Properties of arginase from the sea mollusc Concholepas concholepas

Comp. Biochem. Physiol. VoL 78B, No. 3, pp. 591-594, 1984 Printed in Great Britain

0305-0491/84 $3.00+ 0.00 © 1984 Pergamon Press Ltd

PROPERTIES OF ARGINASE FROM THE SEA MOLLUSC CONCHOLEPAS CONCHOLEPAS N. CARVAJAL, M. BUSTAMANTE,P. HINRICHSEN and A. TORRES Departamento de Biologia Molecular, Facultad de Ciencias Biol6gicas y de Recursos Naturales, Universidad de Concepci6n, Casilla 2407, Concepci6n, Chile

(Received 4 January 1984) Abstract--1. Arginase activity was detected in homogenates prepared from the gill of the sea mollusc Concholepas concholepas. From this tissue a partially purified preparation (sp. act. 30 units/mg of protein) was obtained and characterized. 2. The metal ion requirement of the enzyme is satisfied by Mn 2÷, Co 2÷ and Ni 2+ and a significant inhibition of the Mn2+-activated enzyme is caused by Cd 2+, Mg 2+ and Zn 2+. 3. The enzyme exhibits Michaelis-Menten kinetics and at the pH optimum of 9.5 the Km for arginine was found to be 25 mM. Lysine and the product ornithine are competitive inhibitors while the inhibition caused by branched chain amino acids and proline is non-competitive. 4. The enzyme has a molecular weight of about 27,500, which is in the order of the molecular weight of the subunits of oligomeric arginases. The molecular weight of the enzyme from Concholepas concholepas is not altered by addition or withdrawal of metal ions.

in the presence and absence of 1 mM MnCI2. The column was standardized with Blue Dextran 2000, human liver arginase, bovine serum albumin, ovalbumin and myoglobin.

INTRODUCTION Arginase (EC 3.5.3.1.) catalyzes the hydrolysis of arginine into ornithine and urea, which is the final step in the urea cycle of the liver of ureotelic organisms. The enzyme is not exclusive, however, of organisms with a ureotelic pattern of nitrogen excretion but it is widely distributed in biological systems. A m o n g marine invertebrates, highest activities of arginase have been found in the Crustacea and particularly in the digestive gland of Carcinus maenas (Hanlon, 1975). Low levels were found in the gastropods Nassa obsoleta, Busycon canaliculatum and Littorina litorrea (Hanlon, 1975) but significant activities were observed in other species of Littorina (Gaston and Campbell, 1966). This paper shows the presence of arginase in the gill of the sea mollusc Concholepas concholepas and compares its properties with those of the enzyme in other systems.

Materials Amino acids, proteins used as molecular weight markers, Trizma Base, DEAE-cellulose and p-hydroxymercuribenzoate (PHMB) were purchased from Sigma Chemical Co. Sephadex G-200 and Blue Dextran 2000 were products of Pharmacia, Uppsala. Human liver arginase was purified as described by Bascur et al., (1966). All other chemicals were analytical grade. RESULTS AND DISCUSSION

MATERIALS AND METHODS Animals were obtained from commercial fishermen and homogenates were prepared and fractionated at 4°C. Arginase activity was determined by measuring the formation of urea (Archibald, 1945) from arginine. The standard reaction mixture contained 50 mM glycine (pH 9.5), 1 mM MnCI2 and 125 mM arginine, and reactions were initiated by adding the enzyme. A preincubation of enzyme with metal ions was not required for maximal activation. For pH optimum, the buffers used were: 50mM Tris-HCl (pH 7.0-8.7) and 50raM glycine-NaOH (pH 8.7-10.5). One unit of arginase is defined as the amount of enzyme that produces I #mole urea per rain at 37°C. Protein concentrations were determined by the method of Lowry et al. (1951). Treatment with EDTA at a concentration of 30 mM in 50mM Tris-HCl (pH 8.0) was performed as described (Carvajal et al., 1971). Molecular weights (Mr) were determined by gel filtration on Sephadex G-200 columns (60 cm x 1.2 cm). Elution was performed with 10mM Tris-HC1 (pH 7.5) containing 20 mM KC1 and the activity of the fractions was determined 591

Tissue distribution and isolation o f arginase Tissues of Concholepas concholepas were homogenized with 50 m M Tris-HCl (pH 7.5) containing I m M MnCI2 and 100mM KC1. Significant activity (12.5 units per gram (wet wt) of tissue) was associated with the gill but the enzyme was absent or almost undetectable in other tissues. This activity is considerably higher than that found in other marine invertebrates (Hanlon, 1975). Arginase was partially purified by fractionation of the homogenate with (NH4)2 SO4 (35-70~ saturation) followed by chromatography on a DEAE-cellulose column equilibrated with 5 m M Tris-HCl (pH 7.5). After washing with this buffer, the enzyme was eluted with 0.1 M arginine. Active fractions were pooled, dialyzed and finally concentrated by lyophilization. This enzyme preparation with a sp. act. of 30 units/mg of protein was used for the experiments that follow. Effect o f metal ions Arginase requires a divalent metal cation for activity (Greenberg, 1960) and studies with the enzyme from different sources indicate that the requirement is satisfied mainly by M n 2+, Co 2+ and Ni 2+ (HirschKolb et al., 1971; Baret et al., 1972). As shown in

