Purification and characterization of the endonuclease present in Physalia physalis venom

Comp. Biochem.Physiol.,Vol.6711,pp. 155 to 158 PergamonPressLtd 1980.Printedin Great Britain

PURIFICATION AND CHARACTERIZATION OF THE ENDONUCLEASE PRESENT IN PHYSALIA PHYSALIS VENOM ISHAK NEEMANt, GARY J. CALTON1 and JOSEPH W. BURNETT1'2 tDepartment of Pharmacognosy and Division of Dermatology, University of Maryland Schools of Pharmacy and Medicine, Baltimore, Maryland, U.S.A., and 2U.S. Veterans' Administration Hospital, Baltimore, Maryland, U.S.A.

(Received 2 January 1980) Abstract--1. Physalia physalis nematocyst venom contains a DNase which has a non-specific endonucleolytic action. 2. This enzyme has an approximate molecular weight of 75,000 daltons. 3. The enzyme can cleave DNA over a wide pH range with an optimum near neutrality. 4. The enzyme is thermolabile and its activity can be stimulated by 80 mM NaCI or 10 mM MgC12. INTRODUCTION

Specimens of Physalia physalis were collected off the Florida Atlantic coast during the winter of 1976. The tentacles were removed manually, shipped to Baltimore in dry ice, and stored at - 70°C. A suspension of nematocyst was prepared from these tentacles by homogenization and centrifugation a s described by Calton & Burnett (1973).

The nematocyst venom of the Portuguese man-o'war (Physalia physalis) contains a n u m b e r of toxic proteins a n d injurious enzymes which exhibit multiple actions including mouse lethality, dermonecrosis, neurotoxicity, hemolysis and cardiotoxicity (Burnett & Calton, 1977). The enzymatic content of the venom was determined by Burnett & Calton (1974). A m o n g the deleterious enzymes, a D N a s e was present in the venom of the man-o'war as well as in that of the sea nettle (Chrysaora quinquecirrha). D N a s e is also present in the venom of other animals such as snakes (Pritchard et al., 1977) and wasps (Slor et al., 1978). The sea nettle D N a s e has recently been further purified and characterized (Neeman et al., 1980). The present investigation was undertaken to characterize Physalia D N a s e in order to compare it with those found in other venoms and thus to obtain a better understanding of the possible routes of venom action.

DNase Assays Acid-soluble fraction assay. The assay was based on the conversion of thermally denatured or native [3H]DNA, to acid soluble products, Friedberg et al. (1975). The standard assay mixture of 0.2 ml contained 0.3 nmol DNA in 10 mM Tris pH 7.0 buffer with 20 mM NaC1. The test sample was added and the reaction topped after 30 min incubation at 37°C by the addition of 0.1 ml of cold 20% trichloracetic acid and 0.005 ml of calf thymus DNA solution (1 mg/ml). The sample was immersed in ice for 10 min then centrifuged at 7000 @for 15 min at 4°C. The radioactivity present in 0.2 ml of supernatant was counted in 10 ml Biofluor scintillation counting fluid (New England Nuclear Co., Boston, MA). The supernatant fluid obtained from reagent control incubations contained 0.05% of the added radioactivity. One unit of DNase converted 1 nmol. of denatured E. coil DNA to an acid soluble form, under the conditions described. Incision assay. The incision assay was done according to the method of Braun & Grossman (1974). A mixture containing [3HI 0X 174 RFI DNA, and 0.2 ml 10 mM Tris (pH 7.0) buffer with 10 mM NaCI, was incubated at 37°C for 30 min. The nucleic acid was denatured by the addition of 2 ml Tris (pH 12) buffer containing 100 mM sodium phosphate, 30 mM sodium chloride and 25 mM EDTA. This mixture was later neuuralized to pH 7.6 by adding 0.4 ml of 25 mM Tris-HCl (pH 4.0) solution. Under these conditions [3HI 0X RFI DNA was conserved, but the incised DNA was converted to at least tw.o elongated single stranded pieces. The RFI DNA passed through nitrocellulose membrane filters (B-6 filters, Scleier and Schnell) (Center & Richardson, 1970) whereas, the single stranded DNA remained on the nitrocellulose filters. The filters were washed twice with 4 ml NaCI citrate solution (300 mM NaCI and 30 mM citrate), dried and counted in 10 ml Instatiuor counting fluid (Packard Instrument Co., Downers Grove, IL).

