Purification to Homogeneity of Pyrroline-5-Carboxylate Reductase of Barley

Abstract

An enzyme has been purified to homogeneity from barley seedlings which has 'proline dehydrogenase' and the pyrroline-5-carboxylic acid reductase activities. The purification achieved is 39,000-fold as calculated from the proline dehydrogenase activity. The subunit molecular weight of the protein is 30 kilodaltons. The native enzyme has molecular weights up to 480 kilodaltons, depending on the buffer environment. From the pH profiles, the specific activities and thermodynamic considerations, it is concluded that the plant proline dehydrogenase functions in vivo as a pyrroline-5-carboxylate reductase. Pyrroline-5-carboxylate reductase (EC 1.5.1.2. L-pro-line:NAD(P)-5-oxidoreductase)catalyzes the final step in proline biosynthesis. The enzyme has been partially purified and characterized from various animal sources (1, 3, 11, 16, 21, 22, 24), bacteria (1, 2, 10, 11, 15), Neurospora (26), and Saccharomyces (6, 7). In higher plants (Pisum and Phaseolus) the reductase was initially detected by Meister et al. (1 1). A partial purification of the enzyme from higher plant sources has been reported subsequently (12, 14, 18, 19, 23). In this paper we describe a procedure which results in the preparation of P5C'-reductase from barley seedlings as a homogeneous single protein. The enzyme is purified 39,000-fold during this process. The purified reductase oxidizes proline under extreme pH conditions (pH 10.3) thus supporting the assumption that in reality the "proline dehydrogenase" (8, 9, 17, 19) ofhigher plants is a P5C-reductase. MATERIALS AND METHODS Barley seedlings (Hordeum vulgare, var. Augusta) were grown in a greenhouse (16 h photoperiod, 20C), watered until the 5th d of growth, and harvested at the age of 10 to 14 d. Six kg of seedlings were frozen in liquid N2 and ground. The resulting powder was homogenized in a total of 31 L of phosphate buffer (0.1 M phosphate (pH 8.0), 1 mM NaEDTA, and 10 mM f,-mercaptoethanol) with an Ultra-Turrax T45 and filtered through cheesecloth. The extract was heated to 60°C for 0.5 to 1 min, immediately cooled, and filtered through Celite 545 (Serva). The filtrate was made 25% saturated with (NH4)2S04 and the result-' Abbreviations: P5C, pyrroline-5-carboxylate; MTT, 344,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide; PMS, phenazine metho-sulfate. ant precipitate discarded after filtration. (NH4)2S04 saturation was increased to 50% and the precipitate collected by centrifugation (22,000g). The sediment was dissolved in 380 ml of phosphate buffer 2 (50 mM phosphate, pH 7.5, and 10 mM ,B-mercaptoethanol), dialyzed against buffer 3 (20 mm piperazine-HCl and 1 mm mercaptoethanol, pH 6.3), and applied to a DEAE-Sephacel column (Pharmacia K 50/30, bed volume 470 ml, flow rate 200 ml/h). The column was rinsed with 1 L of buffer 3 and developed in a 0 to 0.3 M KCI gradient in buffer 3 (1600 ml; fractions of 27 ml were collected). The pooled activity fractions were chromatographed on Ami-con Matrex Gel Red A (Pharmacia K 16/20, bed volume 22 ml). After loading and rinsing the column with buffer 4 (15 mM Tris-HCI, pH 8.0, 5% v/v glycerol, 5 mM MgCl2, and 2 mm DTT) it was developed in a 0 to 1.5 M KCI gradient in the same buffer. Matrex gel was prepared and regenerated with 8 M urea and equilibrated with buffer 4. The final step of the preparation was a chromatography on Amicon Matrex Gel Orange A (bed volume 2.4 ml). The pooled activity fractions from the preceding step were desalted (Sepha-dex G-25 in column PD-10, Pharmacia) and then applied to the Orange A column. The column was rinsed with 5 ml of buffer 4 and 25 ml of buffer 4 + 0.2 M KC1. the elution of the enzyme was achieved with 0.5 M KCI in buffer 4 (flow rate 30 ml/h). Enzyme Assay. The 'proline dehydrogenase' activity was assayed during the isolation procedure. The assay system contained 0.7 ml ofbuffer (7.5 g/L glycine; 5.8 g/L NaCl, 3.0 g solid NaOH resulting in a pH of 10.3); 0.1 ml of proline (0.2 M in water); 0.1 ml of NAD+ (0.15 M NAD+ in water, adjusted to pH 5 to 7 with NaOH); and 0.1 ml of test solution. The reference contained buffer instead of proline. The change of absorbance was measured at 366 nm in an Eppendorf photometer 1 l01M; 1 unit = 16.7 nkat = 1 gmol NADH/min. The assay for P5C-reductase was conducted by monitoring the loss of the absorbance of NADH at 366 nm. Reaction mixtures of 1 ml contained Tris-phosphate buffer (pH 7.5) (0.04 M Tris), 0.095 M NaCl, 0.08 mM MgC92, 0.08% (v/v) glycerol, 0.03 mM DTT, 0.19 to 0.76 mM NADH, and 0.225 to 0.9 mM P5C (stock solution: 9 mM L-P5C, 0.6 to 1 M NaCI). The DL-P5C was synthesized by a slightly modified procedure of Williams and Frank (25). The Dowex column was equilibrated with 0.1 M acetic acid instead of 1 M HCI. Protein concentrations in crude extracts were determined by the procedure of Lowry et al. (5) and in the fmal enriched preparations according to the procedure of Schaffner and Weissmann (20).