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l -leucyl-β-naphthylamidases of the cestode, Moniezia expansa, and the nematode, Ascaris suum
Comp. Biochem. Physiol.. 1978. Vol. 60B. pp. 63 to 66. Pertlamon Press. Printed in Great Britain
L-LEUCYL-fl-NAPHTHYLAMIDASES OF THE CESTODE, MONIEZIA EXPANSA, AND THE NEMATODE, ASCARIS SUUM P. G. C. DoucH Wailaceville Animal Research Centre. Research Division, Ministry of Agriculture and Fisheries. Private Bag, Upper Hutt. New Zealand (Received 13 June 1977)
Abstraet--i. Enzymes hydrolysing L-leucyl-fl-naphthylamide were located in the distal cytoplasm of proglottids of the cestode, Moniezia expansa, and the cytosol of the intestinal epithelial cells and seminal receptacle of the nematode, Ascaris suum. 2. The M. expansa enzyme had a molecular weight of about 200,000, and a pH optimum of 7.2. Activity was stimulated by Mn 2+, Fe 2+ and Co 2+, and inhibited by Ni 2+, Cu 2+, Cd 2+, Hg2. and Zn 2+" 3. The nematode intestinal and seminal receptacle enzymes both had a molecular weight of about 60,000. The pH optimum for the intestinal enzyme was 6.9, and that for the seminal receptacle enzyme, 7.2. The intestinal enzyme was not stimulated by metal ions but the seminal receptacle enzyme was stimulated by C02+; both enzymes were inhibited by Ni 2+. Cu 2+, Cd 2+ and Hg2+. 4. N-Acetylglycine. acetanilide, benzamide, benzanilide and salicylamide were not hydrolysed by the enzymes from either the cestode or the nematode.
anthelmintics (niclosamide, clioxanide and resorantei) which are substituted benzanilide compounds, N-acetylglycine and acetanilide derivatives, might be hydrolysed by such enzymes. In this study the L-leucyl-fl-naphthylamidases of the nematode, A. suum and of the cestode, M. expansa, were examined to determine whether they might be involved in the metabolism of anthelmintics.
INTRODUCTION Arylamidases, a group of enzymes that hydrolyse aminoacyl-fl-naphthylamides, have been demonstrated in many organisms (Brecher & Suszkiw, 1969). They are distinct from the well-defined peptidases and proteases. Patterson et al. (1963) have shown that the enzyme hydrolysing leucinamide, leucine aminopeptidase, is distinct from that in ascites tumour cells which hydrolyses L-leucyl-fl-naphthylamide (LNA). The latter enzyme has been referred to as leucine aminopeptidase, but for clarity will be designated "leucine aminopeptidase" in this paper. Histochemical techniques have been used to demonstrate the presence of enzymes capable of hydrolysing LNA in Several invertebrate species. These enzymes have been found in the webbing clothes moth larvae (Ward, 1975a, b, c), the rhabdocoel turbellarians, Temnocephala brenesi and T. novae-zealandiae (Jennings, 1968), the triclad turbellarians, Polycelis cornuta and Orthodemus terrestris (Jennings, 1962), the trematode, Philophthalmus oralli (Cheng & Yee, 1968), rhynchocoelan worms (Gibson & Jennings, 1967) and leeches (van der Lande, 1972). The nematode, Ascaris lumbricoides, has LNA-hydrolysing enzymes in intestinal cells, anterior excretory canals and hypodermis (Lee, 1962). An enzyme hydrolysing DL-leucylglycine, and thought to be leucine aminopeptidase, which was activated by Mn 2+, has also been described in this species (Carpenter, 1952). The exsheathing fluids of juvenile nematodes, Haemonchus contortus and Trichostrongylus colubriformis, contain a leucine aminopeptidase which appears to attack the sheath (Rogers, 1965). Aminoacyi-fl-naphthylamidases occur widely in invertebrates, particularly parasitic helminths, and could be involved in the metabolism of xenobioties such as anthelmintics (Nagasawa et al., 1976). Several
MATERIALS AND METHODS
Animals and enzyme preparations A. suum and M. expansa were obtained from the intestines of swine and sheep, respectively, and maintained in a balanced salt solution as described previously (Douch & Blair, 1975). A. suum worms were held for 12hr in this salt solution which was changed three times to allow for the removal of intestinal contents. The cuticle, mesenchyme fluid, reproductive tissues and intestine were dissected from A. suum and assayed for L-leucyl-~-naphthylamidase activity. Proglottids of various developmental stages from M. expansa were also assayed. Tissues from both heiminth species were homogenised (50% w/v in phosphate buffer, 7.0, 0.I mM, containing 0.25M sucrose) and centrifuged to give 2000g, 10,000g: and 75,000g fractions as described previously (Douch, 1975a, b). Supernatants and sediments were assayed. For most experiments, the dialysed or desalted I0,000 g supernatants were used as enzyme source. To remove salts enzymes were either dialysed against phosphate buffer (0.I mM, pH 7.0) or passed through a Sephadex G25 column (Pharmacia Fine Chemicals, A.B., Uppsala, Sweden) (Douch, 1975b). Incubation procedures
Tissues, tissue homogenates or the centrifuged fractions prepared as described above were incubated in duplicate in phosphate buffer (pH 7.0, 0.I M) with the substrate," LNA (0.5mM) in a total volume of 10ml at 37°C for 15 min. Experiments were conducted with three separate 63
64
P.G.C. DoucH
