Preparation and properties of L-asparaginase from green chillies ( Capsicum annum L.)

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1 J. Biosci., Vol. 2, Number 4, December pp Printed in India Preparation and properties of L-asparaginase from green chillies ( Capsicum annum L.) MOZEENA BANO and V. M. SIVARAMAKRISHNAN Isotope Division, Cancer Institute, Madras MS received 29 October 1979; revised 21 July 1980 Abstract. Green chillies (Capsicum annum L.) and tamarind (Tamarindus indica) contain appreciable amount of L-asparaginase. The enzyme was purified 400-fold from green chillies, by successive precipitations with ammonium sulphate and sodium sulphate, Sephadex-gel filtration and affinity chromatography and the purified enzyme was homogenous on gel electrophoresis. The enzyme exists in two forms, only one having antitumour activity. The purified enzyme has a molecular weight of 120,000 ±500. The N-terminal and the C- terminal amino acids are alanine and phenylalanine, respectively. The enzyme has a sharp optimum ph of 8.5 and a temperature optimum of 37 C. It is stable upto 40 C. The energy of activation is 3 kilo calories. The Km value for the enzyme is 3.3. mm. The enzyme has little action on D-asparagine, which is a strong inhibitor. The enzyme has inseparable glutaminase ctivity and is thus an asparaginase glutaminase. In addition, it possesses urease activity. Keywords. Purification; Capsicum annum; L-asparaginase; asparaginase-glutaminase. Introduction L-Asparaginase (L-asparagine amidohydrolase, E.C ) acquired some clinical importance in 1961, when the antitumour effect of guinea pig serum, originally discovered by Kidd (1953), was traced by Broome (1961) to the presence of this enzyme. The enzyme from Escherichia coli was clinically used to treat patients suffering from asparagines dependent leukemias and lymphomas. A number of undesirable side effects, ascribed to the presence of contaminating bacterial endotoxins in the enzyme preparations were observed (Burchenal et al., 1970). Hence, we attempted to prepare this enzyme from an entirely different source plant materials and study its biochemical and biological properties. Very little work has been carried out on L-asparaginase from higher plants. Chibnall and Grover (1927) demonstrated the presence of an amidase in barley roots capable of hydrolyzing L-asparagine. Lees and Blackeney (1970) studied the distribution of L-asparaginase in Lupinus leuteus and Dolichos lab lab seedlings. A survey of several plants revealed that green chillies (Capsicum annum L.) contained appreciable amounts of this enzyme. In this paper, we report the partial purification and properties of the enzyme from green chillies. 291

2 292 Bano and Sivaramakrishnan Materials and methods The assay system (Meister, 1955) consisted of 0.5 ml of enzyme extract, 0.5 ml of substrate (L-asparagine, 0.04 M) and 1 ml of sodium borate buffer, 0.01 M, ph 8.5. After incubation for 1 h at 37 C, 1 ml of 15% trichloroacetic acid was added to stop the reaction. The ammonia formed was estimated by the method of Greenstein and Leuthardt(1958) by liberating it by the addition of a saturated solution of K 2 CO 3 and aerating it into sulphuric acid traps (10 ml of 0.1 ΝH 2 SO 4 ). To 5 ml of the resulting ammonium sulphate solution, 1 ml of Nessler' s reagent was added. The intensity of colour was measured in a Klett-Summerson colorimeter using the 480 nm filter. Protein was determined by the method of Lowry (1951) using bovine serum albumin as the standard. One unit of L-asparaginase activity was defined as the amount of enzyme required to liberate 1 µmol of ammonia per mg protein per min. Purification of the enzyme from green chillies Fresh green chillies (250 g) about 5-6 cm long and about 1-2 cm broad at the thick end, were homogenized with three volumes of 0.15 Μ KCl solution, centrifuged and the supernatant separated. This was designated as the crude extract. The residue was re-extracted with 0.15 Μ KCi solution and the total volume made upto 200 ml. All the steps were carried out at 4 C. To the crude extract, solid ammonium sulphate was added with stirring to give 60% saturation. The enzyme was collected by centrifugation at 7000 g, dissolved in 20 ml water and reprecipitated by the gradual addition of sodium sulphate to 30% concentration. The precipitate was dissolved in water and further purified by gel filtration through Sephadex G-75 using 0.15 Μ KCl as eluant(sachs et al., 1972). The gel filtration yielded two isoenzymes corresponding to two peaks. As peak I enzyme was in larger amounts and also had a considerably higher specific activity (figure 1), it was taken for further purification by affinity chromatography. Figure 1. Sephadex G-75 chromatography of L-asparaginase

