Properties and Regulation of Aspartate Kinase from Barley Seedlings ( Hordeum vulgare L.)

Abstract

Aspartate kinase (EC 2.7.2.4) has been purified 8-fold and characterized from germinating barley (Iordeum vulgare) seedlings. The enzyme is inhibited 50% by 0.7 mM L-lysine and almost completely at 5 mM. L-Methionine does not affect the enzyme on its own, but at low concentrations (0.1-1 mM) increases the inhibition in the presence of lysine, dicatig that the two amino acds act as cooperative feedback regulators. Aspartate kinase (ATP: L-aspartate 4-phosphotransferase, EC 2.7.2.4) catalyzes the ATP-dependent conversion of aspar-tate to f-aspartylphosphate, the first reaction in the biosynthesis of the aspartate family of amino acids-lysine, methionine, thre-onine, and isoleucine (16). The enzyme is subject to feedback control by its amino acid end products, and a variety of regulatory patterns have been demonstrated for the enzyme from different plant tissues. The enzyme from maize is sensitive to L-lysine (5, 6, 14), whereas in Pisum sativum, the main effector is L-threonine and lysine is inactive (3). In contrast, Wong and Dennis (22, 23) suggested that the enzymes from wheat germ and Lemna minor were inhibited cooperatively by lysine and threonine. In both cases, however, their experiments were carried out with very low activity preparations, and high (1-3 mM) concentrations of thre-onine were required to cause appreciable inhibition. The wheat enzyme was inhibited 60% by 1 mm lysine on its own. Recently, Aarnes (2) studied the enzyme from four green plants and two algae and demonstrated that all were inhibited to varying extends by lysine and/or threonine. In the present paper, we describe the partial purification and properties of an enzyme from barley which is sensitive to inhibition by lysine and to cooperative inhibition by lysine and methio-nine. Methionine has not previously been demonstrated to have a role in the regulation of aspartate kinase in higher plants. MATERIALS AND METHODS Chemicals. Amino acids and cofactors were purchased from Sigma. Amino acid analogues were purchased from Sigma, Cal-biochem, Aldrich Chemical Co. Inc., and R. N. Emanuel Ltd. Those not commercially available were generous gifts from L. Fowden and P. J. Lea (Rothamsted). L-[U-_4C]aspartic acid (180 ,Ci/j,mol) was obtained from the Radiochemical Centre, Amersham. Enzyme Extrction and Purification. Untreated seeds of barley (Hordeum vulgare [L] cv. Julia) were germinated in the dark in moist vermiculite at 20 C for 7 days. Shoots were blended at 5 C with a 1:1 (w/v) ratio of 0.2 M tris-HCl (pH 8) containing 0.1 M KCI, 1 mm EDTA, 10 mm 2-mercaptoethanol, and 30% (v/v) glycerol. After filtration through gauze, the extract was centrifuged at 2,500g for 45 min. Solid (NH4)2SO4 was added to the extract to 60% saturation and the precipitate was collected by centrifugation. This was dissolved in 0.05 M K-phosphate buffer (pH 7.5) with 1 mm EDTA, 5 mm 2-mercaptoethanol, and 20% (v/v) glycerol, and dialyzed overnight against the same buffer. The dialyzed extract was shaken with DEAE-cellulose which was then eluted batchwise with phosphate buffer + 0.2 M NaCl (4 ml/g DEAE-cellulose). The active eluate was concentrated by (NH4)2SO4 precipitation (60%), and the precipitate was redissolved in phosphate buffer and dialyzed overnight. The extract was finally centrifuged at 70,000g for 60 min. The preparation was stable at 5 C for several days and at-15 C for at least a week. Enzyme Assays. The hydroxamate assay for aspartate kinase was based on that of Bryan et al. (5). The standard assay contained 50 mm L-aspartate, 20 mM MgSO4, 20 mm ATP, 125 mM NH2OH HCl (adjusted to pH 7 with KOH immediately prior to the assay), 100 mm K-phosphate (pH 7.5), and enzyme in a total volume of 1 ml. The aspartate concentration was 25 mm in the amino acid feedback experiments. The reaction was linear for 60 min and over the range of enzyme concentration used (0.8-2.2 mg/assay). After incubation at 30 C for 60 min, the reaction was terminated by the addition of 0.5 ml of 15% trichloroacetic acid. The precipitated protein was removed by centrifugation, and 1 ml acid ferric chloride reagent was added to the supernatant. The absorbance at 505 nm was measured after 20 min, and the concentration of 13-aspartyl hydroxamate was calculated from a standard curve prepared using the authentic compound. Coupled assays for aspartate kinase were based on those of Cheshire and Miflin (6) and Bryan et al. (5). Aspara-gine synthetase was determined by the specific radiochemical assay of Lea and Fowden (12) using NH4+ and glutamine as amino (amide) donors. Protein was determined by the method of Lowry et al. (13). RESULTS Chacteristics of the Enzyme. In typical experiments, 2 kg of fresh tissue yielded approximately 125 mg of protein with a specific activity of 2 to 4 nmol/min-mg protein under standard assay conditions. Modifications to the buffer systems, including alterations in pH, ionic strength, buffer type, and the inclusion of 1 mm lysine and 1 mm threonine had no effect on the amount of activity extracted. The inclusion of phenylmethylsulfonyl-fluoride, a protease inhibitor, in the extraction buffer was also without effect. We suggest, therefore, that the amount of activity extracted may have represented a fairly low endogenous level of active enzyme. The overall purification was 8-to 9-fold, and the specific activity of the final preparation was similar to those of the preparations used by some other workers (3, 5, 6). The results are from 20 separate enzyme isolations, and the proper-69