7iaxleae, 1976, Vol. 14. pp. 389-391. Parinm Pnr. Prinhd in QiaatHrlWn.
SHORT COMMUNICATIONS MULTIPLE MOLECULAR FORMS OF SNAKE VENOM PHOSPHODIESTERASE FROM VIPERA PALASTINAE Z1vA LEVY and AVNER BDoLAH Department of Zoology, The George s. Win Center for Life SciencM Tel Aviv University, Tel Aviv, Ierael (AcceptedforpxbUcation 27 Jammy 1976)
I (EC 3.1.4.1) is one of the most common enzymes of snake venoms. It is found in the venoms of the two families of venomous snakes, the Viperidae and Elapidae, and also in the oral secretion of non-venomous snakes of the Colubridae (MEBS, 1970). In this laboratory the enzyme has been used as one of the markers for study of secretion in the venom gland of V. palaestinae (SHmLtm et al., 1974). Several procedures for the purification of this enzyme by conventional methods (RAzzE1.L and KHoRANA, 1959; FELDC et al., 1960 ; WII.LIAb1.s et al., 1961 ; BARY, 1963; RicHAPDs et al., 1967) and more recently by affinity chromatography (FhiscHAuF and EcKSmN, 1973 ; Do1.APCHw et al., 1974) have been described. From data that have been published by several laboratories, it becomes apparent that more than one peak of phosphodiesterase appears when venom is fractionated by ion exchange chromatography (BomAN and KALm TA, 1957; Runt et al., 1960; WnLuw et al., 1961). In the present communication we use electrophoresic methods and isoelectrofocusing to show the existence of multiple molecular forms of phosphodiesterase in the venom of V. palaestinae. PHoSPHoDIEsTußAsE
PURIFICATION OF THE ENZYME
The enzyme was purified by a modification of the procedure reported by F)BLnt et al. (1960), which included fractionation with acetone and ethanol and ion exchange chromatography. The DEAF-cellulose eluate was chromatographed twice on hydroxylapatite resulting in the removal of contaminating proteins and a higher specific activity of the enzyme (Table 1). The elution pattern of the phosphodiesterase (Fig. 1) demonstrated several peaks and thus indicated that the enzyme was a non-homogeneous protein. The sedimentation pattern of the purified enzyme showed a single boundary with an S value of 6-2 (D,o .,, = 4-8 x 107). A molecular weight of about 130,000 was estimated (FR scHAuF and EclcsmN, 1973). POLYACRYLAMIDE ELECTROPHORESIS
When crude venom was subjected to disc electrophoresis on acrylamide a group of bands, which reacted positively with a specific staining for phosphodiesterase (S11aeAKowsKA and SHuGAR, 1963), were evident near the cathodic origin of the gel. The purified enzyme showed a similar pattern (Fig. 2). TOJaWN 1976 VaL 14
389
390
Short Communications TAuz
1.
PuRmcATmN oP PsosPHODIFsxzRAw PRom V. palassttnae vwox
Total activity Yield of activity Speck activity step (Units') M (U/ing prot.t) 2 Crude venom 13,500 100 10 Acetone (40-44%0 X00 60 35 22 Ethanol (40-60 %O 4700 21 75 CM-cellulose 2850 2150 15 97 DPAE-cellulose 1800 13 172 Hydroxylapatite I 1400 11 240 Hydroxylapatite II enzyme was assayed according to the method of R Azz L and KHoRANA *The (1959) with 0"5 mM thymidine 5'-phosphop-nitrophenyl in 0-05 M Tris buffer (pH 9-0); 1 mM Car,. A unit of phosphodiesterase is defined as the amount of enzyme which catalyzes the liberation of 1 Pmole product per min at 37°C. tProtein was estimated by the method of Loway et al. (1951).