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Table 1. Effect of metal ions on arginase Enzyme species and salt added

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The enzymeswere incubated with the metal ions at a concentration of 5raM for 15rain at 37°C and then assayed for enzymatic activity. In experiment A, 100% represents the activity of arginase before the treatment with EDTA. Table 1, Concholepas concholepas arginase previously inactivated by EDTA regained activity in the presence of Mn 2+, Co z+ or Ni 2÷ and the Mn2+-activated enzyme was inhibited by Cd 2+, Mg 2÷ and Zn 2+. Staphylococcus aureus arginase is also inhibited by these cations (Soru and Zaharia, 1976). This is interesting because M f + and Zn 2÷ have no effect on rat liver (Bond et al., 1983), rat kidney (Kayser and Strecker, 1973) and beef liver arginases (Bond, 1973) and there are instances of activation by Cd 2÷ (HirschKolb et al., 1971; Campbell, 1966). It appears, therefore, that apart from the general activating effect of Mn 2+, Co 2+ and Ni 2+, the effects of other metal ions will depend on the sources of the enzyme. Due to its stronger activating effect, Mn 2+ was selected for subsequent studies. p H optimum The pH optimum was 9.5. This is close to the pH optima for several other arginases which are in the range from 9.3 to 10.5 (Soru and Zaharia, 1976; Campbell, 1966; O'Malley and Terwilliger, 1974; Bascur et al., 1966). Substrate kinetics Studies at pH 7.5, 8.7 and 9.5 revealed Michaelis-Menten kinetics even in the presence of ornithine and also indicated the absence of inhibition by excess substrate. By contrast, the enzymes from the marine crustacea Carcinus maenas (Hanlon, 1975), Neurospora crassa (Carlisky et al., 1968) and some vertebrates (Tarrab et al., 1974; Carlisky et al., 1972) are significantly inhibited by excess arginine. On the other hand, human liver arginase exhibits cooperative effects at pH 7.5 (Carvajal et al., 1982) and beef liver arginase is allosterically affected by ornithine (Bedino, 1977). From the results in Fig. 1 the Km for arginine of Concholepas concholepas arginase was found to be 17 mM at the pH optimum of 9.5. Published values are in a wide range. A value of 1.8 mM was obtained for the enzyme from Staphylococcus aureus (Soru and Zaharia, 1976) whereas values from 3 to 20 mM were obtained for mammalian arginases (Hirsch-Kolb et al., 1970; Bascur et al., 1966) and many uricotelic species have arginases with Km from 100 to 200 mM (Mora et al., 1965). Among invertebrate systems, Campbell (1966) reported that arginase from the hepatopancreas of Otala lactea have a Km of 3-9 mM

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and a value of about 40mM was estimated by Hanlon (1975) for the enzyme from Carcinus maenas. The data in Fig. 1 also show that ornithine is a linear competitive inhibitor with a K, value of 6.2 raM. This amino acid is a competitive inhibitor for most arginases (Roche et al., 1954; Bond, 1973: Campbell, 1966; Soru and Zaharia, 1976). Exceptions are one of the molecular forms of human liver arginase (Bascur et al., 1965) and the bullfrog renal enzyme (Carlisky et al., 1968) which are inhibited non-competitively by ornithine. Effects of amino acids In addition to ornithine, lysine was also a competitive inhibitor while valine, isoleucine, leucine and proline behaved as non-competitive inhibitors of arginase from Concholepas concholepas. Other amino acids tested (alanine, serine, cysteine, tryptophan, norleucine, phenylalanine, glutamate and aspartate) showed no inhibition or activation of the enzyme. In general, these results are in accord with those commonly observed with the enzyme from other species (Bascur et al., 1966; Bond, 1973; Kayser and Strecker, 1973; Soru and Zaharia, 1976). Effect o f p-hydroxymercuribenzoate The activity of arginase from Concholepas concholepas was not affected by PHMB at concentrations up to 2 mM. Other arginases are inhibited (Mora et al., 1966; Soru and Zaharia, 1975), unaffected (Mora et al., 1966; Carvajal et al., 1982) or slightly stimulated (O'Malley and Tergeilliger, 1974) by reaction with PHMB. Effect o f temperature on reaction rate Under standard assay conditions maximum activity was observed at 43°C and from the Arrhenius plot of the data the energy of activation was found to be 7.8 kcal/mole (32.6 kJ/mole). This value is close to those reported for the enzyme from several other