enzyme preparations of the test tissue. Metal ions (as CI-, NO~-, or SO~-), EDTA, thiols or inhibitors were preincubated with enzyme for up to 15 rain before addition of substrate. Enzyme activity was terminated by the addition of 2 ml of 40% (w/v) trichloroacetic acid or by placing the test tubes into iced water. Assay procedures In whole tissues fl-naphthylamine liberated by hydrolysis was determined by coupling with Fast red B salt (stabilized diazonium salt of 5-nitro-2-aminoanisole). The redcoloured complex obtained was observed in frozen thin sections of tissue under the microscope. The same reaction was used to measure enzyme activity in incubates by extraction of the red-coloured complex into 10 ml volumes of butan-l-ol for colorimetric determination. For experiments in which the effects of thiols were investigated, the method of Goldbarg & Rutenberg (1958) was used. Glycine liberated from N-acetylglycine was determined by the method of Troll &Cannan (1953). Hydrolysis of benzamide and salicylamide was determined by measuring benzoic acid and salicylic acitl formed, respectively. Hydrolysis of acetanilide and benzanilide was measured by the determination of the aniline formed (Douch & Gahagan, 1977). Protein concentrations were determined by the method of Lowry et al. (1951). Molecular weight estimations Molecular weights of the enzymes were estimated by gel filtration on a Sephadex G200 column (Pharmacia Fine Chemicals A.B., Uppsala, Sweden) as described previously (Douch, 1975c), RESULTS
Localization of "leucine aminopeptidase" activity
Histochemical examination of incubated tissues showed that "leucine aminopcptidase" activity was present only in the distal (tegumental) cytoplasm of M. expansa proglottids. Activity was evenly distributed along the length of the tapeworm. In A. suum, the enzymic activity was located in the intestinal brush border epithelial cells and in the seminal receptacles of adult female worms. "Leucine aminopeptidase" activity appeared to be evenly distributed along the intestine but the pharyngeal and rectal regions showed very low activity. No activity was observed in the cuticle, mesenchyme fluid, male reproductive tract or the excretory canals. Experiments in which enzymes had been prepared from tissues by homo-
genization and centrifugation yielded similar results for the distribution of the enzyme. Distributions of the enzyme in centrifugal fractions of homogenates of M. expansa and of A. suum intestine and seminal receptacles are given in Table 1. Differential centrifugation of tissue homogenates showed that "leucine aminopeptidase" activity was present in the 75,0000 supernatant (cytosol) fractions of both helminths. The molecular weight of the "leucine aminopeptidase" from M. expansa was estimated to be about 200,000 and that from both intestine and seminal receptacles of A. suum, 60,000. Optimum assay conditions
The optimum pH of the "ieucine aminopeptidase" from M. expansa 10,000g supernatant preparations in phosphate buffer was 7.2. For the nematode 10,000 g supernatant preparations, the intestine enzyme and the seminal receptacle enzyme had pH optima of 6.9 and 7.2 respectively. The enzymes from all three sources showed linear reaction rates for at least 1 hr. Reaction rates after 15 min incubation at 37°C are given in Table 1. Optimum temperature of hydrolysis for all three enzymes was in the range of 35-40°C; above 45°C enzyme activity fell sharply. Hydrolytic activity increased linearly between 5° and 40°C. A similar linear response was obtained by increasing the protein concentration to 0.3 mg protein/ml for nematode intestine enzyme and 0.6 mg protein/ml for both the nematode seminal receptacle enzyme and the tapeworm enzyme. The K,.s, determined from graphical plots of 1/v against 1/IS] for the three enzymes with LNA as substrate were in the range 0.2-0.35 raM. The substrate concentration that gave maximum enzyme activity was 0.5 mM. Effects of cations and anions on hydrolytic activity
The enzyme from the tapeworm was stimulated by addition of Mn 2+, Fe 2+ or Co 2+, whereas the nematode seminal receptacle enzyme activity was enhanced by Co 2 + only (Table 2). Nematode intestinal enzyme was not stimulated by metal ions. The enzyme from all three sources was inhibited by 1 mM Ni 2+, Cu 2+, Cd 2+ or Hg 2+. In addition, tapeworm enzyme was inhibited by Zn 2+.
Table 1. Subcellular distribution of "leucine aminopeptidase" in homogenates of A. suum intestine and seminal receptacle and in M. expansa A. suum
Fraction Whole homogenate 2000 g supernatant 2000 g sediment 10,000g supernatant 10,000g sediment 75,000g supernatant 75,0000 sediment
M. expansa nmol/hr/mg protein
13.3 + 1.0 24.6 __+0.6 3.9 + 0.2 33.8 4- 0.6 2.4 + 0.3 41.9 + 1.1 1.3 + 0.1
%
Intestine nmol/hr/mg protein
%
Seminal receptacle nmol/hr/mg protein %
100 93 6 89 3 87 1
316.4 _ 2.3 628.7 ___1.8 49.3 ___0.9 845.0 4- 2.2 7.3 4- 0.6 968.2 __+2.8 2.1 4- 0.3
100 95 4 91 2 88 2
191.8 _ 1.9 301.2 4- 2.4 20.3 ___0.4 470.1 + 1.7 3.9 4- 0.6 593.6 4- 1.9 1.7 _ 0.2
Homogenates of tissues were centrifuged and assayed as described in the text. Means rates of hydrolysis (_ S.E.M;) are given for three separate enzyme preparations. Total activity in a fraction is given as a percentage of that in whole homogenate.
