Isolation and characterization of arginine ester hydrolase from Heloderma horridum (beaded lizard) venom

Inf. J. Biochem. Vol. 24, No. 3, pp. 415-420, Printed in Great Britain. All rights reserved

1992 Copyright

0

OC20_711X/92 $5.00 + 0.00 1992 Pergamon Press plc

ISOLATION AND CHARACTERIZATION OF ARGININE ESTER HYDROLASE FROM HELODERMA HORRIDUM (BEADED LIZARD) VENOM TOSHIAKI

NIKAI, KUNIKO IMAI, YUMIKO KOMORI and HISAYOSHISUGIHARA*

Department of Microbiology, Faculty of Pharmacy, Meijo University, Tenpaku-Ku, 468 Nagoya, Japan (Received 30 April 1991) Abstract-l. An arginine ester hydrolase was isolated from Heloderma horridurn (headed lizard) venom by Sephadex G-75, DEAE-Sephacel and Q-Sepharose column chromatography, resulting in 5.4 mg of purified enzyme from 320.0 mg of crude venom. 2. The enzyme was shown to be homogeneous by both SDS and non-SDS disc electrophoresis on polyacrylamide gel at pH 8.3. 3. The enzyme possesses arginine ester hydrolase and transglutaminase-like activities, but did not exhibit clotting activity. 4. Molecular weight was determined to be IX 29 kDa, with an isoelectric point of 4.4. 5. The enzyme was stable to heat treatment (95”C, 10 min) and to pH changes over the range 2-l 1. 6. The arginine ester hydrolase was inactivated by diisopropylfluorophosphate (DFP), b-mercaptoethanol and N-bromosuccinimide, suggesting that serine, disulfide bonds and tryptophan are involved in enzymatic activity. 7. Amino terminal sequences were determined and appear to be similar to porcine pancreatic kallikrein.

INTRODUCTION

Assay for arginine ester hydrolase

Arginine ester hydrolase activity was assayed using benzoyl-L-arginine ethyl ester as the substrate. The reaction mixture contained 0. I ml of arginine esterase, 0.8 ml of 0.4 M Tris-HC1 buffer @H 8.5), and 1OmM benzoyl-r_arginine ethyl ester in a total volume of 1.Oml. The sample was incubated for 15 min at 37°C and the amount of benzoyl+arginine ethyl ester hydrolyzed was determined by the hydroxamate method of Roberts (1958). One unit of benzoyl+arginine ethyl ester hydrolase activity was defined as the amount of enzyme that hydrolyzed 1pmol of substrate per minute.

Lizard venom is known to contain biologically active proteins. Hendon and Tu (198 1) and Tu and Hendon (1983) isolated a lethal protein and hyaluronidase from the venom of Heloderma horridum. Mebs (1969) isolated a bradykinin-releasing enzyme from H. suspectum and Alagon et al. (1986) isolated a kallikrein-like, hypotensive enzyme from the venom of H. horridum. This paper reports the purification of an arginine ester hydrolase from the venom of H. horridum and compares the enzymes with arginine ester hydrolases from snake venom.

MATERIALS AND

Assay for smooth muscle contracting activity

METHODS

Lyophilized crude venom was purchased from the Miami Serpentarium Laboratories (Salt Lake City, Ut., U.S.A.). Diethylaminoethyl-Sephacel, quaternary amine-Sepharose, Sephadex G-75, and a molecular weight standard kit were purchased from Pharmacia (Uppsala, Sweden). Ampholyte (PH range 3.5-10) was obtained from LKB-Produkter (Stockholm, Sweden). Bovine fibrinogen was supplied by Daiichi Pure Chemical Co. Insulin B chain was obtained from Sigma Chemical Co. (St Louis, MO., U.S.A). Benzoyl-t,-arginine ethyl ester, tosyl-L-arginine methyl ester, z-Phe-Arg-4-methylcoumaryl-7-amide, Glt-Gly-Arg4-methyl-coumaryl-7-amide, ProPhe-Arg4methylcoumaryiFamide, Sue-Arg-ProPhe-His-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide, Boc-IleGluGly-Arg4-methylcoumaryl7-amide, BocVal-ProArg-4-methylcoumaryl-7-amide, and SucGly-Pro-Leu-Gly-Pro4methylcoumaryl-7-amide were purchased from the Peptide Institute (Osaka, Japan). Other chemicals used were of analytical grade from commercial sources.