MATERIALS AND METHODS The materials used in these investigations were obtained as follows: Tritium labeled 0X 174 RFI DNA was prepared by the method of Feldberg & Grossman (1976). The phage had a specific activity of 2-3 x 105 cpm/pg. Tritium labeled E. coil DNA was prepared according to the method of Mahler (1967) with a specific activity of 36,000 cpm/nmol. Calf thymus DNA and transfer RNA were obtained from Sigma Chemical Co., St Louis, MO; phage DNA was purchased from Bethesda Biochemical Laboratory, Bethesda, MD. Whatman DEAE-cellulose (DE-52) was used after precycling. DNA cellulose was prepared according to the technique of Riazuddin (1977) as follows. Cellulose (Munktell No. 410) was washed several times with boiling ethanol to remove pyridine, reduced at 60°C for 60 min with 0.2 M sodium borohydride, washed with water to neutrality and finally lyophilized. Treated cellulose (20 g) was mixed into a slurry with 40 ml of calf thymus DNA (2 mg/ml) and spread in a thin layer on a glass surface to dry slowly at room temperature. After drying, the DNA cellulose was resuspended in 100 ml of 95~o ethanol, irradiated with u.v. light for 10 rain at a dose of 15 J/m/see, then filtered and washed exhaustively with "10mM sodium chloride. The final product was air dried and stored at -20°C.

Protein determination Protein was determined by the method of Lowry et al. (1951) or Groves et al. (1968).

155

156

ISHAK NEEMAN, GARY J. CALTON a n d JOSEPH W. BURNETT

Table 1. Purification scheme for Physalia endonuclease

Fraction Extract Streptomycin sulfate supernatant DEAE cellulose Concentration with Sephadex G-200 DNA cellulose

Volume (ml) 40 70 90 5 7

Total protein (mg)

Total DNase* Specific activity activity (units per mg (units) protein)

300 190 45 40 1.2

7000 6200 4800 4500 3100

23.3 32.6 106.6 112.5 2583

* DNase activity is defined as the amount of enzyme necessary to convert 1 nmol of denatured E. coli DNA to an acid soluble form.

Ultraviolet irradiation Tritium labeled 0X 174 DNA in 1 mM Tris Buffer (pH 7.3) containing 1 mM EDTA was irradiated in a 2 mm path length. Radiation was emitted primarily at 254 nM from a low pressure mercury lamp (Sylvania). The amount of incident radiation, 2500 erg/mm 2, was determined by a Black Ray ultraviolet monitor (Ultra-Violet Products, Inc., San Gabriel, CA). Molecular weight determination Molecular weights were determined using Biogel A 0.5M (Bio Rad Lab, Richmond, CA) column (2.6 x 37.6 cm). The eluent was 50mM potassium phosphate buffer pH 7.0 containing 5% glycerol. The standards were bovine serum albumin, soybean lipoxidase, yeast alcohol dehydrogenase and catalase. DNA denaturation All DNA denaturation was done by boiling the DNA solution for 10 min before immediate cooling. Isolation and purification of Physalia physalis endonuclease Frozen nematocyst suspensions (20 g) were thawed and suspended in 20 ml buffer containing 10 mM PO4 (pH 7.0), 2 mM 2-mercaptoethanol and 5% glycerol. This mixture was homogenized manually and then sonicated with four pulses of 45 sec each in a Branson sonifier (Branson Instrument, Danbury, CT). All operations were performed at 4°C. After sonication, the tissue particles were removed by centrifugation (90 min at 18,000 g). 30 ml buffer containing 3.3% w/v of streptomycin sulfate was slowly added to the clarified solution and 1 hr later the precipitate was removed by centrifugation. This final supernatant was dialyzed for 24 hr against buffer A (10 mM potassium phosphate buffer, pH 7.3; 1 mM 2-mercaptoethanol; 5% glycerol). DEAE-cellulose chromatography A column of precycled Whatman DE-52 (1.6 × 20 cm) was prepared and equilibrated with 3 liters of buffer A. The dialyzed crude extract was loaded on the column with a flow rate of 1 ml/min and washed with 160 ml of Buffer A. The protein was collected in 5 ml fractions in a 450 ml linear gradient of Buffer A containing 30-90 mM KCI. Concentration of the active fractions from the DE-52 column was performed by solvent absorption into dry Sephadex G-200 beads. DNA cellulose chromatography A column of DNA cellulose (0.8 × 4 cm) was equilibrated with 200 ml of Buffer B, (10 mM Tris, pH 7.0); 1 mM 2-mercaptoethanol; 5% glycerol). 5 ml of the inoculation to be introduced was initially dialyzed for 6 hr against Buffer B before being applied to the column at a rate of 1 ml per 15 min. The column was then washed with 5 ml Buffer B and eluted with a 40 ml linear gradient of Buffer B containing 0-1 M NaC1. All fractions were collected in 2.2 ml aliquots.