Flo. 1. CHROMATooRAPHY oN HYDROxyLAPATnm. Ten mg protein of the DEAF-cellulose elate wen dialyzed in 5 mM phosphate buffer (pH 6-8) and applied to a 10 ml hydroxylapatite-column equilibrated with the same buffer. The enzyme was eluted with a linear gradient of 0-005-0"32 M phosphate buffer (20 ml of each). Solid line, Also ; dotted line, enzyme activity. Fractions of 0"5 ail each were collected . ISOELECTROFOCUSING Crude venom that was subjected to isoelectrofocusing in acrylamide gel with carrier ampholytes at a pH range of 3-10 showed multiple phosphodiesterase-positive bands spread over a wide range (Fig. 3 c and d). This pattern was completely different from that obtained after staining for protein, thus excluding the possibility that the multiple phosphodiesterase bands resulted from non-specific absorption of the diazonium salt by the venom proteins. Isofocusing of purified phosphodiesterase obtained by stepwise elution from a hydroxylapatite column showed different profiles for aliquots of enzyme that were eluted by 0-05 M or by 0-1 M phosphate buffer (Fig. 3 a and b). IMMUNOELECTROPHORESIS Specific antiserum against phosphodiesterase was prepared by immunizing rabbits with the enzyme isolated in a preparative acrylamide electrophoresis (SHAHAM et al., TD%ICON 1976 Vol. 14
FIG. 2. DISC ELECrROPHORESIS OF CRUDE VENOM AND PURIFIED PHOSPHODIIMERAM.
Electrophoresis was carried out on discontinuous gel ; running gel, 7-5%, pH 8-6 and spacer gel, 4%, pH 6-7. (a) and (b) 100 pg crude venom; (c) and (d), 50 Flg purified enzyme (the upper portion of the gel) . (a) and (c) stained for phosphodiesterase ; (b) and (d) stained for protein (Coomasie brilliant blue). FIG. 3. ISOELECIROFOCUSING OF PURngED ENZYME AND CRUDE VENOM.
The isofocusing was carried out in 7-5 % acrylamide (CATsimpooLAs, 1968) with 1 % carrier ampholytes at a pH range of 3-10. The protein samples were mixed into themonomer solution and photopolymerized with riboflavin . Gels were stained for protein with CoOmasie after the ampholytes had been removed by repeated washing with 10% trichloroacetic acid. (a) and (b) 0-7 units of phosphodiesterase eluted from hydroxylapatite with 0-05 M and 0-1 M phosphate respectively (see text). (c) and (d) 100 Vg crude venom (0-2 units of enzyme). (a), (b) and (c) stained for phosphodiesterase ; (d) stained for protein. FIG. 4. IMMUNOELECTROPHORESIs . Troughs, antisera against the enzyme from two different rabbits. Central well, 8 mg per ml of purified enzyme. Outer wells, 10 mg per ml of crude venom. (a) Stained for protein; (b) stained for phosphodiesterase . f.p .
390
Short Communicattooe
391
1973). When the purified enzyme was tested against this antiserum, four precipitin arcs were evident (Fig. 4). The specific staining for phosphodiesterase was not clear enough, but several phosphodiesterase positive arcs could again be revealed . From the results presented above we conclude that phosphodiesterase exists in the venom of V. palaestinae in multiple molecular forms. This was clearly demonstrated by the elution pattern of the enzyme from hydroxylapatite and by the electrophoretic and iso electrofocusing profiles . In the crude venom phosphodiesterase with a wide range of isoelectric points was found. Employing the purification procedure described above an enrichment of enzyme species with alkaline isoelectric points was achieved. Acknowledgements-The authors thank Prof. E. KocHvA for his encouragement daring the preparation of this paper and to Mrs. S. KmtAwN for performing the isoelectrofocusing. REFERENCES Bj6RK, W. (1963) Purification of phosphodieste aw from Bothrops atrox venom, with special consideration of the elimination of monophosphatase. J. biol. Clam. 238, 2487. BOMAN, H. O. and KArsrrA, U. (1957) ChromatograPhY of rattlesnake venom. A separation of three Phosphodiestma9es . Biochim. biophys. Acts 24, 619. CATSMIPOOLAS, N. (1968) Micro isoeixtrk focusing in polyacrylamide gel columns. Analyt. Btochen 26, 480. Dot.ApcEnv, L. B., SuLKowsa, E. and LAswwsa, M., SR. (1974) Purification of exonuclease (phospho. diesterase) from the venom of Crotalw adamanteus . Biochem. btophys. Res. CommwL 61, 273. Fir, F., Porrim, J. L. and LASKOwsK4 