3 L -Asparaginase from green chillies 293 CH-Sepharose-4B, covalently linked to D-asparagine was prepared according to the method of Kristiansen et al. (1970). The enzyme solution was applied to the affinity column (1 5 cm) and the unadsorbed proteins were washed away with the equilibrating buffer. The above procedure resulted in a 400-fold purification with 28% recovery. The enzyme moved as a single band on polyacrylamide gel electrophoresis at ph 8.5. The enzyme was dialyzed against distilled water and concentrated by slow evaporation in a vacuum desiccator. The enzyme powder thus obtained was stored at 4 C (table 1). Table 1. Purification of L-asparaginase from green chillies (Capsicum annum L.). Properties Molecular weight determination: Molecular weight of the enzyme was determined using Sephadex G-100 column calibrated with bovine serum albumin, cytochrome-c from horse heart, ribonuclease, aldolase and chymotrypsinogen. Determination of N-terminal and C-terminal aminoacids in the purified L-asparaginase: The N-terminal amino acid was determined using l-fluoro-2,4-dinitrobenzene (Sanger. 1945). The C-terminal amino acid was identified by hydrazinolysis (Akabori- Ohno, 1952). Results The occurrence of L-asparaginase in various plant sources is presented in table 2. Chillies, tamarind, tomato etc. contain appreciable amounts of the enzyme; lemons, onions and potatoes have only trace amounts, while the enzyme could not be detected in drumsticks and ginger.

294 Bano and Sivaramakrishnan Table 2. L-Asparaginase in various vegetables. (Activity expressed as µmol of ammonia liberated by 1 g of fresh tissue in 1 min.

4 294 Bano and Sivaramakrishnan Table 2. L-Asparaginase in various vegetables. (Activity expressed as µmol of ammonia liberated by 1 g of fresh tissue in 1 min.) Physico-chemical' and kinetic properties of the enzyme The molecular weight of the enzyme was determined to be approximately 120,000 ±500 daltons, with alanine as the N-terminal amino acid and phenylanine as the C- terminal amino acid. The enzyme functioned optimally at ph 8.5 and temperature 37 C. The enzyme activity was linear for 60 min. The K m value for L-asparagine was 3.3 mm. The enzyme hydrolyzed L-glutamine also, the activity being 50% of the activity with L-asparagine (table 3). On the other hand, D-asparagine was a potent inhibitor (table 3). Table 3. Substrate specificity of L-asparaginase. The activity using L-asparagine was taken as 100. The enzyme hydrolyzed urea also. In order to determine whether these activities are inherent properties of the enzyme or due to contaminating enzymes, the glutaminease and urease activities were determined at each step of the purification (table 1).

5 L-Asparaginasefrom green chillies 295 The ratio of L-asparaginase to L-glutaminase activities remained constant at a value of 2.0 (within limits of experimental errors). On the other hand, the ratio of L-asparaginase activity to urease activity increased gradually from 2.56 to 3.93, showing that the urease activity is at least partially separable. At 20 mm, ions of heavy metals like silver, mercury and copper inhibited the enzyme activity very strongly (more than 90%). The enzyme was also inhibited by ferric chloride (90%) and to a lesser extent by zinc and cobalt ions (about 80%). On the other hand, the addition of magnesium chloride to the incubation mixture slightly enhanced the enzyme activity (20%). The enzyme was also inhibited by EDTA (20%), manganese and magnesium ions, magnesium being the most effective. Discussion In higher plants, L-asparaginase has been found to be present in vegetables and we have achieved a 400-fold purification of the enzyme from green chillies. This enzyme was purified from many bacteria and from guinea pig serum through diverse procedures, (Wriston et al. 1973). We adopted affinity chromatography as the final step in the purification procedure, since it is highly specific and can be expected to give considerable enrichment of the enzyme. The homogeneity of the purified enzyme from green chillies is shown by a single band in polyacrylamide and agar gel electrophoreses. In existing in two forms, the green chilli asparaginase resembles more the bacterial enzymes than mammalian. Asparaginases from E. coli Β (Mashburn and Wriston, 1964); E. coli K 12 (Schwartz et al., 1966) and Acinetobacter calcoaceticus, all exist in two forms, but only one having antitumor potential. On the other hand, the guinea pig serum enzyme yields only one sharp peak. The exact differences between the different forms of the same enzyme have not been worked out. Table 4. L-Asparaginase, L-glutaminase and urease activities of the green chilli enzyme at various stages of purification. (The substrates L-asparagine, L-glutamine and urea were used at a concentration of 40 mm. The enzyme activity is expressed as mg ammonia liberated per min.).