RESULTS Enzyme purification Physalia DNase was purified a total of ll0-fold by a four-step procedure (Table 1). D E A E cellulose chromatography resulted in a three-fold purification of the endonuclease (Fig. 1). Seventy-seven per cent of the enzyme was recovered and pooled for the next procedure. Subsequent affinity chromatography with D N A cellulose allowed a 23-fold purification (Fig. 2) with a 68% recovery of enzyme. Peak enzymatic activity in the eluate from this column was detected at 0.4 M NaC1.

Properties of the enzyme The molecular weight of the enzyme was estimated to be 75,000 daltons according to Biogel column chromatography. At least half the enzyme activity was lost after storage in 50% glycerol for 4 weeks at - 2 0 ° C . The addition of 2-mercaptoethanol (2mM) was partially protective and reduced this loss of enzyme potency to only 10% under the same conditions. The enzyme had a pH optimum of 6.5-7.1 in either 10 m M Tris or sodium phosphate buffers. At pH 3.5 and 8.5 it retained only 30 and 10% of its maximal activities respectively. The endonuclease possessed maximal potency at 80 m M sodium chloride (Fig. 3). This salt concentration was optimal for the enzyme with substrates of either native E. coli D N A or heat denatured DNA. The enzyme is strongly stimulated by the addition of MgCI2 with maximum augmentation appearing at concentration of 10 m M (Fig. 4). POOLED FRACTION

IOC

mM KCI 3OO


°

i

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N~ 5c

J ss1 IS

Om

~$I~~"

200 I00'

N ~/ o,i., [

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7:5

8~, 97

Fig. 1. Elution pattern of DNase from man-o'war venom on DEAE c e l l u l o s e - - - KCI concentration. The crude venom was treated with streptomycin sulfate and the supernatant placed on the column.

Physalia physalis endonuclease

I001

/ I

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~z

75

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/

90

a

z

80 7°

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20

FRACTION Fig. 2. Elution pattern of DNase activity of man-o'war venom on DNA cellulose. The inoculum had previously been chromatographed on DEAE and concentrated. - - NaC1 concentration.

The endonucleolytic activity of the enzyme was demonstrated by the 0X incision assay and by the acid soluble fraction release from native or heat denatured 0X D N A (Table 2). The activity was equal for both single and double stranded DNA. Irradiation of the substrate with ultraviolet light did not affect the activity of the endonuclease (Table 2). A competition experiment using unlabeled D N A (15 fold concentration) in the presence of either labeled native, heat denatured or irradiated D N A indicated that the endonuclease affected all three substrates equally (Table 2). DISCUSSION Physalia DNase was equally active on native and heat denatured 0X RFI D N A thus indicating a nonspecific endonucleolytic action. Activities such as this are not common since most venoms possess exonucleolytic actions. A small amount of endonuclease is found in some snake venoms, e.g. Crotalus adamanteus. Pritchard et al. (1977) reported that this enzyme 10(