100 93 5 88 4 85 1
65
Helminth "leucine aminopeptidases" Table 2. Metal ion requirement of L-leucyl-fl-naphthylamidases of helminths A. suum enzymes prepared from
enzyme
intestine
seminal receptacle
100 99 97 98 131 126 99 152 72 (5.5 mM) 6 (6.0/aM) 71 (5.0 raM) 26 (0.75 raM) 39 (0.85 raM) 87
100 102 106 l 12 116 115 110 110 71 (5.3 mM) 45 (1.1 mM) 109 51 (1.0 mM) 6 (5.7/aM) 109
100 101 104 103 97 102 106 198 53 (0.95 mM) 12 (13.0#M) 102 25 (0.6 mM) 5 (3.9/aM) 107
M. expansa
Addition None EDTA Mg 2÷ Ca 2÷ Mn 2+ Fe z+ Fe a+ Co 2+ Ni 2+ Cu 2+ Zn 2+ Cd ~+ Hg 2+ Pb 2÷
Enzymes prepared as dialysed lO,O00g supernatants were incubated in triplicate with substrate metal ions (1 mM) or EDTA (1 mM) as described in the text. Figures are percentages of the reaction rate of incubations without additions. 150 values are given in parentheses. None of the enzymes was affected by most anions tested (SO~-, CI-, F - , PO~-, P 2 0 ~ - , N O r , MoO~-), but the nematode intestine enzyme was stimulated by C N - .
the Sephadex G200 column. The column eluates containing "leucine aminopeptidase" activity were unable to hydrolyse acetanilide, benzamide, benzanilide or salicylamide. Similarly N-acetylglycine was not hydrolysed by these enzymes,
Effects of thiols, thiol group rectoents, and other compounds
DISCUSSION
None of the t h i o l s stimulated the hydrolysis of LNA by any of the enzymes (Table 3). 2-Mercaptoethanolamine strongly inhibited the reaction with all three enzymes (/so, 20-28/aM) while cysteine and glutathione inhibited the nematode intestinal enzyme to a small extent, p-Chloromercuribenzoate also strongly inhibited tapeworm and nematode seminal receptable enzymes whereas nematode intestine enzyme was only slightly inhibited. Parathion and the anthelmintic organophosphates (haloxon, naphthalophos and trichlorfon) did not affect the hydrolysis. Similarly, SKF-525-A showed no inhibitory effects. Acetanilide, benzamide, benzanilide and salicylamide at concentrations up to 10 mM did not inhibit the hydrolysis of LNA by any enzyme eluted .from
Aminopeptidase activity towards LNA was found in the intestine of A. suum and the seminal receptacle of adult females. Carpenter (1952) found no DL-leucylglycine hydrolase activity in the latter tissue. The enzyme from both tissues was soluble and the washed 10,000g and 100,000g sediments did not show aminopeptidase activity. Plasma membranes isolated from rat and mouse liver are reported to possess bound L-leucyl-fl-naphthylamidase activity (Emmelot et al., 1968). The distribution of aminopeptidase activity in M. expansa is similar to that previously obtained for nitroreductase and acetanilide N-deacetylase activities (Douch, 1975; Douch & Gahagan, 1976). The trematode, Philophthalmus gralli, also has aminopeptidase
Table 3. The effects of thiols and thiol-group reagents on hydrolytic activity M. expansa
Addition None Thioglycollic acid Ditbiothreitol Cysteine Glutathione 2-Mercaptoethanol 2-Mercaptoethanolamine N-Ethylmaleimide p-Chloromereuribenzoate
A. suum enzyme prepared from
enzyme
intestine
seminal receptacle
1O0 94 93 98 89 103 18 101 23
1O0 86 90 74 79 91 9 92 73
1O0 103 82 90 86 88 10 86 11
Dialysed i0,000g supernatant enzyme was incubated in triplicate with substrate (0.5mM) and additions of the compounds listed (l mM) as described in the text. Figures are percentages of the mean reaction rates of triplicate incubations with no addition.
c.a.p.60/IR--E
66
P . G . C . Doucn
activity associated with its body surface and like M. expansa its tegument probably has an absorptive
function (Cheng & Yee, 1968). The "leucine amlnopeptidases" examined in the present study and others from various species and tissues show similar wide variations in optimum pH, metal ion requirements and responses to activating and inhibiting substances (Brecher & Suszkiw, 1969). Carpenter (1952) found that A. lumbricoides intestinal DL-leucylglycine hydrolase was activated by Mn 2+. The exsheathing "leucine aminopeptidases" from T. colubriformis and H. contortus require Mg 2+ or Mn 2+ for activity and it has been suggested that they may be zinc-containing metaUo-enzymes (Rogers & Brooks, 1976). In the present study the aminopeptidase activity of M. expansa was also enhanced by Mn 2+ and Co 2+ whereas the enzyme from A. suum seminal receptacle was stimulated by Co 2+. In contrast, the aminopeptidase activity of the trematode P. ~lralli was inhibited by Mg 2+ and Mn 2+ (Cheng & Yee, 1968). Inhibition by heavy metals indicates that the enzyme activity is dependent on sulphydryl groups. N-Ethylmaleimide slightly inhibited A. suum seminal receptacle enzyme but had no effect on the other enzymes tested, p-Chloromercuribenzoate strongly inhibited the cestode enzyme and the nematode seminal receptacle enzyme whereas the intestine enzyme was only slightly inhibited. Cysteine and glutathione inhibited the enzyme activities slightly; no activation by thiols was observed. 2-Mercaptoethanolamine, however, inhibited all three enzymes. These observations indicate either that the thiol group in the enzyme is a disulphide or that maintenance in a reduced state is not necessary for activity. A wide range of molecular weights (70,000300,000) has been indicated for enzymes hydrolysing LNA (Ward 1975b, c). The M. expansa enzyme with molecular weight of about 200,000 is within this range, whereas those of A. suum intestine and seminal receptacle are less than those reported above. None of the helminth enzymes hydrolysing LNA could effect the hydrolysis of acetanilide, benzamide, benzanilide or salicylamide. These compounds did not inhibit aminopeptidase activity, indicating that they are not alternative substrates. It would appear, therefore, that although enzymes hydrolysing L-leucyl-fl-naphthylamide are present in both M. expansa and A. suum, their substrate specificity does not extend to analogues of anthelmintics.