6 296 Bano and Sivaramakrishnan The properties of L-asparaginase from green chillies show a number of similarities and some sharp differences when compared with the properties of L-asparaginases from other sources (summarised in table 5). The molecular weight of the enzyme Table 5. Properties of L-asparaginases from different sources. N. D. = Not determined; N.A. = Information not available from green chillies was found to be 120,000 ±500; which closely resembled the enzymes from guinea pig serum or E. coli, most of which have a molecular weight around 130,000 daltons. L-asparaginase from green chillies had L-alanine at the N- terminal end and L-phenylalanine as the C-terminal amino acid and resembled the enzyme in Erwinia aroides (Staerk et al., 1971) which had alanine as the N-terminal amino acid. The optimum ph of 8.5 for the green chilli enzyme resembled those of E. coli Β and A. calcoaceticus which also had ph optima around 8.5 but differed from the guinea pig serum enzyme which showed a wide range of optimal ph from The K m value for the chilli enzyme is 3.3 mm. This was considerably larger than the K m values for most of the asparaginases from other sources, which are of the order of 0.01 mm. The enzyme from green chillies has thus a lesser affinity for the substrate L-asparagine. The enzyme from green chillies is actually an 'asparaginaseglutaminase enzyme. A number of such enzymes with both glutaminase and asparaginase activities were purified from Pseudomonas (Greenberg et al., 1964),

7 L-Asparaginase from green chillies 297 from A. glutaminasificans (Roberts et al., 1972) and from A. eutrophus (Allison et al., 1971). It is not certain whether the presence or absence of L-glutaminase activity in L-asparaginase is an advantage or hindrance in the treatment of cancer. It is believed that L-glutaminase by itself is an anti-tumour agent (Roberts et al., 1970). The presence of urease activity in the purified L-asparaginase preparation was totally unexpected. The urease activity was separable, at least partially. As such, the enzyme may not be a general amidase. We have examined a few commercial preparations of L-asparaginase (all from E. coli) and found urease activity. The presence of urease activity can have very adverse effects on patients, By hydrolyzing blood urea, it can give rise to ammonia toxicity. The experiments reported here show that magnesium is an activator for L-asparaginase from green chillies. The inhibition of the enzyme activity by heavy metal ions like Ag +, Hg 2+ ; and Cu 2+ is to be expected. Acknowledgements We thank Dr S. Krishnamurthi for his keen interest and constant encouragement. The financial support from the Council of Scientific and Industrial Research, New Delhi for this project is acknowledged. References Akabori-Ohno and Narita (1952) Bull. Soc. Chem. Jpn., 25, 214. Allison, J. P., Mandy, W. J. and Kitto. G. Β. (1971) FEBS Lett., 14, 107. Broome, J. D. (1961) Nature, 191, Burchenal, J. Η. and Kanofsky, D. A. (1970) Cancer, 25, 241. Chibnall, A. C. (1939) Protein metabolism in the plant, (New Haven:Yale Univ. Press) Davidson, L. (1977) Biochim. Biophys. Acta, 480, 282. Greenberg, D. M., Ramadan, M. E. A. and Asmar, F. E. (1964) Arch. Biochem. Biophys., 108, 143, 150. Greenstein, J. P. and Leuthardt, F. M. (1958) Arch. Biochem. Biophys., 17, 105. Kidd, J. G. (1953) J. Exp. Med., 98, 565, 583. Kristiansen, Τ., Einarsson, M., Sundberg, Μ. and Porath, J. (1970) FEBS Lett.,7, 294. Lees, E. M. and Blackeney, A. B. (1976) Biochim., Biophys. Acta., 215, 145. Lowry, O. H., Rosenbrough, N. J., Farr, L. A. and Randal, R. J. (1951) J. Biol. Chem., 193, 265. Mashburn, L. T. and Wriston, J. C. (1964) Arch. Biochem. Biophys. 105, 450. Meister, A. (1955) Methods Enzymol., 2, 383. Roberts, J., Holcenberg, J. S. and Dolwy, W. C. (1970) Nature, 227, Roberts, J., Holcenberg, J. S. and Dolowy, W. C. (1972) J. Biol. Chem., 247, 84. Sachs. D. H. and Painter, E. (1972) Science, 175, 781. Sanger, F. (1945) Biochem. J., 39, Staerk, J. Zwisler, Ο. and Ronneebercger, H. (1971) Experientia. 27, 250. Schwartz, J. Η., Reeves, J. Υ. and Broome, J. D. (1966) Proc. Nat. Acad. Sci. USA. 56, Wriston, J. C., Jr. and Yellin, T. O. (1973) Adv. Enzymology, 39, 185.

Control of ornithine decarboxylase activity in jute seeds by antizyme

J. Biosci., Vol. 15, Number 2, June 1990, pp. 83-91. Printed in India. Control of ornithine decarboxylase activity in jute seeds by antizyme MALABIKA PANDIT and BHARATI GHOSH Department of Botany, Bose