‘To whom all correspondence

should be addressed. 415

The assay for smooth muscle contracting activity was originally developed by Rocha e Silva et al. (1949) using the ileum, but was later modified by Erspamer and Erspamer (1962) and Trautschhold (1970), using the rat uterus. A section of rat uterus l-l.5 cm long was suspended in a bath containing De Jalon’s solution (NaCl, 9 g; KCl. 0.4 g; CaCI,, 0.06 g; NaHCO,, 0.15 g; glucose, 1 g/l). Heatdenatured plasma (1 ml) containing I mM o-phenanthroline was incubated for 5 min at 37°C with arginine esterase. Disc polyacrylamide gel electrophoresis

Disc polyacrylamide gel electrophoresis was carried out on 8.5% polyacrylamide gels (0.5 cm x 12.5 cm, 2 mA, 4’C), using 40mM Tris-glycine @H 8.3) as the running buffer. Gels were stained with 0.25% Coomassie Blue R-250 in 7.5% acetic acid containing 5% methanol for 1 hr at 48°C followed by diffusion destaining. Determination of molecular weight

The molecular weight was determined by polyacrylamide gel electrophoresis (PAGE), using the method of Weber and Osbom (1969). Protein standards included phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa) and lactalbumin (14 kDa). Samples and

TOSHIAKI NIKAI et al.

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Arginine ester hydrolase from lizard venom

Fig. 2. Non-denaturant PAGE of the purified arginine ester hydroiase from the venom of Heloderma horr~d~rn.

Fig. 3. Insulin B chain clot formation resulting from activity of arginine ester hydrolase from the venom of ~e~odermu horridurn.

M.W

cx -polymer

94k

Y -dimer

67k 43k

30k 20.lk 14.4k

minutes Fig 4 Elect.rojphoresis of reduced fibrinogen and fibrin on SDS-polyacrylamide gels follow ring action by 1Ihe argini ine ester hydrolase from the venom of Heloderma horridurn. (A) Molecular weight stamdards (see text for details); (B) fibrinogen; (C), (D) and (E) fibrin.

TOSHIAKI NIKAI et al.

418

standards were reduced with 4% mercaptoethanol and 4% sodium dodecyl sulfate for 3 min at ~100°C. Sodium dodecvl sulfate (SDS)-PAGE was carried out on 7.5% polyacrylamide gels (0.5 cm x 12.5 cm), using a constant current of 8 mA at 20°C.

Table I. Amino acid composition of arginine ester hydrolase Amino Thr” SO” au Gly Ala Cysh val Met Ile LU T’yr Phe Lys HIS

Amino acid composition The amino acid composition of the carboxyamidemethylated arginine esterase was determined with a Hiatachi Mode1 835 high-speed automatic analyzer. Carboxyamide methylation was carried out according to the method of Hendon and Tu (1981). Samples were hydrolyzed with constant boiling HCI at llo”C. A minimum of three analyses from each of the 24-, 48- and 72-hr hydrolysates was used. Tryptophan content was determined by the method of Edelhoch (1967). Isoelectric point

Arg Pro Trph Total

Isoelectric focusing was performed on an LKB ampholine electrofocusing column of 110 ml, using carrier ampholyte (pH 3.5510). Arginine esterase (1 mg), previously dialyzed against a 1% solution of glycine, was placed in the center of the column. Isoelectric focusing was performed for 72 hr

Arginine ester hydrolase (3.2 p(g) was dissolved in 10 mM TrissHCl buffer (DH 7.5). The oxidized insulin B chain was dissolved in 1 ml-of the’same buffer at a concentration of I mg/ml. The arginine ester hydrolase was then added to the substrate.

