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

Abstract

Ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase, and arginase activity were measured in extracts from cotyledons of developing and germinating seeds of Pisum sativum L. The course of activity of these four urea cycle enzymes showed a similar pattern during seed development. The activity per cotyledon increased sharply initially and reached a maximum about 5 weeks after anthesis, when the relative water content of the seeds was about 60%. About 8 weeks after anthesis, the seeds were mature (air-dry) and had enzyme activities which were much lower. The activities of the enzymes differed considerably. Ornithine carbamoyltransferase showed the highest activity, followed in order of decreasing activity by arginase, argininosuccinate lyase, and finally argininosuccinate synthetase.The course of the activity of the four enzymes was different during germination. Arginase activity increased sharply 7 hours after the onset of germination and remained at a constant level during the folowing days.Argininosuccinate synthetase activity decreased; the other enzymes showed a small increase in activity and a subsequent decrease. Results are discussed in relation to the regulation of the arginine metabolism during pea seed development and germination.Developing pea seeds accumulate arginine-rich storage proteins. During seed germination, the arginine released from these storage proteins is either directly translocated to the growing axis, incorporated into proteins or degraded in situ as has been shown for broad beans (3) and pumpkins (17).The final steps of the synthesis and the break down of arginine in higher plant tissues occurs in a similar way to that in mammalian tissues (urea cycle), lower plants, and in bacteria. The enzymes involved have all been demonstrated in extracts of several higher plants. The enzymes are OCT,2 ASA synthetase, ASA lyase, and arginase.Data on the changes in the activity of these enzymes during seed development (4, 5) and germination (1, 4-6, 14, 18) Preparation of Extracts. About 12 g of cotyledons were ground in a mortar and pestle with 5 to 8 g of sand and 20 ml extraction medium containing 400 mm mannitol, 50 mm morpholinopropane sulfonic acid buffer (pH 7.4), and 0.25% (w/v) BSA. It contained also 30 mm MgCl2 when used for the isolation of ASA synthetase.After passing through a Perlon screen (mesh width, 45 ,um), the homogenate was centrifuged at 2,500g for 5 min. The pellet obtained after centrifugating the 2,500g supernatant at 40,000g for 10 min was washed twice and resuspended in extraction medium. This suspension was used as the main source for arginase. The 40,000g supernatant was used as the main source for OCT, ASA synthetase, and ASA lyase, but the latter were further partially purified.ASA Synthetase. Solid (NH4)2SO4 was added to the 40,000g supernatant to 30% saturation (at 0WC). The precipitate was removed by centrifugation (10 min at 40,000g), and additional (NH4)2SO4 was added to make a final concentration of 70% (at 0°C). The preci...

Amino Acids Content in Germinating Seeds and Seedlings from Castanea sativa L

“…An important role of arginine in the metabolism of the seedling has been claimed (2) (14), in pumpkins (22), in peas (6,7) and in grapevine (20) (3). An increase of activity of OAT in early stage of germination has been demonstrated in pea seeds (7) and also observed in cotyledons of pumpkins seedling (23).…”

Arginine Catabolism in the Cotyledons of Developing and Germinating Pea Seeds

“…In developing pea seeds (4) and cotton seeds (5), arginine formation is considered to be located in the cotyledons themselves.…”

“…Arginine-carbon is principally respired, whereas arginine-nitrogen may be transported to the axis tissue (24). Arginase and urease activities have been demonstrated in germinating seeds of several species and in cotyledons of developing seeds (4,5,12,20). It is unknown whether the presence of arginase in developing cotton seeds (5) and urease in developing soybeans (20) A powder sample equivalent to 10 cotyledons was extracted with 80% (v/v) ethanol.…”

Arginase, Arginine Decarboxylase, Ornithine Decarboxylase, and Polyamines in Tomato Ovaries (Changes in Unpollinated Ovaries and Parthenocarpic Fruits Induced by Auxin or Gibberellin)

“…Arg is usually a major nitrogen storage compound, consisting of approximately 65% of the free amino acid nitrogen in young pea cotyledons (De Ruiter and Kolloffel, 1983), so any enzymes involved in its metabolism should be regulated to control its catabolism or its accumulation as a free amino acid or in reserve proteins (De Ruiter and Kolloffel, 1982). In pea fruits high Orn transcarbamylase activity, a high Arg/ Orn ratio, the presente of ADC activity, and the absence of arginase and ODC activities (García-Espafia et al, 1989;Pérez-Amador and Carbonell, 1995) indicate that Arg acts as a substrate for putrescine biosynthesis and as a source of nitrogen for seed development (Pate and Flinn, 1977).…”