M. (1960) Action of phosphodiesteraw on deo yribo-oligonudeotides carrying amonoesterißed Phosphate on carbon 3'. J. blot. Chum. 234 1150. FRHG$AuF, A. M. and EczsrsN, F. (1973) Purification of ph~phodiestaase from Bothrops atrox by amity chromatography. Eur. J. Biodum 32, 479. LOWRY, O. H., AosmRoUßH, N. J., FARR, À. L. and RANDALL, RJ. (1951) Protein measurement with folin phenol reagent. J. b1ol. Chem 1A 265. Mrs, D. (1970) A comparative studyof enzyme activities in snake venomL Int. J. Biochem,1, 335. RAZZES., W. E. and KEoRANA, H. (l. (1959) Studies on polynucleotides.111 . Enzymic degradation, substrate specificity and properties of snakevenom phosphodieaterase. J. blot. Chem. 234, 2105. RiamARUS, 0. M., TuTAS, D. J., Wecwnm, W. J. and LmzowsKr, M., SR. (1967) Hydrolysis of dinucleoside monophosPhates containing arabinow in various intemurleotide linkages by exonuclease from venom of Crotaho adamanteus. Biochemistry 6, 2908. SHAHAm, N., BDoLAH, A. and KocavA, E. (1973) Isolation of lamino add oxidaw from Ylpera palaesthw venom and preparation of a monospeciflc antiserurn in rabbits. In : Toxins of Animal and Plant Origin. VOL 3, p. 919, (a VRms, A. and KocHvA, E., Eds.) . New York : Gordon & Breach. SHAHAm, N., IxvY, Z., KocavA, E, and BDor AE4 A. (1974) Localization of venom antigens in the venom gland of Vipers paAhwstinae. Hlstodum. J. 6, 98. SoneAKOwsKA, H. and SHUCux, D. (1963) Cytochemical localization of phosphodiesteraw by the azo dye simultaneous coupling method . Bioc~ biophys. Res. ComnuuL 11, 70. WIImASO, E. J., SuNo, S. and LAmowsa, M., SR . (1%1) Action of phosphodiesterase on DNA. J. b1ol. Chem. 236,1130.
T027CON 1976 YoL 14
SHORT COMMUNICATIONS MULTIPLE MOLECULAR FORMS OF SNAKE VENOM PHOSPHODIESTERASE FROM VIPERA PALASTINAE Z1vA LEVY and AVNER BDoLAH Department of Zoology, The George s. Win Center for Life SciencM Tel Aviv University, Tel Aviv, Ierael (AcceptedforpxbUcation 27 Jammy 1976)
I (EC 3.1.4.1) is one of the most common enzymes of snake venoms. It is found in the venoms of the two families of venomous snakes, the Viperidae and Elapidae, and also in the oral secretion of non-venomous snakes of the Colubridae (MEBS, 1970). In this laboratory the enzyme has been used as one of the markers for study of secretion in the venom gland of V. palaestinae (SHmLtm et al., 1974). Several procedures for the purification of this enzyme by conventional methods (RAzzE1.L and KHoRANA, 1959; FELDC et al., 1960 ; WII.LIAb1.s et al., 1961 ; BARY, 1963; RicHAPDs et al., 1967) and more recently by affinity chromatography (FhiscHAuF and EcKSmN, 1973 ; Do1.APCHw et al., 1974) have been described. From data that have been published by several laboratories, it becomes apparent that more than one peak of phosphodiesterase appears when venom is fractionated by ion exchange chromatography (BomAN and KALm TA, 1957; Runt et al., 1960; WnLuw et al., 1961). In the present communication we use electrophoresic methods and isoelectrofocusing to show the existence of multiple molecular forms of phosphodiesterase in the venom of V. palaestinae. PHoSPHoDIEsTußAsE
PURIFICATION OF THE ENZYME
The enzyme was purified by a modification of the procedure reported by F)BLnt et al. (1960), which included fractionation with acetone and ethanol and ion exchange chromatography. The DEAF-cellulose eluate was chromatographed twice on hydroxylapatite resulting in the removal of contaminating proteins and a higher specific activity of the enzyme (Table 1). The elution pattern of the phosphodiesterase (Fig. 1) demonstrated several peaks and thus indicated that the enzyme was a non-homogeneous protein. The sedimentation pattern of the purified enzyme showed a single boundary with an S value of 6-2 (D,o .,, = 4-8 x 107). A molecular weight of about 130,000 was estimated (FR scHAuF and EclcsmN, 1973). POLYACRYLAMIDE ELECTROPHORESIS
When crude venom was subjected to disc electrophoresis on acrylamide a group of bands, which reacted positively with a specific staining for phosphodiesterase (S11aeAKowsKA and SHuGAR, 1963), were evident near the cathodic origin of the gel. The purified enzyme showed a similar pattern (Fig. 2). TOJaWN 1976 VaL 14
389
390
Short Communications TAuz
1.