REFERENCES BRECHERA. S. & SUSZKlWJ. B. (1969) Brain arylamidase. Purification and characterization of the soluble bovine enzyme. Biochem. J. 112, 335-342. CARPENTER M, F. P. (1952) The digestive enzymes of Ascaris lumbricoides var. suum; their properties and distribution in the alimentary canal. Ph.D. Thesis, University of Michigan. University Microfilms: Publ No. 3729, Ann Arbor, Michigan. CHENGT. C. & YEEH. W. F. (1968) Histochemical demonstration of aminopeptidase activity associated with the intramolluscan stages of Philophthalmus flralli Mathis & I.,6ger. Parasitolooy 5g, 473-480.
Doucx P. G. C. (1975a) 4-Nitrobenzoic acid reductase of the nematode Ascaris lumbricoides vat. suum. Localization of the enzyme and optimum assay conditions. Xenobiotica 5, 293-302. Doucrt P. G, C, (1975b) Azo- and nitro-reductases of the cestode Moniezia expansa. Localization of the enzyme activities and optimum assay conditions. Xenobiotica 5, 773-780. Douex P. G, C. (1975c) 4-Nitrobenzoic acid reductase of Ascaris lumbricoides vat. suum. Substrate specificity and reaction products. Xenobiotica 5, 401-406. DOUCHP. G. C. & GA~IAGANH. M. (1977) The metabolism of niclosamide and related compounds by Moniezia expansa, Ascaris lumbricoides var. suum, mouse- and sheep-liver enzymes. Xenobiotica 7, 301-307. EMMELOTP., VISSERA. & BENEDETTIE. L. (1968) Studies of plasma membranes--VII. A leucyl-fl-naphthylamidasecontaining repeating unit on the surface of isolated liver and hepatoma plasma membranes. Biochim. biophys. Acta 150, 364-375. GmsoN R. & JENmNGSJ. B. (1967) "Leucine aminopeptidase" activity in the blood system of rhynchocoelan worms. Comp. Biochem. Physiol. 23, 645-651. GOLDBARGJ. A. & RUTENBERGA. M. (1958) The colorimetric determination of leucine aminopeptidase in urine and serum of normal subjects and patients with cancer and other diseases. Cancer 11, 283-291. JENNINGSJ. B. (1962) Further studies on feeding and digestion in triclad turbellaria. Biol. Bull. 123, 571-581. JENNINGSJ. B. (1968) Feeding, digestion and food storage in two species of temnocephalid flatworms (TurbeUaria: Rhabdocoela). J. Zool., Lond. 156, 1-8. LEE D. L. (1962) The histochemical localization of leucine aminopeptidase in Ascaris lumbricoides. Parasitology 52, 533-538. LOWRYO. H., ROSEBROUGHN. J., FARRA. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. NAGASAWAH. T., KUO T. H., SHIROTAF. N. & ALEXANDER C. S. (1976) An intestinal arylamidase that selectively hydrolyses certain aromatic amides. Biochem. Pharmac. 25, 855-858. PATTERSONE. K., HSlAOS. H. & KEPPELA. (1963) Studies on dipeptidases and aminopeptidases. I. Distinction between leucine aminopeptidase and enzymes that hydrolyse Ldeucyl-fl-naphthylamide. J. biol. Chem. 238, 3611-3620. ROGERS W. P. (1965) The role of leucine aminopeptidase in the moulting of nematode parasites. Comp. Biochem. Physiol. 14, 311-321. ROGERS W. P. & BROOKSF. (1976) Zinc as a co-factor for an enzyme involved in exsheathment of Haemonchus coft~tortus. Int. J. Parasitol. 6, 315-319. TROLLW. & CANNANR. K. (1953) A modified photometric ninhydrin method for the analysis of amino and imino acids. J. biol. Chem. 200, 803-811. VAN DER LANDEV. M. (1972) Observations on the histochemical "aminopeptidase" reaction in the intestine of certain species of leech (Annelida: Hirudinae), with particular reference to Erpobdella octoculata (L). Comp. BiDchem. Physiol. 41A, 813-824. WARD C. W. (1975a) Aminopeptidases in webbing clothes moth larvae. Properties and specificity of the major enzyme of low electrophoretic mobility. Int. J. Biochem. 6, 765-768. WARD C. W. (1975b) Resolution or proteases in the keratinolytic larvae of the webbing clothes moth. Aust. J. biol. Sci. 28, 1-23. WARD C. W. (1975c) Aminopeptidases in webbing clothes moth larvae. Properties and specificities of enzymes of highest electrophoretic mobility. Aust. J. biol. Sci. 28, 447-455.