Residues

21 14 25 25 29 17 14 22 4 14 21 9 7 II I 6 I3 4 269

“The hvdrolvsis value of threonine and serl’ne were extrapolated lo time zero. ‘Tryptophan content was determined by the spectroscopx method of Edelhoch (1967).

at 4’C by applying 700 V/column. Two-milliliter fractions were collected and benzoyl-L-arginine ethyl ester hydrolase activity and the pH of each fraction were measured at room temperature immediately after the experiment. Insulin B chain clot formation activity

acid

ASX

(5.4 mg) demonstrated benzoyl-L-arginine ethyl ester hydrolytic activity at a level of 191.0 U/mg. Homogeneity

Casein hydrolytic activity was determined by the method of Murata et al. (1963). Phosphodiesterase activity was

Homogeneity of the final preparation was established by PAGE. The final preparation gave a single band both on disc PAGE at pH 8.3 (Fig. 2). and SDS-PAGE. When antivenin-H. horridurn was tested against the purified arginine ester hydrolase by immunodiffusion, a single band was detected.

determined by the method of Suzuki and Iwanaga (1958). Fluorogenic peptide 4-methylcoumarine amide (MCA) was

Biochemical

Other methods

used to determine substrate hydrolysis the method of Morita et al. (1977).

activity,

according

to

RESULTS AND DISCUSSION Isolation procedure Heloderma horridum venom (320 mg) was dissolved at a concentration of 20% in 50 mM Tris-HCl buffer (pH 8.5) containing 0.1 M NaCl, and the insoluble material was removed by centrifugation (2000g) for 20min at 4°C. The supernatant was applied to a Sephadex G-75 column (Fig. 1, first step). Benzoyl-Larginine ethyl ester hydrolytic activity was found in fractions 3 and 4. Fraction 4 was further fractionated as follows. It was equilibrated by dialysis with 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM NaCl, and loaded on a DEAE-Sephacel column. The column was eluted using a 600-ml 0.01-0.5 M NaCl linear gradient in the equilibration buffer (Fig. 1, second step). Benzoyl-L-arginine ethyl ester hydrolytic activity was again found in fraction 4. Fraction 4 from the DEAE-Sephacel chromatography was dialyzed against 10 mM Tris-HCl buffer (pH 7.5) containing 10mM NaCl. The dialyzate was subjected to column chromatography on Q-Sepharose. The column was eluted with a 600-ml 0.01-0.5 M NaCl linear gradient in equilibration buffer (Fig. 1, third step). Benzoyl-L-arginine ethyl ester hydrolytic activity was found in fraction 2. The final preparation

properties

The molecular weight of arginine ester hydrolase was determined to be 29 kDa by SDS-PAGE. The isoelectric point was found to be 4.4. The amino acid composition of the reduced and carboxymethylated purified preparation is shown in Table 1. Arginine ester hydrolase is composed of 269 amino acid residues, based on the molecular weight of 29 kDa. The purified arginine ester hydrolase possesses a much higher benzoyl-L-arginine ester hydrolytic activity (191.0 Ujmg) in comparison with crude venom (10.6 Ujmg). However, no z-PheeArg-4methylcoumaryl-7-amide hydrolytic, Glt-Gly--Arg-4methylcoumaryl-7-amide hydrolytic, Pro-Phe-Arg4-methylcoumaryl-7-amide hydrolytic, Sue-Arg-ProPhe-His-Leu-Leu-Val-Tyr-4-methylcoumaryl-7amide hydrolytic, Boc-Ile-GluGly-Arg-4-methylcoumaryl-7-amide hydrolytic, Boc-Val-Pro-Arg-4methylcoumaryl-7-amide hydrolytic, Sue-GlyyProLeu-Gly-Pro-4-methylcoumaryl-7-amide hydrolytic. caseinase, fibrinogenase, phosphodiesterase, or phospholipase Az activities were observed. The effects of various inhibitory reagents (15 min, 37°C) on the benzoyl-L-arginine ethyl ester hydrolytic activity of the final preparation (2 pgg/ml) were examined. The enzymatic activity was inhibited by diisopropyl fluorophosphate, /I’-mercaptoethanol and N-bromosuccinimide. However, ethylenediaminetetraacetic acid, soybean trypsin inhibitor or p-tosyl-~-phenylalanine chloromethyl ketone (Table 2) had no effect.

