Purification and General Properties of Argininosuccinate Lyase from Jack Bean, Canavalia Ensiformis (L.) DC

Biochem. J. (1969) 112,415 415 Printed in Great Britain

Purification and General Properties of Argininosuccinate Lyase from Jack Bean, Canavalia ensiformis (L.) DC

BY GERALD A. ROSENTHAL* AND AUBREY W. NAYLOR Department of Botany, Duke Univer8ity, Durham, N.C. 27706, U.S.A. (Received 23 December 1968)

1. Argininosuccinate lyase (EC 4.3.2.1) from jack bean [Canavalia ensiformi8 (L.) DC] seeds was purified 532-fold from an acetone-butanol-dried powder. 2. The enzyme functions reversibly and exhibits maximum stability at 160. 3. At 16° it has a half-life (ti) of 263min. 4. The enzyme is both cold-labile (t, 131min. at 00) and heat-inactivated (t, 74min. at 380); inactivation appears to be irreversible. 5. Treatment of the acetone-butanol-extracted powder with sodium dodecyl sulphate increased the sensitivity of the enzyme to temperature (ti 70min. at 00; t, 23min. at 380). 6. Addition, to the purified enzyme, of a fraction containing lipid from the seed increased the half-life to about 5lOmin. at either 00 or 380. 7. Arginine or homoarginine, and to a smaller extent some other amino acids or fumarate, protected the enzyme from cold-inactivation. 8. Reactivation attempts with both the cold- and heat-inactivated enzyme failed. 9. The Km value for argininosuccinate at pH7-5 is 1-3 x 10-4. 10. The enzyme was inactivated completely within 15min. at 16° by 0-5mM-p-hydroxymercuribenzoate, and subsequent exposure to 5mM- cysteine had no restorative effect.

Argininosuccinate lyase (L-argininosuccinate buffer systems, but was reversibly cold-labile in arginine-lyase, EC 4.3.2.1) catalyses a reversible tris buffer. Thermal reactivation of the cold- non-hydrolytic cleavage of L-argininosuccinate to inactivated enzyme occurred after several minutes L-arginine and fumarate (Ratner, Anslow & at 380. Arginine and argininosuccinate (2mM) and Petrack, 1953), and is operative in the ornithine- phosphate (50mM) prevented cold-inactivation. urea cycle. Isolation of the enzyme from plant Thiol-group titration studies of the fully active material was first achieved by Davison & Elliott crystallized enzyme demonstrated the presence of (1952), by isoelectric precipitation of an aqueous one freely available thiol group/enzyme molecule. extract of dry pea meal. Walker & Myers However, the inability of a wide variety of reagents (1953), demonstrated the presence of arginino- to inhibit the enzyme led Havir et al. (1965) to succinate lyase in commercial jack-bean meal and in suggest that this thiol group is not essential for Chlorella pyrenoido8a. Subsequently, Buraczewski, enzymic activity. Kleczkowski & Reifer (1960) reported increased This paper summarizes results from our studies of amounts of arginine in wheat and rye-grass the argininosuccinate-cleaving enzyme present in seedlings that had been supplied with L-arginino- vascular-plant material. Our investigations reveal succinate. Although Ennis & Gorini (1961) several differences between the argininosuccinate demonstrated the occurrence of this enzyme in lyase isolated from jack bean and that from ox liver. various strains of E8cherichia coli, their study did not include a description ofpurification procedures. EXPERIMENTAL While our investigation was in progress, Cohen & Bishop (1966) published a procedure for the Material8 isolation of argininosuccinate lyase of high specific L-Argininosuccinate (barium salt) was obtained from activity from Neuro8pora crassa. Calbiochem, Los Angeles, Calif., U.S.A. Free arginino- Havir, Tamir, Ratner & Warner (1965) developed succinic acid was prepared from the barium salt by the a procedure for preparing crystalline arginino- addition of 1 0ml. of 0-5M-K2SO4/50,umoles of substrate. succinate lyase from ox liver. Their crystallized Sodium dodecyl sulphate was purchased from Matheson, enzyme was stable at 380 in both tris and phosphate Coleman and Bell, East Rutherford, N.J., U.S.A., and free base L-arginine from Sigma Chemical Co., St