L-LEUCYL-fl-NAPHTHYLAMIDASES OF THE CESTODE, MONIEZIA EXPANSA, AND THE NEMATODE, ASCARIS SUUM P. G. C. DoucH Wailaceville Animal Research Centre. Research Division, Ministry of Agriculture and Fisheries. Private Bag, Upper Hutt. New Zealand (Received 13 June 1977)
Abstraet--i. Enzymes hydrolysing L-leucyl-fl-naphthylamide were located in the distal cytoplasm of proglottids of the cestode, Moniezia expansa, and the cytosol of the intestinal epithelial cells and seminal receptacle of the nematode, Ascaris suum. 2. The M. expansa enzyme had a molecular weight of about 200,000, and a pH optimum of 7.2. Activity was stimulated by Mn 2+, Fe 2+ and Co 2+, and inhibited by Ni 2+, Cu 2+, Cd 2+, Hg2. and Zn 2+" 3. The nematode intestinal and seminal receptacle enzymes both had a molecular weight of about 60,000. The pH optimum for the intestinal enzyme was 6.9, and that for the seminal receptacle enzyme, 7.2. The intestinal enzyme was not stimulated by metal ions but the seminal receptacle enzyme was stimulated by C02+; both enzymes were inhibited by Ni 2+. Cu 2+, Cd 2+ and Hg2+. 4. N-Acetylglycine. acetanilide, benzamide, benzanilide and salicylamide were not hydrolysed by the enzymes from either the cestode or the nematode.
anthelmintics (niclosamide, clioxanide and resorantei) which are substituted benzanilide compounds, N-acetylglycine and acetanilide derivatives, might be hydrolysed by such enzymes. In this study the L-leucyl-fl-naphthylamidases of the nematode, A. suum and of the cestode, M. expansa, were examined to determine whether they might be involved in the metabolism of anthelmintics.
INTRODUCTION Arylamidases, a group of enzymes that hydrolyse aminoacyl-fl-naphthylamides, have been demonstrated in many organisms (Brecher & Suszkiw, 1969). They are distinct from the well-defined peptidases and proteases. Patterson et al. (1963) have shown that the enzyme hydrolysing leucinamide, leucine aminopeptidase, is distinct from that in ascites tumour cells which hydrolyses L-leucyl-fl-naphthylamide (LNA). The latter enzyme has been referred to as leucine aminopeptidase, but for clarity will be designated "leucine aminopeptidase" in this paper. Histochemical techniques have been used to demonstrate the presence of enzymes capable of hydrolysing LNA in Several invertebrate species. These enzymes have been found in the webbing clothes moth larvae (Ward, 1975a, b, c), the rhabdocoel turbellarians, Temnocephala brenesi and T. novae-zealandiae (Jennings, 1968), the triclad turbellarians, Polycelis cornuta and Orthodemus terrestris (Jennings, 1962), the trematode, Philophthalmus oralli (Cheng & Yee, 1968), rhynchocoelan worms (Gibson & Jennings, 1967) and leeches (van der Lande, 1972). The nematode, Ascaris lumbricoides, has LNA-hydrolysing enzymes in intestinal cells, anterior excretory canals and hypodermis (Lee, 1962). An enzyme hydrolysing DL-leucylglycine, and thought to be leucine aminopeptidase, which was activated by Mn 2+, has also been described in this species (Carpenter, 1952). The exsheathing fluids of juvenile nematodes, Haemonchus contortus and Trichostrongylus colubriformis, contain a leucine aminopeptidase which appears to attack the sheath (Rogers, 1965). Aminoacyi-fl-naphthylamidases occur widely in invertebrates, particularly parasitic helminths, and could be involved in the metabolism of xenobioties such as anthelmintics (Nagasawa et al., 1976). Several
MATERIALS AND METHODS
Animals and enzyme preparations A. suum and M. expansa were obtained from the intestines of swine and sheep, respectively, and maintained in a balanced salt solution as described previously (Douch & Blair, 1975). A. suum worms were held for 12hr in this salt solution which was changed three times to allow for the removal of intestinal contents. The cuticle, mesenchyme fluid, reproductive tissues and intestine were dissected from A. suum and assayed for L-leucyl-~-naphthylamidase activity. Proglottids of various developmental stages from M. expansa were also assayed. Tissues from both heiminth species were homogenised (50% w/v in phosphate buffer, 7.0, 0.I mM, containing 0.25M sucrose) and centrifuged to give 2000g, 10,000g: and 75,000g fractions as described previously (Douch, 1975a, b). Supernatants and sediments were assayed. For most experiments, the dialysed or desalted I0,000 g supernatants were used as enzyme source. To remove salts enzymes were either dialysed against phosphate buffer (0.I mM, pH 7.0) or passed through a Sephadex G25 column (Pharmacia Fine Chemicals, A.B., Uppsala, Sweden) (Douch, 1975b). Incubation procedures
Tissues, tissue homogenates or the centrifuged fractions prepared as described above were incubated in duplicate in phosphate buffer (pH 7.0, 0.I M) with the substrate," LNA (0.5mM) in a total volume of 10ml at 37°C for 15 min. Experiments were conducted with three separate 63
64
P.G.C. DoucH