Arginine

ester hydrolase

Table 2. Effects of various inhibitors on arginine ester hydrolase

Inhibitor (final concentration)

from lizard

Table 3. Fibrin clot solubility by monochloroacetic Clotting time (W (Thrombin)

Benzoyl-L-arginine ethyl ester hvdrolvtic activity (%)

None

100

Di-isopropyl fluorophosphate (I. 1 mM) p-Mercaptoethanol (9.0 mM) N-Bromosuccinimide (2.0 mM) Ethylenediaminetetraacetic acid (I .OmM) Soybean trypsin inhibitor (10.0 mM) p-Tosyl+phenylalanine chloromethvl ketone (0.5 mM)

0 0 0 100 100 100

Arginine ester hydrolase was stable to heating at 95°C for 10 min (pH 8.5) and the enzyme demonstrated a wide pH stability (over the range from 2 to 11). Enzymatic properties

The Michaelis constant (K,,,) for benzoyl-Larginine ethyl ester of arginine ester hydrolase at pH 8.5 was 6.2 x lo-‘M. The inhibition constant (K,) of diisopropyl fluorophosphate (0.15 mM) for this preparation was determined by measuring the initial rate of hydrolysis of benzoyl+arginine ethyl ester at pH 8.5. The inhibition of diisopropyl fluorophosphate was competitive, with an inhibition constant of 1.9 x 10m4M. Smooth muscle contracting activity

One milliliter of heat-treated plasma (70°C 12 min) containing 1 mM o-phenanthroline was incubated for 5 min at 37°C with arginine ester hydrolase. After 5 min, 0.1 ml of the reaction mixture was transferred to a lo-ml bath. Arginine ester hydrolase was observed to liberate smooth muscle contracting material from the plasma within 25 set, and when this enzyme was transferred to the bath, smooth muscle contractions were detected in 120 sec. Transglutaminase-like activity

Oxidized insulin B chain (1.0 mg in I.0 ml of 10 mM ammonium acetate buffer, pH 9.0) was incubated with 3.2 pg of arginine ester hydrolase at 37°C for 24 hr. As can be seen from Fig. 3, oxidized insulin B chain coagulated. XIIIa factor deficient fibrinogen was prepared by the method of Shulman (1953). A solution containing 500~1 of fibrinogen [2mg/ml in 0.05 M phosphate buffer (pH 6.2) containing 0.9% NaCl], 50 ul of 0.025 mM CaCl, and arginine ester hydrolase (25.8 pg) was incubated at 37°C. After

419

venom

Saline + fibrinogen Saline + Ca2+ + fibrinogen Arginine ester hydrolase + Ca’+ + fibrinogen

acid

Monochloroacetic acid

89 66

Soluble within 25 set Soluble within 29 set

34

Insoluble

40 min, 10 ~1 of thrombin (250 U/ml) was added and incubated at 37°C for 12 hr. The solubility of the fibrin clot formed by thrombin was determined by using 1.Oml of 0.44 M monochloroacetic acid, which causes fibrinogen containing arginine ester hydrolase and calcium to be insoluble (Table 3). Fibrin clots formed by thrombin were treated with 4% mercaptoethanol and 4% SDS in 10 M urea at 37°C for 15 hr and then subjected to SDS-PAGE. As can be seen in Fig. 4, y-dimer and a-polymer appeared after 5and 20-min incubations, respectively, with thrombin. During this same time interval the y and Au chains begin to disappear. From these results it is concluded that isolated arginine ester hydrolase possesses a XIIIa-like activity. Sequence analysis of arginine ester hydrolase