Louis, Mo., * Present address: Department of Biology, Case Western U.S.A. Crystalline fumarase, disodium fumarate, and all Reserve University, Cleveland, Ohio 44106, U.S.A. amino acids, except L-arginine, were obtained from Cal- 416 G. A. ROSENTHAL AND A. W. NAYLOR 1969 biochem. Jack-bean (Canavalia en8iformi8) seeds were The acetone-butanol-dried cake was spread out bought from Mr Ernest Nelson, Waldron, Ark., U.S.A. at room temperature until the residual butanol Calcium phosphate gel was prepared by the procedure of evaporated. After drying, the meal was ground Keilin & Hartree (1938) as outlined by Dixon & Webb into a fine powder in a mortar and stored at - 200. (1964), and was aged for 6 months in the dark at 20. The weight of the final powder was 40-45% of that of the hydrated seeds. Methods Step I. Preparation of the homogenate. Unless Arginine assay. Arginine assays were conducted with otherwise indicated, the buffer system was 50mm- double-strength reagents in a final volume of 6-0ml. by the potassium phosphate buffer, pH7 5, and centri- Sakaguchi (1925) method as modified by Van Pilsum, fuging was conducted at 12000g for 13min. Martin, Kito & Hess (1956). Over the range 0-02-02mm, Acetone-butanol-dried powder (45g.) was extracted E516 was proportional to arginine concentration. with 250ml. of buffer for 60min. at 2°. After Definition of activity. One unit of enzyme catalyses the formation of 1 ,mole of arginine/min. under the described filtration through four layers of cheesecloth and conditions. Specific activity is defined as milliunits/mg. of centrifuging, the supernatant solution was diluted protein. Protein content was determined by the method of with buffer to 215ml. Lowry, Rosebrough, Farr & Randall (1951), with crystal- To minimize inhibition by arginine and canavan- lized bovine plasma albumin as a standard. ine, the activity was determined after dialysing Enzyme a88ay. The standard assay mixture contained lOml. samples of the supernatant solution for 8hr. 4 0umoles offreshly prepared argininosuccinate, 100 moles at 16°. The main solution was dialysed against of potassium phosphate buffer, pH7 5, and excess of approx. 600ml. of buffer containing 0-1mM-EDTA. fumarase. After 2min. equilibration at 30°, the total II. Ammonium fractionation. The volume was adjusted to 2-0ml. by the addition of not more Step sulphate than 35 milliunits of enzyme. Similarly incubated reaction homogenate was adjusted to 25% saturation by mixtures containing all ofthe components except substrate adding saturated ammonium sulphate at pH6-6. were used as controls. Argininosuccinate itself does not After standing at 20 for lOhr., the turbid solution produce a significant colour in the modified Sakaguchi was centrifuged for 20min. (After lOhr. a gradual reaction. decrease in the enzyme activity of the solution After incubation for 20min. at 300, 3-0ml. of 10% (w/v) occurred.) The precipitate was discarded. The trichloroacetio acid was added. After centrifuging at supernatant solution, adjusted to 37% saturation 20000g for 10min., duplicate samples (I.Oml.) of the with ammonium sulphate, was kept for 90min. and supernatant solution were assayed for arginine. Care was centrifuged. taken to prevent contamination of the arginine-assay tubes with precipitated protein. At this stage of purification the enzyme became Argininosuccinate lyase isolated directly from hydrated cold-labile, and, unless otherwise indicated, all seeds showed far less activity than an equivalent sample of subsequent purification steps were conducted at acetone-butanol-dried powder, and it was suspected that room temperature. The precipitate was suspended the very high concentration of lipid present interfered with in 20mm-phosphate buffer, pH 6-8, homogenized enzymic activity and its measurement. Experiments and diluted to 100ml. supported this conclusion. Further, lipids interfered with all Step III. Negative adsorption with calcium procedures used to purify this enzyme directly from the phosphate gel. Calcium phosphate gel suspension hydrated seed. An acetone-butanol-dried