PuRmcATmN oP PsosPHODIFsxzRAw PRom V. palassttnae vwox
Total activity Yield of activity Speck activity step (Units') M (U/ing prot.t) 2 Crude venom 13,500 100 10 Acetone (40-44%0 X00 60 35 22 Ethanol (40-60 %O 4700 21 75 CM-cellulose 2850 2150 15 97 DPAE-cellulose 1800 13 172 Hydroxylapatite I 1400 11 240 Hydroxylapatite II enzyme was assayed according to the method of R Azz L and KHoRANA *The (1959) with 0"5 mM thymidine 5'-phosphop-nitrophenyl in 0-05 M Tris buffer (pH 9-0); 1 mM Car,. A unit of phosphodiesterase is defined as the amount of enzyme which catalyzes the liberation of 1 Pmole product per min at 37°C. tProtein was estimated by the method of Loway et al. (1951).
Flo. 1. CHROMATooRAPHY oN HYDROxyLAPATnm. Ten mg protein of the DEAF-cellulose elate wen dialyzed in 5 mM phosphate buffer (pH 6-8) and applied to a 10 ml hydroxylapatite-column equilibrated with the same buffer. The enzyme was eluted with a linear gradient of 0-005-0"32 M phosphate buffer (20 ml of each). Solid line, Also ; dotted line, enzyme activity. Fractions of 0"5 ail each were collected . ISOELECTROFOCUSING Crude venom that was subjected to isoelectrofocusing in acrylamide gel with carrier ampholytes at a pH range of 3-10 showed multiple phosphodiesterase-positive bands spread over a wide range (Fig. 3 c and d). This pattern was completely different from that obtained after staining for protein, thus excluding the possibility that the multiple phosphodiesterase bands resulted from non-specific absorption of the diazonium salt by the venom proteins. Isofocusing of purified phosphodiesterase obtained by stepwise elution from a hydroxylapatite column showed different profiles for aliquots of enzyme that were eluted by 0-05 M or by 0-1 M phosphate buffer (Fig. 3 a and b). IMMUNOELECTROPHORESIS Specific antiserum against phosphodiesterase was prepared by immunizing rabbits with the enzyme isolated in a preparative acrylamide electrophoresis (SHAHAM et al., TD%ICON 1976 Vol. 14
FIG. 2. DISC ELECrROPHORESIS OF CRUDE VENOM AND PURIFIED PHOSPHODIIMERAM.
Electrophoresis was carried out on discontinuous gel ; running gel, 7-5%, pH 8-6 and spacer gel, 4%, pH 6-7. (a) and (b) 100 pg crude venom; (c) and (d), 50 Flg purified enzyme (the upper portion of the gel) . (a) and (c) stained for phosphodiesterase ; (b) and (d) stained for protein (Coomasie brilliant blue). FIG. 3. ISOELECIROFOCUSING OF PURngED ENZYME AND CRUDE VENOM.
The isofocusing was carried out in 7-5 % acrylamide (CATsimpooLAs, 1968) with 1 % carrier ampholytes at a pH range of 3-10. The protein samples were mixed into themonomer solution and photopolymerized with riboflavin . Gels were stained for protein with CoOmasie after the ampholytes had been removed by repeated washing with 10% trichloroacetic acid. (a) and (b) 0-7 units of phosphodiesterase eluted from hydroxylapatite with 0-05 M and 0-1 M phosphate respectively (see text). (c) and (d) 100 Vg crude venom (0-2 units of enzyme). (a), (b) and (c) stained for phosphodiesterase ; (d) stained for protein. FIG. 4. IMMUNOELECTROPHORESIs . Troughs, antisera against the enzyme from two different rabbits. Central well, 8 mg per ml of purified enzyme. Outer wells, 10 mg per ml of crude venom. (a) Stained for protein; (b) stained for phosphodiesterase . f.p .