The amino terminal sequence of the arginine ester hydrolase was determined using an Applied Biosystems Model 477A sequencer. The phenylthiohydantoin derivatives of the amino acid were identified with an Applied Biosystems Model 120A PTH analyzer. Table 4 shows the amino terminal sequence of arginine ester hydrolase compared with thrombin-like and kallikrein-like enzymes. From these amino terminal sequences arginine ester hydrolase appears to be most similar to porcine pancreatic kallikrein. Recently, a number of arginine ester hydrolases have been isolated from snake venoms (Markland et al., 1971; Bjarnason et al., 1983; Nikai et al., 1983; Sugihara et al., 1984; Sekoguchi et al., 1986). These enzymes, and the arginine ester hydrolase from H. horridum venom, were found to be inhibited by diisopropyl fluorophosphate but were not affected by EDTA. These results suggest that the enzymatic biological activity is not dependent on metal ions, and further indicates that the serine residue may be involved in the biological action. The arginine ester hydrolase from H. horridum venom is similar to

Table 4. Amino-terminal sequences of arginine ester hydrolases, kallikrein-like and thrombin-like proteinases Arginine ester hydrolase 5 IO 15 (A) H,N-Ile-Ile-Gly-Gly~Gln~lu-?-As~Glu-Thr~ly-His-Pr~T~Leu~Aia~Leu-Leu-His-Arg~ 25 30 35 Ser-Glu-Gly-Ser? -?-?-Gly-Val-Leu-Leu-Asn-?-?-AspIle-LeuPorcine pancreatic kallikrein (cc-chain) (B) H,N-Ile-Ile-Gly-Gly-Arg-Glu-Cys-Glu-Lys-Asn-Ser-Hi~Pr~T~Gln-Val~Ala-Il~Tyr-

20

C. atrox. EI (C) H,N~His~Val-Gly-Gly-As~Glu-Cys-Asn-Ile-Asn-Glu~His-Arg-Ser-Leu-Val-Ala-ll~PheC. adamanreus, crotalase (D) H,N~Val-Ile-Gly-Gly-AspClu-Cys-Asn-Ile-Asn-Glu-His-Arg-Ph~Leu~Val-Ala-Leu-Phe B. arierans

(E) H,N-Val-Ile-Gly-Gly-AspGlu-CysAs~Il~Asn~lu-Hi~Pr~Ser-Leu-Ala-Leu-Il~Tyr(A) This paper. (B) Tschesche et al. (1979). (C) Bjamason et al. (1983). (D) Markland and Damus (1971). (E) Joubert and Merrifield (1985).

420

TOSHIAKINIKAI et al.

thrombin-like and kallikrein-like acidic proteins with molecular weights of ca 30 kDa. Purified arginine ester hydrolase from H. horridurn is shown to exhibit XIIIa-like activity, although this activity has not been found in arginine ester hydrolases from other sources. To elucidate this finding further, it would be necessary to investigate the relationship of the purified arginine ester hydrolase with the XIIIa-like activity under in vitro conditions.

Murata Y., Satake M. and Suzuki T. (1963) Studies on snake venom. Distribution of proteinase activities among Japanese and Formosan snake venoms. J. B&hem., Tokyo 53, 431-437.

Nikai T., Kito R., Mori N., Sugihara H. and Tu A. T. (1983) Isolation and characterization of fibrinogenase from western diamondback rattlesnake venom and its comparison to the thrombin-like enzyme, crotalase. Camp. Biochem. Physiol. 76, 679-686.

Roberts P. S. (1958) Measurements of the rate of plasmin action on synthetic substrates. J. hiol. Chem. 232, 285-291.

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Bjarnason J. B., Barish A., Direnzo G. S., Campbell R. and Fox J. W. (1983) Kalhkrein-like enzymes from Crotalus atrox venom. J. biol. Chem. 258, 12566612573. Edelhoch H. (1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 194881954. Erspamer V. and Erspamer F. (1962) Pharmacological actions of eledoisin on extravascular smooth muscle. Br. J. Pharmac. 19, 337-354.

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Joubert F. J. and Merifield E. H. (1985) Purification and properties of arginine esterases from Bitis arierafls (puff adder) venom. Int. J. Biochem. 17, 1293-1298. Markland F. S. and Damus P. S. (1971) Purification and properties of a thrombin-like enzyme from the venom of Crotalus adamanteus (eastern diamondback rattlesnake). J. biol. Chem. 246, 6460-6473.

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