powder therefore (10ml., 32-1mg. dry wt./ml.) and of served as the starting material for the enzyme used in most 440,umoles of the studies reported here. L-arginine were added to the suspended residue of step II. The resulting mixture was stirred mechanically for 15min. and centrifuged at 31 OOOg for RESULTS 12min. Purification of the enzyme. Jack-bean seeds, Step IV. Second ammonium sulphate fractiona- submerged in water for 24hr., were peeled and then tion. Saturated ammonium sulphate, pH 7 75, was stored overnight at -20°. Freezing of the seeds, added to the supernatant solution to make it 35% before the preparation ofthe acetone-butanol-dried saturated. After 45min., the precipitated protein powder, increased the yield of enzyme. The frozen was removed by centrifugation at 31 OOOgfor 15min. seeds were blended with 2vol. of freshly distilled and dissolved in 15-16ml. of buffer. Sometimes the acetone at - 200. The resulting mash was stirred precipitate was stored overnight at -20° in with an additional 3vol. of cold acetone, and the ammonium sulphate solution. suspension filtered by suction. Step V. Sephadex gel filtration. The dissolved Freshly distilled butan-l-ol (3vol.) at -20° protein from step IV was passed through a column was mechanically stirred with the acetone-extracted (2.5cm. x 45cm.) of Sephadex G-200. The enzyme cake for 4min. in a sodium chloride-ice bath. The was eluted with buffer at 160 containing 4mM-L- suspension was filtered (20-30min.) at 20. Butanol arginine hydrochloride, at a flow rate of 36ml./hr. treatment removes acetone-insoluble phospholipids Fractions (3ml.) were collected (Fig. 1) and the (Morton, 1955). contents of the three tubes containing the most Vol. 112 JACK-BEAN ARGININOSUCCINATE LYASE 417 enzyme activity were pooled for further purification. this manner is referred to below as 'treated' As shown in Fig. 1, excellent separation of the enzyme. enzyme from other proteins was achieved with Characterization of the reaction. Identification of Sephadex. fumarate as one of the reaction products was Step VI. Third ammonium sulphate fractiona- established by incubating in a spectrophotometer tion. The pooled eluate was adjusted to 25% cuvette 3,umoles of L-argininosuccinate, 25 milli- saturation with ammonium sulphate solution units of enzyme and 100lmoles of buffer, pH7-5, (pH7.75). After 30min. the precipitated protein (final vol. 3 0ml.). A linear increase in E240 was collected by centrifugation at 31 0OOg for occurred, due to fumarate formation (Racker, 13min. and the supernatant solution was adjusted 1950). The addition of fumarase resulted in a rapid to 33% saturation with ammonium sulphate. The decrease in E240. protein precipitate was allowed to form for 45min., To determine whether the enzyme catalyses collected by centrifuging for 13min. at 31 OOOg and arginine-dependent fumarate utilization, 3-0ml. of dissolved in 2-3ml. of buffer. A summary of the a mixture containing 1.2 ,moles of fumarate, purification procedure for the isolation of the 100,umolesofphosphate buffer, pH 7 5,25 milliunits enzyme is given in Table 1. ofenzyme and either water or 5 0 ,umoles of arginine In certain experiments enzyme treated with was monitored at 240nm. A linear decrease in sodium dodecyl sulphate was used. Such treated extinction occurred only when arginine was enzyme was prepared by the addition of 3'5g. of present, indicating that the argininosuccinate- sodium dodecyl sulphate to the acetone-butanol- cleaving reaction is reversible. dried powder before extraction with the buffer Effect of 8ub8trate concentration. The Km value (see Step 1). Argininosuccinate lyase prepared in for argininosuccinate at pH 7-5 was about

W 0 0-8 0.8 0-7 0-7 1-g- .n 0-6 0-6

° 0-5 0-5 - .9 *1- 0 4 do 0 03 0- 3 003°> '4-, Cs 0-2 0-2 ._1 0 0.1 0 4 II Tube no. Fig. 1. Elution ofprotein from step IV from Sephadex G-200 (see the text). Argininosuccinate lyase activity was assayed in the standard system, with 0 2ml. of each fraction. El'm- was measured on fractions diluted 16-fold. *, Arginine formed; o, E280.

