Agric. Biol. Chem., 46 (3), 739-743, 1982 739 Purification and Partial Characterization of Arginine Decarboxylase from Rice Embryos (Oryza sativa L.) Murari Mohan Choudhuri and Bharati Ghosh Plant Physiology Laboratory, Botany Department, Bose Institute, Calcutta-700009, West Bengal, India Received July 30, 1981 Arginine decarboxylase (E.C. 4.1.1) after purification from rice seedlings was separated into fractions A (MW88000) and B (MW174000) by gel chromatography. Fraction B was much more active than A. After DEAEcellulose chromatography, the active fraction of the enzyme (B) was purified to homogeneity, which appeared as a single band in gel electrophoresis. The optimal pH and temperature for the enzyme were8.0 and 45°C, respectively. The enzyme followed typical Michaelis-Menten kinetics with a Kmvalue of 0.28mM. It had no dependence on a metal, and consisted of 16 amino acids of which proline was prominent. Pyridoxal-5-phosphate acted as a co- factor of the enzyme. The enzyme activity was inhibited by various amines and inhibitors, of which the highest inhibition was obtained with spermine and hydroxylamine. The plant hormones played a vital role in regulating the activity of the enzyme which was promoted by kinetin and inhibited by abscisic acid. The diamine, putrescine, serves as the obli- MATERIALS AND METHODS gatory precursor of polyamines, spermidine Chemicals. L-[U-14C] Arginine (Spec. act. 66 mCi/mmol) and spermine.1'2) In animals, bacteria and cer- was purchased from Bhaba Atomic Research Centre tain plants, putrescine is formed from or- (Trombay, Bombay, India). Sepharose 4B and DEAE- nithine by ornithine decarboxylase, a rate cellulose are the products of Sigma Chemical Co. limiting factor in the biosynthesis of poly- Inhibitors and promoters are purchased from Sigma Co., U.S.A. All other chemicals used were of analytical reagent amines. In E. coli putrescine is formed from grade. ornithine and arginine via agmatine.3) However in higher plants putrescine is ob- Materials. Rice seeds were obtained from the experi- tained solely from arginine by decarboxylation mental garden of Bose Institute and germinated in dark at 37± 1°C as detailed elsewhere.8) with arginine decarboxylase in which agmatine Assay of arginine decarboxylase (arginine carboxylase is first produced and later converted to putres- E.C. 4.1.1). cine.24) This enzyme has been purified to The assay mixture5) of arginine decarboxylase consisted homogeneity from Lathyrus sativus5) and to an of 160 /unol Tris-HCl (pH 8.0), protein 0.5~2.0 mg, apparent homogeneity from oat seedlings6* 50/imol pyridoxalphosphate, 2 /miol dithiothreitol in a though in barley it is purified only 10 fold.7) total volume of 1ml and was incubated in Dubnoff Our previous results8} showed that in rice, metabolic shaker at 45°C for 1 hr. The incubations were carried out in Pyrex tube (18 x 50 mm)with disposable agmatine and putrescine accumulate in a co- center tube attached to rubber stopper. The released ordinated fashion during development and 14CO2 was trapped with 0.5ml of (n)KOH in the center germination, so the importance of arginine tube. The reaction was terminated by injecting 0.6ml of decarboxylase in this system raises no ques- 4(n)H2SO4 through the rubber stopper. The tubes were tion. In this paper wehave communicatedthe than incubated further for 30 min at 37°C to confirm that isolation and purification of arginine decar- all the dissolved CO2 was released from the acidified medium. The center tube with its contents was directly boxylase in a homogeneous form and tried plunged into a scintillation vial containing 5 ml of dioxane to compare it from enzyme of other plant based scintillant fluid. In all experiments 2 or 3 non- sources. enzymatic controls were used containing standard in- 740 M. M. Choudhuri and B. Ghosh cubation mixtures but enzymewas replaced by water or with stirring at 0°C and centrifuged. Further blank values were obtained by terminating the reaction acetone at -20°C was added (150ml/100ml with acid immediately after extract addition. Protein was determined by Lowry's method9) after precipitation with enzyme solution) in the same manner to the trichloroacetic acid and solubilization in (n)KOH. above soup and the precipitate recovered by centrifugation at 10,000xg for 30 min was Determination of amino acid composition. The purified dissolved in Tris-HCl buffer (0.1 m, pH 7.0), enzyme was hydrolysed with HC1and analysed for amino dialysed and centrifuged at 5,000 x g for 5 min acid composition in an automatic amino acid analyser. [Acetone fraction]. The number of amino acid residues shown in the Table II were calculated on the basis of molecular weight, 88000. Step 4. The acetone extract was applied to Tryptophan content was determined by the spectropho- the Sepharose 4B column (16 cmxl7 cm) tometric method of Edelhoch.10) The content ofcysteine in equilibrated with 50mMTris-HCl buffer (pH the enzymeprotein was not determined. 