Properties of arginase from the sea mollusc Concholepas coneholepas

593

In conclusion, arginase from Concholepas concholepas is a low molecular weight enzyme which exhibits many of the general properties of arginase in other systems. We are now initiating experiments to

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study the role of arginase in Concholepas concholepas and, consequently, to understand its localization in the gill of the mollusc. In any case, the comparison of arginase from Concholepas concholepas with the enzymes from many ureotelic and non-ureotelic species supports the notion that there is no direct correlation between the general properties of arginase and the particular pattern of nitrogen excretion of a given organism (O'Malley and Terwilliger, 1974).

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Acknowledgements--This investigation was carried out with financial support from the Departamento de Biologia Molecular, Facultad de Ciencias Biol6gicas y de Recursos Naturales, Universidad de Concepci6n.

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Fig. 2. Gel filtration of Mn2÷-activated (A) and EDTAinactivated (B) arginase on Sephadex G-200. The activity of the fractions was assayed in the absence (O) and presence (O) of I mM MnCl2. The inset shows the estimation of the molecular weight of the enzyme. Molecular weight markers used were myoglobin (1) ovalbumin (2), bovine serum albumin (3) and human liver arginase (4). Ve is the elution volume of the proteins and Vo the void volume of the column. The points in the inset represent the average value of three determinations. sources (Soru and Zaharia, 1976; Tarrab et al., 1974; Campbell, 1966).

Molecular weight Arginase was activated with 1 m M MnC12 for 15 min at 37°C, and then filtered into a Sephadex G-200 column. After chromatography the enzyme showed a mol. wt of about 27,500 (Fig. 2A) and exhibited essentially the same activity in the absence and presence of added M n 2+. The enzyme was also treated with E D T A and then examined by gel filtration. Such preparation exhibited a single activity peak corresponding also to Mr 27,500 but detected only in the presence of M n 2÷ (Fig. 2B). This molecular weight is close to the values described for the subunits of arginase from other sources (Hirsch-Kolb and Greenberg, 1968; Carvajal et al., 1971; O'Malley and Terwilliger, 1974). However, whereas the molecular weight of arginase from Concholepas concholepas is not altered by addition or withdrawal of metal ions, subunits of oligomeric arginases are inactive in the absence of M n 2+ and reassociation to active species occurs in the presence of the metal ion. The molecular weight of the arginases so far studied are about 120,000 to 260,000 (Hirsch-Kolb and Greenberg, 1968; O'Malley and Terwilliger, 1974; Reddy and Campbell, 1970; Hirsch-Kolb et al., 1970). One exception is the enzyme from the terrestrial oligochaete, Lumbricus terrestris (Reddy and Campbell, 1968) with a molecular weight which is almost identical to that found here for the enzyme from Concholepas concholepas.

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mammalian arginases. Comp. Biochem. Physiol. 37, 345-359. Hirsch-Kolb H., Kolb H. J. and Greenberg D. M. (1971) Nuclear magnetic resonance studies of manganese binding of rat liver arginase. J. biol. Chem. 246, 395-401. Kayser G. A. and Strecker H. J. (1973) Purification and properties of arginase of rat kidney. Biochem. J. 133, 779-788. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Mora J., Martuscelli J., Ortiz-Pineda J. and Sorber6n G. (1965) The regulation of urea biosynthesis in vertebrates Biochem. J. 96, 28-35. Mora J., Tarrab R. and Bojalil L. F. (1966) On the structure and function of different arginases. Biochim. biophys. Acta 118, 206-209. O'Malley K. and Terwilliger R. C. (1974) Structure and

properties of arginase from the polychaete annelid Pista pacificia Berkeley. Biochem. J. 143, 591-597. Reddy S. R. R. and Campbell J. W. (1968) A low molecular weight arginase in the earthworm. Biochim. biophys. Acta 159, 557-559. Reddy S. R. R. and Campbell J. W. (1970) Molecular weights of arginase from different species. Comp. Biochem. Physiol. 32, 499-509. Roche J., van Thoai N. and Verrier J. (1954) Sur l'inhibition de l'arginase h6patique par concurrence avec le substrat. C.R. Sbanc. Soc. Biol. 148, 782-784. Soru E, and Zaharia O. (1976) Staphylococcal arginase. Purification and properties. Rev. Roum Biochim. 13, 49-60. Tarrab R., Rodriguez J., Huitr6n C., Palacios R. and Sober6n G. (1974) Molecular forms of rat liver arginase. Isolation and characterization. Eur. J. Biochem. 49, 457-468.