enzyme preparations of the test tissue. Metal ions (as CI-, NO~-, or SO~-), EDTA, thiols or inhibitors were preincubated with enzyme for up to 15 rain before addition of substrate. Enzyme activity was terminated by the addition of 2 ml of 40% (w/v) trichloroacetic acid or by placing the test tubes into iced water. Assay procedures In whole tissues fl-naphthylamine liberated by hydrolysis was determined by coupling with Fast red B salt (stabilized diazonium salt of 5-nitro-2-aminoanisole). The redcoloured complex obtained was observed in frozen thin sections of tissue under the microscope. The same reaction was used to measure enzyme activity in incubates by extraction of the red-coloured complex into 10 ml volumes of butan-l-ol for colorimetric determination. For experiments in which the effects of thiols were investigated, the method of Goldbarg & Rutenberg (1958) was used. Glycine liberated from N-acetylglycine was determined by the method of Troll &Cannan (1953). Hydrolysis of benzamide and salicylamide was determined by measuring benzoic acid and salicylic acitl formed, respectively. Hydrolysis of acetanilide and benzanilide was measured by the determination of the aniline formed (Douch & Gahagan, 1977). Protein concentrations were determined by the method of Lowry et al. (1951). Molecular weight estimations Molecular weights of the enzymes were estimated by gel filtration on a Sephadex G200 column (Pharmacia Fine Chemicals A.B., Uppsala, Sweden) as described previously (Douch, 1975c), RESULTS
Localization of "leucine aminopeptidase" activity
Histochemical examination of incubated tissues showed that "leucine aminopcptidase" activity was present only in the distal (tegumental) cytoplasm of M. expansa proglottids. Activity was evenly distributed along the length of the tapeworm. In A. suum, the enzymic activity was located in the intestinal brush border epithelial cells and in the seminal receptacles of adult female worms. "Leucine aminopeptidase" activity appeared to be evenly distributed along the intestine but the pharyngeal and rectal regions showed very low activity. No activity was observed in the cuticle, mesenchyme fluid, male reproductive tract or the excretory canals. Experiments in which enzymes had been prepared from tissues by homo-
genization and centrifugation yielded similar results for the distribution of the enzyme. Distributions of the enzyme in centrifugal fractions of homogenates of M. expansa and of A. suum intestine and seminal receptacles are given in Table 1. Differential centrifugation of tissue homogenates showed that "leucine aminopeptidase" activity was present in the 75,0000 supernatant (cytosol) fractions of both helminths. The molecular weight of the "leucine aminopeptidase" from M. expansa was estimated to be about 200,000 and that from both intestine and seminal receptacles of A. suum, 60,000. Optimum assay conditions
The optimum pH of the "ieucine aminopeptidase" from M. expansa 10,000g supernatant preparations in phosphate buffer was 7.2. For the nematode 10,000 g supernatant preparations, the intestine enzyme and the seminal receptacle enzyme had pH optima of 6.9 and 7.2 respectively. The enzymes from all three sources showed linear reaction rates for at least 1 hr. Reaction rates after 15 min incubation at 37°C are given in Table 1. Optimum temperature of hydrolysis for all three enzymes was in the range of 35-40°C; above 45°C enzyme activity fell sharply. Hydrolytic activity increased linearly between 5° and 40°C. A similar linear response was obtained by increasing the protein concentration to 0.3 mg protein/ml for nematode intestine enzyme and 0.6 mg protein/ml for both the nematode seminal receptacle enzyme and the tapeworm enzyme. The K,.s, determined from graphical plots of 1/v against 1/IS] for the three enzymes with LNA as substrate were in the range 0.2-0.35 raM. The substrate concentration that gave maximum enzyme activity was 0.5 mM. Effects of cations and anions on hydrolytic activity
The enzyme from the tapeworm was stimulated by addition of Mn 2+, Fe 2+ or Co 2+, whereas the nematode seminal receptacle enzyme activity was enhanced by Co 2 + only (Table 2). Nematode intestinal enzyme was not stimulated by metal ions. The enzyme from all three sources was inhibited by 1 mM Ni 2+, Cu 2+, Cd 2+ or Hg 2+. In addition, tapeworm enzyme was inhibited by Zn 2+.
Table 1. Subcellular distribution of "leucine aminopeptidase" in homogenates of A. suum intestine and seminal receptacle and in M. expansa A. suum
Fraction Whole homogenate 2000 g supernatant 2000 g sediment 10,000g supernatant 10,000g sediment 75,000g supernatant 75,0000 sediment
M. expansa nmol/hr/mg protein
13.3 + 1.0 24.6 __+0.6 3.9 + 0.2 33.8 4- 0.6 2.4 + 0.3 41.9 + 1.1 1.3 + 0.1
%
Intestine nmol/hr/mg protein
%
Seminal receptacle nmol/hr/mg protein %
100 93 6 89 3 87 1
316.4 _ 2.3 628.7 ___1.8 49.3 ___0.9 845.0 4- 2.2 7.3 4- 0.6 968.2 __+2.8 2.1 4- 0.3
100 95 4 91 2 88 2
191.8 _ 1.9 301.2 4- 2.4 20.3 ___0.4 470.1 + 1.7 3.9 4- 0.6 593.6 4- 1.9 1.7 _ 0.2
Homogenates of tissues were centrifuged and assayed as described in the text. Means rates of hydrolysis (_ S.E.M;) are given for three separate enzyme preparations. Total activity in a fraction is given as a percentage of that in whole homogenate.