Table 1. Purification of argininoauccinate Iya8ee from acetone-butanol dried Canavalia ensiformis 8eed8 For details see the text. Volume Total protein Total enzyme Specific activity Yield Step (ml.) (mg.) (milliunits) (units/mg. of protein) (%) I. Extract 215 9250 10295 1.1 II. First (NH4)2SO4 fraction 100 490 9935 20-3 97 III. Calcium phosphate gel supernatant 110 240 8965* 37-4 87 IV. Second (NH4)2SO4 fraction 16 89 7440 83*6 72 V. Sephadex G-200 eluate 9 5.5 1 695* 308-2 16 VI. Third (NH4)2SO4 fraction 2 1-4 820 585-7 8 * Corrected for arginine inhibition. 418 G. A. ROSENTHAL AND A. W. NAYLOR 1969 Table 2. Effect of various temperature8 on the activity Table 4. Capacity of variowsu reagents to protect of 'treated' (see the text) argininosuccinate Iyase after argininosuccinate lyase from cold-lability and heat- 8tanding for 90min. in 50mM-potassium phosphate inactivation buffer, pH 7-5 Sodium dodecyl sulphate-treated enzyme (0-95ml.) was The reaction mixture contained lOO,umoles of potassium mixed with 41,moles of the indicated reagents in a final phosphate buffer, pH7-5, 4-O,umoles of argininosuccinate, volume of I-Oml. After standing at the given temperature excess of fumarase and 347,Ig. of enzyme solution (specific for 90min., 0-1 ml. samples were removed for assay. The activity 78 milliunits/mg. of protein). The final volume reaction mixture contained 100l moles of potassium was 2-0ml. Incubation was conducted for 20min. at 300. phosphate buffer, pH7.5, 4-O,umoles of argininosuccinate L-Arginine formed and excess of fumarase in a final volume of 2-Oml. Arginine Temp. in 20min. (,umole) was assayed, as described in the text, after 20min. incubation at 30°. Initial activity 0-50 % of initial % of initial 00 0-22 activity activity 11 0-41 remaining after remaining after 16 0-42 Additions 90min. at 00 90min. at 38° 22 0-34 30 0-17 Water 35 8 38 0-02 Arginine 96 Canavanine 67 30 Fumarate 62 26 Table 3. Inactivation rate constants and half-life Arginine+ fumarate* 94 values for argininosuccinate lyase Water 39 6 Citrulline 52 17 See the text for details of treatment. Ornithine 48 Rate constant Half-life Glycine 40 Treatment Temp. (min.-') (min.) Aspartatet 39 6 None 00 5-30 x 10-3 131 Homoarginine 90 16 2-64 x 10-3 263 Water 36 9 38 9-39 x 10-3 74 Cysteine 48 17 Sodium dodecyl 0 9-96 x 10-3 70 Methionine 57 20 sulphate 16 3-02 x 10-3 229 Reduced glutathione 42 12 38 2-95 x 10-2 23 Water 38 Isoleucine 46 Phenylalanine 43 1-3 x 10-4M. The substrate concentration used in Valine 71 the standard assay mixture was virtually saturating. * Cold-lability and heat-inactivation. The effect of Two ,umoles of each. t Aspartic acid (0-lOml.) treated with 0-90ml. of enzyme standing at various temperatures on the activity of solution. 'treated' argininosuccinate lyase is shown in Table 2. Stability of the 'treated' and untreated argininosuccinate-cleaving enzyme is compared at Excellent protection was provided by homo- 00, 160 and 380 in Table 3. arginine and very good protection by valine, The data of Table 3 reveal a decrease in the canavanine, methionine and fumarate. half-life (t2) for the 'treated' enzyme compared Reversal of cold-lability. 'Treated' and untreated with the untreated enzyme. At 160 the half-life enzyme was exposed to 0° for 90 and 110min. difference was small, but at 0° and 380 the difference respectively in the presence or absence of 4mM-L- was about twofold and threefold respectively. In canavanine, and then reactivation at 380 for 8min. contrast, argininosuccinate lyase prepared directly was attempted. No reactivation occurred, in from the hydrated seed rather than from an acetone- contrast with the results of Havir et al. (1965), who butanol-dried powder had a half-life of about found that cold-inactivated argininosuccinate lyase 510min. at either 0° or 38°. from ox liver could be fully reactivated by incuba- Protection against cold-lability and heat- tion for 6min. at 38°. Although canavanine is inactivation. The instability of the 'treated' capable of decreasing cold-inactivation of the enzyme at 00 and 380 could be overcome to different enzyme, this protecting agent does not enhance degrees by the addition of the reagents