390
Short Communicattooe
391
1973). When the purified enzyme was tested against this antiserum, four precipitin arcs were evident (Fig. 4). The specific staining for phosphodiesterase was not clear enough, but several phosphodiesterase positive arcs could again be revealed . From the results presented above we conclude that phosphodiesterase exists in the venom of V. palaestinae in multiple molecular forms. This was clearly demonstrated by the elution pattern of the enzyme from hydroxylapatite and by the electrophoretic and iso electrofocusing profiles . In the crude venom phosphodiesterase with a wide range of isoelectric points was found. Employing the purification procedure described above an enrichment of enzyme species with alkaline isoelectric points was achieved. Acknowledgements-The authors thank Prof. E. KocHvA for his encouragement daring the preparation of this paper and to Mrs. S. KmtAwN for performing the isoelectrofocusing. REFERENCES Bj6RK, W. (1963) Purification of phosphodieste aw from Bothrops atrox venom, with special consideration of the elimination of monophosphatase. J. biol. Clam. 238, 2487. BOMAN, H. O. and KArsrrA, U. (1957) ChromatograPhY of rattlesnake venom. A separation of three Phosphodiestma9es . Biochim. biophys. Acts 24, 619. CATSMIPOOLAS, N. (1968) Micro isoeixtrk focusing in polyacrylamide gel columns. Analyt. Btochen 26, 480. Dot.ApcEnv, L. B., SuLKowsa, E. and LAswwsa, M., SR. (1974) Purification of exonuclease (phospho. diesterase) from the venom of Crotalw adamanteus . Biochem. btophys. Res. CommwL 61, 273. Fir, F., Porrim, J. L. and LASKOwsK4 M. (1960) Action of phosphodiesteraw on deo yribo-oligonudeotides carrying amonoesterißed Phosphate on carbon 3'. J. blot. Chum. 234 1150. FRHG$AuF, A. M. and EczsrsN, F. (1973) Purification of ph~phodiestaase from Bothrops atrox by amity chromatography. Eur. J. Biodum 32, 479. LOWRY, O. H., AosmRoUßH, N. J., FARR, À. L. and RANDALL, RJ. (1951) Protein measurement with folin phenol reagent. J. b1ol. Chem 1A 265. Mrs, D. (1970) A comparative studyof enzyme activities in snake venomL Int. J. Biochem,1, 335. RAZZES., W. E. and KEoRANA, H. (l. (1959) Studies on polynucleotides.111 . Enzymic degradation, substrate specificity and properties of snakevenom phosphodieaterase. J. blot. Chem. 234, 2105. RiamARUS, 0. M., TuTAS, D. J., Wecwnm, W. J. and LmzowsKr, M., SR. (1967) Hydrolysis of dinucleoside monophosPhates containing arabinow in various intemurleotide linkages by exonuclease from venom of Crotaho adamanteus. Biochemistry 6, 2908. SHAHAm, N., BDoLAH, A. and KocavA, E. (1973) Isolation of lamino add oxidaw from Ylpera palaesthw venom and preparation of a monospeciflc antiserurn in rabbits. In : Toxins of Animal and Plant Origin. VOL 3, p. 919, (a VRms, A. and KocHvA, E., Eds.) . New York : Gordon & Breach. SHAHAm, N., IxvY, Z., KocavA, E, and BDor AE4 A. (1974) Localization of venom antigens in the venom gland of Vipers paAhwstinae. Hlstodum. J. 6, 98. SoneAKOwsKA, H. and SHUCux, D. (1963) Cytochemical localization of phosphodiesteraw by the azo dye simultaneous coupling method . Bioc~ biophys. Res. ComnuuL 11, 70. WIImASO, E. J., SuNo, S. and LAmowsa, M., SR . (1%1) Action of phosphodiesterase on DNA. J. b1ol. Chem. 236,1130.
T027CON 1976 YoL 14