100 93 5 88 4 85 1
65
Helminth "leucine aminopeptidases" Table 2. Metal ion requirement of L-leucyl-fl-naphthylamidases of helminths A. suum enzymes prepared from
enzyme
intestine
seminal receptacle
100 99 97 98 131 126 99 152 72 (5.5 mM) 6 (6.0/aM) 71 (5.0 raM) 26 (0.75 raM) 39 (0.85 raM) 87
100 102 106 l 12 116 115 110 110 71 (5.3 mM) 45 (1.1 mM) 109 51 (1.0 mM) 6 (5.7/aM) 109
100 101 104 103 97 102 106 198 53 (0.95 mM) 12 (13.0#M) 102 25 (0.6 mM) 5 (3.9/aM) 107
M. expansa
Addition None EDTA Mg 2÷ Ca 2÷ Mn 2+ Fe z+ Fe a+ Co 2+ Ni 2+ Cu 2+ Zn 2+ Cd ~+ Hg 2+ Pb 2÷
Enzymes prepared as dialysed lO,O00g supernatants were incubated in triplicate with substrate metal ions (1 mM) or EDTA (1 mM) as described in the text. Figures are percentages of the reaction rate of incubations without additions. 150 values are given in parentheses. None of the enzymes was affected by most anions tested (SO~-, CI-, F - , PO~-, P 2 0 ~ - , N O r , MoO~-), but the nematode intestine enzyme was stimulated by C N - .
the Sephadex G200 column. The column eluates containing "leucine aminopeptidase" activity were unable to hydrolyse acetanilide, benzamide, benzanilide or salicylamide. Similarly N-acetylglycine was not hydrolysed by these enzymes,
Effects of thiols, thiol group rectoents, and other compounds
DISCUSSION
None of the t h i o l s stimulated the hydrolysis of LNA by any of the enzymes (Table 3). 2-Mercaptoethanolamine strongly inhibited the reaction with all three enzymes (/so, 20-28/aM) while cysteine and glutathione inhibited the nematode intestinal enzyme to a small extent, p-Chloromercuribenzoate also strongly inhibited tapeworm and nematode seminal receptable enzymes whereas nematode intestine enzyme was only slightly inhibited. Parathion and the anthelmintic organophosphates (haloxon, naphthalophos and trichlorfon) did not affect the hydrolysis. Similarly, SKF-525-A showed no inhibitory effects. Acetanilide, benzamide, benzanilide and salicylamide at concentrations up to 10 mM did not inhibit the hydrolysis of LNA by any enzyme eluted .from
Aminopeptidase activity towards LNA was found in the intestine of A. suum and the seminal receptacle of adult females. Carpenter (1952) found no DL-leucylglycine hydrolase activity in the latter tissue. The enzyme from both tissues was soluble and the washed 10,000g and 100,000g sediments did not show aminopeptidase activity. Plasma membranes isolated from rat and mouse liver are reported to possess bound L-leucyl-fl-naphthylamidase activity (Emmelot et al., 1968). The distribution of aminopeptidase activity in M. expansa is similar to that previously obtained for nitroreductase and acetanilide N-deacetylase activities (Douch, 1975; Douch & Gahagan, 1976). The trematode, Philophthalmus gralli, also has aminopeptidase
Table 3. The effects of thiols and thiol-group reagents on hydrolytic activity M. expansa
Addition None Thioglycollic acid Ditbiothreitol Cysteine Glutathione 2-Mercaptoethanol 2-Mercaptoethanolamine N-Ethylmaleimide p-Chloromereuribenzoate
A. suum enzyme prepared from
enzyme
intestine
seminal receptacle
1O0 94 93 98 89 103 18 101 23
1O0 86 90 74 79 91 9 92 73
1O0 103 82 90 86 88 10 86 11
Dialysed i0,000g supernatant enzyme was incubated in triplicate with substrate (0.5mM) and additions of the compounds listed (l mM) as described in the text. Figures are percentages of the mean reaction rates of triplicate incubations with no addition.
c.a.p.60/IR--E
66
P . G . C . Doucn
activity associated with its body surface and like M. expansa its tegument probably has an absorptive
function (Cheng & Yee, 1968). The "leucine amlnopeptidases" examined in the present study and others from various species and tissues show similar wide variations in optimum pH, metal ion requirements and responses to activating and inhibiting substances (Brecher & Suszkiw, 1969). Carpenter (1952) found that A. lumbricoides intestinal DL-leucylglycine hydrolase was activated by Mn 2+. The exsheathing "leucine aminopeptidases" from T. colubriformis and H. contortus require Mg 2+ or Mn 2+ for activity and it has been suggested that they may be zinc-containing metaUo-enzymes (Rogers & Brooks, 1976). In the present study the aminopeptidase activity of M. expansa was also enhanced by Mn 2+ and Co 2+ whereas the enzyme from A. suum seminal receptacle was stimulated by Co 2+. In contrast, the aminopeptidase activity of the trematode P. ~lralli was inhibited by Mg 2+ and Mn 2+ (Cheng & Yee, 1968). Inhibition by heavy metals indicates that the enzyme activity is dependent on sulphydryl groups. N-Ethylmaleimide slightly inhibited A. suum seminal receptacle enzyme but had no effect on the other enzymes tested, p-Chloromercuribenzoate strongly inhibited the cestode enzyme and the nematode seminal receptacle enzyme whereas the intestine enzyme was only slightly inhibited. Cysteine and glutathione inhibited the enzyme activities slightly; no activation by thiols was observed. 2-Mercaptoethanolamine, however, inhibited all three enzymes. These observations indicate either that the thiol group in the enzyme is a disulphide or that maintenance in a reduced state is not necessary for activity. A wide range of molecular weights (70,000300,000) has been indicated for enzymes hydrolysing LNA (Ward 1975b, c). The M. expansa enzyme with molecular weight of about 200,000 is within this range, whereas those of A. suum intestine and seminal receptacle are less than those reported above. None of the helminth enzymes hydrolysing LNA could effect the hydrolysis of acetanilide, benzamide, benzanilide or salicylamide. These compounds did not inhibit aminopeptidase activity, indicating that they are not alternative substrates. It would appear, therefore, that although enzymes hydrolysing L-leucyl-fl-naphthylamide are present in both M. expansa and A. suum, their substrate specificity does not extend to analogues of anthelmintics.
