N-Acetylglutamate 5-phosphotransferase (acetylglutamate kinase, EC 2.7.2.8) has been isolated from pea (Pisum sativum) cotyledons and purified 312-fold by using heat treatment, (NH4)2SO4 fractionation, affinity chromatography on ATP--Sepharose and ion-exchange chromatography on DEAE-cellulose. This preparation was shown on polyacrylamide-gel electrophoresis to yield one band staining with Coomassie Blue. The enzyme was shown by a variety of techniques to be composed of two different kinds of subunits, of mol.wts. 43000 and 53000 respectively. These subunits are arranged to give either a dimeric or tetrameric enzyme composed of equal numbers of each type of subunit. The dimeric and tetrameric enzyme forms are thought to be interconvertible, the equilibrium between these forms being influenced by the type of ligand bound to the subunits. Kinetic studies performed on the purified enzyme, indicated a random Bi Bi type of mechanism. The enzyme displayed apparent negative co-operativity with respect to one of its substrates, N-acetylglutamate; as a result, two Km values were found for this substrate, one at 1.9 X 10(-3) M and the other at 6.2 X 10(-3) M. A single Km value for ATP was found to be 1.7 X 10(-3) M. Allosteric regulation by arginine was also shown. A model, based on the Koshland, Némethy & Filmer [(1966) Biochemistry 5, 365-385] Sequential model, which adequately describes the kinetic and structural properties of N-acetylglutamate 5-phosphotransferase, is presented.

Studies were carried out to determine the distribution of the following: (1) carbamoyl phosphate synthetase (EC 2.7.2.9), (2) ornithine carbamoyltransferase (EC 2.1.3.3), (3) argininosuccinate synthetase (EC 6.3.4.5), and (4) argininosuccinate lyase (EC 4.3.2.1) in soybean cells grown in suspension culture. Protoplasts were produced from the soybean cells by treatment with cellulase (EC 3.2.1.4) and pectinase (EC 3.2.1.15); the protoplasts were then ruptured by osmotic shock with distilled water. This treatment was followed by differential centrifugation and sucrose density gradient centrifugation to isolate various organelle fractions including mitochondria and plastids.Examination of these fractions using specific enzyme assays showed that carbamoylphosphate synthetase and ornithine carbamoyltransferase were localized in a fraction found to be composed primarily of plastids. Argininosuccinate synthetase and argininosuccinate lyase appeared to be associated with either the cytosol or a membrane fraction in close association with the cytosol such as the endoplasmic reticulum or protoplast membrane.

The levels of the activities of argininosuccinate synthetase and argininosuccinate lyase were measured in soybean (glycine max L. var. Mandarin) cell suspension cultures grown in the presence or absence of exogenous arginine. In some experiments, actinomycin D or cycloheximide were also added to the cultures, at critical stages of their growth. The results obtained led to the conclusion that activity of argininosuccinate synthetase is subject to significant inhibition by levels of arginine similar to those found to occur within the cells. Argininosuccinate lyase activity appeared to be enhanced, when arginine levels were increased above those occurring physiologically. Both enzymes appeared to be subject to inactivation, possibly via proteolysis.The regulation of arginine biosynthesis in microorganisms and in animals has received a considerable amount of attention in the past (8,(13)(14)(15). These studies have shown that in both groups the activity of enzymes involved in the pathway is regulated by the levels of key metabolites and also by the rate of synthesis and degradation of the enzymes. By comparison with these studies, very little is known of the mechanisms used by plants to regulate the biosynthesis of arginine. This communication presents an attempt to demonstrate some of the mechanisms which appear to exist in soybean cells for controlling the activities of two enzymes involved in arginine biosynthesis, argininosuccinate synthetase, and argininosuccinate lyase. MATERIALS AND METHODSThe soybean culture used in these studies (Glycine max L. var. Mandarin), was obtained from Dr. 0. L. Gamborg of the Prairie Regional Laboratory, Saskatoon. Cultures were grown routinely in B5 medium as described by Gamborg (1, 2). The average size of inoculum for the 100-ml cultures used in the experiments described in the text was 2.4 g fresh weight (equivalent to 150 mg dry weight). Additions tained by homogenizing the cells in a Braun MSK homogenizer (Canadian Laboratory Supplies Ltd.). Plant cells were collected on a disk of Miracloth over a Buchner funnel and washed with approximately 100 ml of 0.1 M, pH 7.9, Tricine buffer. They were then homogenized using a 1:2:4 ratio (w/v/w) of plant cells-Tricine buffer-glass beads (1.0-1.05 mm diameter). Homogenization was carried out for 90 sec, during which time the suspension was cooled to approximately 4 C with the aid of liquid CO2. At the end of this time, breakage of the cells appeared complete, when a sample was viewed microscopically. The homogenate was then filtered through a Miracloth disk into a cooled Buchner flask. Finally, it was spun for 10 min at 20,000g in the SS34 head of the Sorval RC2 refrigerated centrifuge, and the supernatant was used for further experiments.Dry weight estimations were made on 2-ml aliquots of the cultures. These were filtered on glass fiber discs, and the discs were washed thoroughly with distilled water. The discs plus cells were then lyophilized for 24 hr and finally weighed to a constant weight.The assay of argininosuccinate...

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SHARGOOL, P. D., and T. T. Nco. 1975. The uptake of sulfate by excised roots of rape seedlings (Brossicn nnprrs var. Target). Can. J. Bot. 53: 914-920. Excised roots from 5-day-old seedlings of Brossico naprrs L. var. Target, which had been grown in 2 x M calcium chloride solution, were incubated in medium containing various concentrations of sulfate. The uptake of this ion by the roots was measured with the aid of [35S]-labelled sulfate and a liquid scintillation counting method. The membrane uptake system was shown to be sensitive to sodium cyanide and dinitrophenol. Kinetic examination of the process demonstrated that it was mediated by two separate systems each of which operated in a specific sulfate concentration range. One system was shown to have a low affinity for sulfate and was thought to remain inoperative until the second high-affinity system became saturated. SHARGOOL, P. D., et T. T. Nco. 1975. The uptake of sulfate by excised roots of rape seedlings (Brassica naprrs var. Target). Can. J. Bot. 53: 914-920. Chez des plantules :gees de 5 jours de Brassica naprrs L. var. Target et cultivees dans une solution 2 x M de chlorure de calcium, les racines ont Cte excisees et incubkes dans un milieu contenant diverses concentrations de sulfate. L'absorption de cet ion par les racines a kt6 mesuree i I'aide de sulfate marque au 35S et d'une mkthode de comptage i scintillation liquide.Les rksultats montrent que le systeme d'absorption de la membrane est sensible au cyanure de sodium et au dinitrophenol. Une etude cinktique a dernontrk que le processus comprend deux systemes differents, chacun etant actifen presence d'une gamme spkcifique de concentrations en sulfate. L'un des systemes a une faible affinite pour le sulfate et les auteurs croient qu'il reste inactifjusqu'a ce que le second systeme, ayant une forte affinitk pourle sulfate, devienne sature.[Traduit par le journal]