.…”

Genes, enzymes and regulation of arginine biosynthesis in plants

“…UMP binding to purified wheat germ ATC decreases its affinity for carbamoyl-P [43]. As for bacterial CPS, UMP inhibition of plant CPS activity is relieved by Orn [45,59]. Orn was also reported to activate squash ATC [47], but this report was probably based upon artefactual OTC activity having been measured, along with ATC activity, upon Orn addition to crude extracts containing both activities.…”

Carbamoyl Phosphate Synthetase, Ornithine Transcarbamylase, and Aspartate Transcarbamylase Activities in the Pea Ovary

“…Although some of the enzymes of the arginine and pyrimidine synthesizing pathways have been assayed in several plant tissues (7,14,(19)(20)(21)(22)24) and in the cotyledons of developing and germinating seeds (10,18), no data have been reported on the activity ofthese enzymes in the ovary during the initial phases of fruit development. Such data would be important in that fruit set constitutes a good example of rapid induction of increase in weight and differentiation (25).…”

“…The number of ovaries used per extraction, corresponding to 0.2 to 1 g fresh weight, varied within the ranges of 4 to 24 and 1 to 10 for the nontreated and GA3-treated groups, respectively. The ovaries were homogenized in a mortar at 0 to 4°C with 5 volumes of a solution (10) of 0.4 M mannitol, 50 mM N-morpholinopropane-sulphonic acid buffer (pH 7.4), 2.5 mg/mL bovine serum albumin, 10 ,M leupeptin (Sigma), and 5 mm 2-mercaptoethanol. The homogenates were centrifuged 15 min at 30,000 g and 4°C.…”