Mitochondrial Arginase Activity from Cotyledons of Developing and Germinating Seeds of Vicia faba L

Abstract

Differential and sucrose density gradient centrifugation established that about 80% of the total arginase activity (EC 3.5.3. 1) in cotyledons of germinating broad bean seeds (Vicia faba L.) was present in the mitochondrial fraction. The mitochondrial arginase activity was enhanced considerably by exposure to osmotic shock, by freezing and thawing, or by Triton X-100 treatment. About 10% of the total arginase activity was recovered from the 40,000g supernatant fraction. During seed maturation, arginase activity in the cotyledons decreased to about one-third of its maximal activity, while increasing over 10-fold during subsequent germination. The time courses of mitochondrial arginase, succinate oxidase, and succinate dehydrogenase activities differed considerably during germination.Cotyledons of developing Vicia faba (L.) seeds accumulate large amounts of arginine-rich protein (2, 13). During subsequent germination these storage proteins are hydrolyzed (1) and the amino acids released are either metabolized in situ or directly translocated to the growing axis tissue. It has been shown with '4C-labeled arginine (9) that in broad bean cotyledons arginine is degraded mainly in situ by an arginasecatalyzed formation of ornithine and urea. Pumpkin cotyledons also contain appreciable amounts of protein-bound arginine which is metabolized primarily in situ (20); less than 1 % of the arginine is translocated to the axis tissue (5). In these pumpkin cotyledons (19), as in seeds of Dolichos lablab (21), the arginase activity increased during germination. This paper shows that this enzyme is already present in developing broad bean cotyledons.Although arginase has been isolated from many plant tissues (3,6,9,14,19,21), its subcellular distribution is unknown. In mammalian tissues (7,8,15) and in Neurospora (22), arginase activity was located in the cytosol. This paper reports that in germinating broad bean cotyledons arginase activity was present mainly in the mitochondrial fraction. MATERIALS AND METHODSPlant Material. Seeds of Vicia faba L. cv. R 35 were planted in the field and 3 to 4 months later pods were taken from the plants and selected for equal thickness, color, and turgor. The seeds were removed from the pods; the cotyledons were separated and divided into two portions, one for the extraction of the enzymes and the other for the determination of fresh and dry weight (24 hr at 105 C.). The relative water content of the seeds, defined as mg water/mg fresh weight x 100%, was taken as the criterion for their physiological state. During the growth period studied, the relative water content decreased from about 70% to about 12% (air-dry mature seeds).Germination Conditions. Air-dry mature seeds were harvested and soaked in tap water under aeration for 18 to 24 hr. They were allowed to germinate further by transferring them subsequently to moist filter paper in large Petri dishes in darkness at 23 C for the appropriate time. The period between the beginning of soaking and the moment the cotyledons were used for...

Purification and Properties of Arginase from Soybean, Glycine max, Axes

“…We could not find any evidence of the presence of arginase isozymes in purification steps and purity tests including pore gradient gel electrophoresis. Such results are in good agreement with the previous reports (4,14,25,28). We suggest that our purification protocol is effective, compared with other methods, and it may be useful in studies where purified arginase is required for further enzyme studies, production of antibodies, etc.…”

Purification, Properties and Subunit Structure of Arginase from Iris Bulbs

“…Unlike arginase from animal or microbial sources, plant arginase is poorly known. Only its cellular localization, probably in the mitochondria [8,9], and its Mn2+ requirement [lo] are established. Recently, Wright et al [ I l l presented some properties of an arginase purified 450-fold from Jerusalem artichoke tubers.…”

Developmental Biochemistry of Cotton Seed Embryogenesis and Germination

“…ARG concentrations higher than 125 mm inhibit the arginase activity. Kolloffel and van Dijke (14) reported that arginase activity from germinating cotyledons of Vicia faba is largely found in isolated mitochondria. In cotton cotyledons, however, a reduction in centrifugation time and speed of the homogenate to 2,500g for 5 min or addition of 0.5% Triton X-100 to the extraction medium did not increase the recovery of enzyme activity, indicating that cotton arginase may not be particulate or that osmotic rupture of mitochondria under the extraction procedure was complete.…”