listed in thermal reactivation of either the treated or the Table 4. A high degree of protection against untreated enzyme. cold-lability was conferred by addition of substrates Lipid recombination study. A single experiment fostering the reversal of argininosuccinate cleavage. was performed to ascertain if highly purified Complete protection against cold-lability was argininosuccinate lyase could be protected against provided by arginine or by arginine plus fumarate. heat-inactivation by the addition of lipid material Vol. 112 JACK-BEAN ARGININOSUCCINATE LYASE 419 from hydrated seeds. The lipid material was extracted as follows. DISCUSSION Approx. 50g. of hydrated seeds were blended This is the first report of highly purified arginino- with 125ml. of 50mi-potassium phosphate buffer, succinate lyase from seed plants. The 523-fold pH 75. After centrifugation, the supernatant purified enzyme appeared to contain only minor solution was adjusted to 40% saturation with contamination as judged by disc electrophoresis at saturated ammonium sulphate, pH 6-6. After pH7*2 and pH8-6. With increasing purity a 90min. the suspension was centrifuged. Most of decrease in stability was noted, and a lipid- the lipid floated as a cake; this material was containing fraction derived from the seed restored dialysed exhaustively against water at 00. A stability. Jack-bean argininosuccinate lyase puri- suspension of the enzymically inactive 'lipid fied from an acetone-butanol-dried powder is a fraction' (containing some protein) in 50mM- cold-labile and heat-sensitive enzyme (Table 2). potassium phosphate was added to an equal Loss of activity is apparently irreversible. It is volume of highly purified enzyme, and the mixture suggested that this enzyme is, in situ, associated was exposed to 380 for 60min. The enzyme, thus with and stabilized by lipid material. protected, synthesized nearly five times as much arginine as did enzyme similarly warmed in the absence of the 'lipid fraction'. Disc electrophoresis (Davis, 1964) of the third REFERENCES ammonium sulphate fraction at pH 7 2 and pH8 6 Buraczewski, S., Kleczkowski, K. & Reifer, I. (1960). revealed a single, minor, contaminant band Bull. Acad. polon. Sci., Ser. sci. biol. 8, 93. migrating above the argininosuccinate lyase. Cohen, B. B. & Bishop, J. 0. (1966). Genet. Res. 8,243. Effect of thiol reagents. Incubation of a control Davis, B. J. (1964). Ann. N. Y. Acad. Sci. 121, 404. sample of enzyme for 30min. at 160 caused a Davison, D. C. & Elliott, W. H. (1952). Nature, Lond., 169, decrease in activity of 7%; with 5mM-iodoacetate 313. and 5mM-N-ethylmaleimide the decrease was 23% Dixon, M. & Webb, E. C. (1964). Enzymes, 2nd ed., p. 950. and 57% respectively. p-Hydroxymercuribenzoate London: Longmans, Green and Co. Ltd. was much more effective, causing complete loss of Ennis, H. L. & Gorini, L. (1961). J. molec. Biol. 3, 439. Havir, E. A., Tamir, H., Ratner, S. & Warner, R. C. (1965). activity within 15min. at a concentration of J. biol. Chem. 240, 3079. 0 05mM; this was not reversed by 5mM-cysteine. Keilin, D. & Hartree, E. F. (1938). Proc. Roy. Soc. B, 124, Both untreated and 'treated' enzymes were 397. inhibited similarly. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, Enzyme storage. Normally enzyme preparations R. J. (1951). J. biol. Chem. 193, 265. were stored as a pellet in ammonium sulphate Morton, R. K. (1955). In Methods in Enzymology, vol. 1, solution, after the final centrifugation at step IV p. 25. Ed. by Colowick, S. P. & Kaplan, N. 0. New or VI, at - 200 for 2-4 days with little loss in York: Academic Press Inc. activity. Dissolved enzyme, however, was stored Racker, E. (1950). Biochim. biophys. Acta, 4, 211. - Ratner, S., Anslow, W. P., jun. & Petrack, B. (1953). in the presence of 4mM-L-canavanine at 200. In J. biol. Chem. 204, 115. both cases the enzyme was rapidly frozen in an Sakaguchi, S. (1925). J. Biochem., Tokyo, 5, 133. acetone-solid carbon dioxide bath before storage. Van Pilsum, J. F., Martin, R. P., Kito, E. & Hess, J. (1956). The enzyme can also be stored as a freeze-dried J. biol. Chem. 222, 225. preparation. Walker, J. B. & Myers, J. (1953). J. biol. Chem. 203, 143.