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~m

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I 40

Fig. 4. Effect of MgCI2 on endonuclease activity. The reaction was carried out in 10 mM Tris buffer, pH 7.0. One unit of enzyme was reacted with 1 #g of E. coli DNA. could cleave single stranded and superhelical DNA. An endonuclease was detected in the venom of the oriental hornet (Slor et al., 1970). The Physalia endonuclease can cleave D N A over a wide pH range with an optimum near neutrality. The D N A digesting enzyme is stimulated by 75 m M NaC1 yet can be inhibited by 200 m M NaC1. The single stranded D N A purified from sea nettle (Chrysaora quinquecirrha) venom was stimulated by low salt concentration and active even at 300 m M NaC1 Neeman et al. (1980). Physalia endonuclease also differed from sea nettle endonuclease in that low concentrations of NaC1 augmented the action of the former enzyme but inhibited that of the sea nettle. The equal activity of the Physalia endonuclease on ultraviolet light irradiated D N A of both native and heat denatured D N A excluded an inhibiting effect of thymine-thymine dimer on this enzyme. Such an inhibition had been previously reported on some exonucleases (Small, 1971).

Table 2. Relative activity of Physalia endonuclease on various substrates DNA substrate

Q o<~ z _o_

I 30

Mg CIz CONCENTRATION mM

t'z~ i0

157

3bo

No CI CONCENTRATION mM.

Fig. 3. Effect of NaC1 concentration on endonuclease activity. The reactions were carried out in 10 mM Tris buffer, pH 7.0. One unit of enzyme was used with 1 #g of E. coli DNA or 1/~g of denatured E. coli DNA. • • Native E. coli DNA; @ • Denatured E. coil DNA.

Yo Relative enzyme activity

Native 0X RFI Heat denatured OX RFI UV denatured OX RFI Heat and u.v. denatured 0X RFI

100 100 100 100

Native E. coli Heat denatured E. coli Native labeled and native unlabeled E. coil Native labeled and heat denatured unlabeled E. coli Heat denatured labeled and native unlabeled E. coil Heat denatured labeled and heat denatured unlabeled E. coli

100 100 5

8 3 4

DNA was denatured by irradiation with a dose of 250 J/m 2. The endonuclease activities of the substrates in the upper half of the table were performed by the incision assay, whereas, those reported in the lower half were determined by the release of acid soluble products.

158

ISHAK NEEMAN, GARY J. CALTON and JOSEPH W. BURNETT

Acknowledgements--These investigations were funded by grants-in-aid ES01474 from the National Institutes of Health and the Brandywine Foundation.

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GROVES W. E., FAVIS F. C. JR & SELLS B. H. (1968) Spectrophotometric determination of microgram quantities of protein without nucleic acid interference. Analyt. Biochem. 22, 195-210. LOWRY O. H., ROSEBROUGHN. J,, FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MAHLER I. (1967) Preparation of tritium-labeled DNA from Bacillus subt_..s and Escherichia coli. In Methods of Enzymology (Edited by GROSSMAN L. & MOLDAVE K.), Vol. 12, Part A, p. 693. Academic Press, New York. NEEMAN I., CALTON G. J. & BURNETTJ. W. (1980) Purification of a novel endonuclease present in Chrysaora quinquecirrha venom. Proc, Soc. exp. Biol. Med. In press. PRITCHARD A. F., KOWALSKI D. t~ LASKOWSKI M. SR (1977) An endonuclease activity of venom phosphodiesterase specific for single stranded and superhelical DNA. J. biol. Chem. 252, 8652-8659. R1AZUDDIN S. • GROSSMAN L. (1977) Micrococcus luteus correndonucleases--I. Resolution and purification of two endonucleases specific for DNA containing pyrimidine dimers. J. biol. Chem. 252, 6280-6286. SLOR H., RING B. & ISHAYJ. (1976) Nucleases of the oriental hornet (Vespa orientalis) venom sac extract I acid, neutral and alkaline deoxyribonucleases and their pharmacological effect on cat blood in vitro. Toxicon 14, 427-433. SMALL G. n., TAO M. ~,~ GORDON M. P. (1971) Pyrimidine hydrates and dimers in ultraviolet irradiated tobacco mosaic virus. J. molec Biol. 38, 75-87.