7) and eluated with the same buffer containing 0.1 m KC1. As shown in Fig. 1, on gel filtration RESULTS AND DISCUSSION with Sepharose 4B column, two arginine de- A five step purification procedure has been carboxylase were obtained, fraction B having adopted in the present investigation for exten- a specific activity higher than fraction A. The sive purification of arginine decarboxylase from seeds of Oryza sativa L. and all the operations were carried out at 4°C. /\ 8? Step 1. llhx germinated rice embryos were 8" i'A\ ° homogenised with 2 volumes of 0.05m Na2HPO4containing 5 mM2-mercaptoethanol and 20jim pyridoxal phosphate with a chilled I i/ P -5à" mortar and pestle. The homogenate was fil- tered through cheese cloth and centrifuged at *6 f Vi R 10,000xg for 30 min. The supernatant was designated as crude extract. Step 2. The crude extract was subjected to ammoniumsulphate fractionation and the ^o^ «_^±£*> 0 10 20 protein precipitating at 25~55% saturation No of Tubes was dispersed in Tris-HCl (0.1 m pH 7.0) and dialysed overnight against this buffer Fig. 1. Fractionation ofAcetone Fraction in Sepharose 4B Column Using 0.1 m KC1 in 50niM Tris-HCl (pH 7). [(NH4)2SO4 fraction]. 5ml fraction collected. Twofractions Aand B were Step 3. Precooled (-20°C) acetone (50ml) obtained and their activities determined. ( #-#), ODat was added to the above dialysed soup (100ml) 280nm; (O-O), activity of the enzyme (nKat). Table I. Purification of Arginine Decarboxylase from 72 hr Germinated Rice Seedlings T o ta l S p . a ctiv ity P u rifi c a tio n Y ield S te p s a c tiv ity (in k at /m g p ro te in ) (fo ld ) (% ) C ru d e 9 2 9 0 6 0 0 .69 1 1 0 0 ( N H4 ) 2 S O 4 4 6 4 5 3 0 1. 3 7 2 5 0 A c eto n e 3 15 8 8 0 2. 6 4 34 S e p h a ro se 4 B F ra c tio n -A 1 6 7 2 2 .24 2.3 0 .1 8 S e p h a ro se -4 B F ra c tio n -B 2 0 4 3 9 3 2 54 3 6 6 2 2 D E A E -C ellu lo se 1 1 14 8 7 4 7 5 7 0 0 12 Arginine Decarboxylase from Oryza sativa L. 741 ratio of specific activity of the two fractions Polyaerylamide gel electrophoresis (A:B) is 1 : 113. Further work was done with Disc gel electrophoresis was carried out in this active fraction which were collected and 10% gel at pH 8.5 using Tris-glycine buffer concentrated with saturated (NH4)2SO4. according to the method ofDavis et al.10) Gels Step 5. The enzyme of the fraction B was were stained with Coomassie brilliant blue and applied to DEAE-cellulose column (17 x 1.7 destained with 7.5% acetic acid. cm) equilibrated with Tris-HCl (50mM, pH 7.0). The column was eluated with a linear pHand temperature optima gradient of KC1 (0~1m) and 5ml fractions The enzymeexhibited a single pHoptimum were collected. Active fractions were pooled at 8.0 in Tris-HCl (0.1m) and the optimal and concentrated by (NH4)2SO4 fractionation temperature was found to be 45°C. followed by dialysis. The results of purification are summarized Effect of substrate concentration in Table I and the above procedure afforded at The enzyme exhibited a typical Michaelis- 700 fold purification of the enzyme with a specific activity of 475 nKat/mg protein and a E- yield of 12%. Criteria of purity -\> / The purity of the enzyme was judged by VD_ / observing a sharp single band of protein in polyacrylamide disc gel electrophoresis. > i i Molecular weight / ] QO3 aO7 The molecular weight of the enzyme was determined by reference to standard curves Fig. 3. 1/V (nKat) is plotted against 1/S Om) from with bovine serum albumin, hexokinase and whichKmvalue of the enzymewas determined. lactate dehydrogenase by gel filtration. As shown in Fig. 2, it was estimated to be 88000 Table II. Amino Acids Composition of for fraction B and 174000 for fraction A. Arginine Decarboxylase (Fraction-B) of Rice Seedlings Fraction A may be a dimer offraction B as the . , Numberof residues ratio of their molecular weight is approxi- Ammoacids , c mately 2: 1. per mol of enzyme Phenylalanine 1 3 Tyrosine 7 Leucine 25 Isoleucine 1 3 Valine 1 8 Alanine 35 ~gk /s ss Glycine 30 Proline 52 Glutamic acid 40 Serine 20 Threonine 20 Aspartic acid 34 Arginine 1 7 I I \l Histidine 1*1 3 12 24 Molecular weight ( x10000) Lysine 1 2 Tryptophan* 6.