REFERENCES BRECHERA. S. & SUSZKlWJ. B. (1969) Brain arylamidase. Purification and characterization of the soluble bovine enzyme. Biochem. J. 112, 335-342. CARPENTER M, F. P. (1952) The digestive enzymes of Ascaris lumbricoides var. suum; their properties and distribution in the alimentary canal. Ph.D. Thesis, University of Michigan. University Microfilms: Publ No. 3729, Ann Arbor, Michigan. CHENGT. C. & YEEH. W. F. (1968) Histochemical demonstration of aminopeptidase activity associated with the intramolluscan stages of Philophthalmus flralli Mathis & I.,6ger. Parasitolooy 5g, 473-480.
Doucx P. G. C. (1975a) 4-Nitrobenzoic acid reductase of the nematode Ascaris lumbricoides vat. suum. Localization of the enzyme and optimum assay conditions. Xenobiotica 5, 293-302. Doucrt P. G, C, (1975b) Azo- and nitro-reductases of the cestode Moniezia expansa. Localization of the enzyme activities and optimum assay conditions. Xenobiotica 5, 773-780. Douex P. G, C. (1975c) 4-Nitrobenzoic acid reductase of Ascaris lumbricoides vat. suum. Substrate specificity and reaction products. Xenobiotica 5, 401-406. DOUCHP. G. C. & GA~IAGANH. M. (1977) The metabolism of niclosamide and related compounds by Moniezia expansa, Ascaris lumbricoides var. suum, mouse- and sheep-liver enzymes. Xenobiotica 7, 301-307. EMMELOTP., VISSERA. & BENEDETTIE. L. (1968) Studies of plasma membranes--VII. A leucyl-fl-naphthylamidasecontaining repeating unit on the surface of isolated liver and hepatoma plasma membranes. Biochim. biophys. Acta 150, 364-375. GmsoN R. & JENmNGSJ. B. (1967) "Leucine aminopeptidase" activity in the blood system of rhynchocoelan worms. Comp. Biochem. Physiol. 23, 645-651. GOLDBARGJ. A. & RUTENBERGA. M. (1958) The colorimetric determination of leucine aminopeptidase in urine and serum of normal subjects and patients with cancer and other diseases. Cancer 11, 283-291. JENNINGSJ. B. (1962) Further studies on feeding and digestion in triclad turbellaria. Biol. Bull. 123, 571-581. JENNINGSJ. B. (1968) Feeding, digestion and food storage in two species of temnocephalid flatworms (TurbeUaria: Rhabdocoela). J. Zool., Lond. 156, 1-8. LEE D. L. (1962) The histochemical localization of leucine aminopeptidase in Ascaris lumbricoides. Parasitology 52, 533-538. LOWRYO. H., ROSEBROUGHN. J., FARRA. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. NAGASAWAH. T., KUO T. H., SHIROTAF. N. & ALEXANDER C. S. (1976) An intestinal arylamidase that selectively hydrolyses certain aromatic amides. Biochem. Pharmac. 25, 855-858. PATTERSONE. K., HSlAOS. H. & KEPPELA. (1963) Studies on dipeptidases and aminopeptidases. I. Distinction between leucine aminopeptidase and enzymes that hydrolyse Ldeucyl-fl-naphthylamide. J. biol. Chem. 238, 3611-3620. ROGERS W. P. (1965) The role of leucine aminopeptidase in the moulting of nematode parasites. Comp. Biochem. Physiol. 14, 311-321. ROGERS W. P. & BROOKSF. (1976) Zinc as a co-factor for an enzyme involved in exsheathment of Haemonchus coft~tortus. Int. J. Parasitol. 6, 315-319. TROLLW. & CANNANR. K. (1953) A modified photometric ninhydrin method for the analysis of amino and imino acids. J. biol. Chem. 200, 803-811. VAN DER LANDEV. M. (1972) Observations on the histochemical "aminopeptidase" reaction in the intestine of certain species of leech (Annelida: Hirudinae), with particular reference to Erpobdella octoculata (L). Comp. BiDchem. Physiol. 41A, 813-824. WARD C. W. (1975a) Aminopeptidases in webbing clothes moth larvae. Properties and specificity of the major enzyme of low electrophoretic mobility. Int. J. Biochem. 6, 765-768. WARD C. W. (1975b) Resolution or proteases in the keratinolytic larvae of the webbing clothes moth. Aust. J. biol. Sci. 28, 1-23. WARD C. W. (1975c) Aminopeptidases in webbing clothes moth larvae. Properties and specificities of enzymes of highest electrophoretic mobility. Aust. J. biol. Sci. 28, 447-455.