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A ricin variant [RCA60-(-Tyr)] which lacks tyrosine residues, but retains in vivo and in vitro biological activity
Third Pan-American Symposium--Abstracts
627
In conducting the research described in this report, the investigators adhered to the "Guide for the Care and Use of Laboratory Animals", as prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources Commission on Life Sciences-Narional Research Council. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care. The views of the author(s) do not purport to reflect the positions of the Department of the Army or the Department of Defense. Effect o f a Crotalus basiliseus (Mexican West Coast rattlesnake) venom proteina.se (B2) on complement. ARMANVO VA~LA-R^MIm~.Z' and EpPIE D. IL~L2 (IDepartamento de Invesrigacion, Facultad de Medicina, Universidad Autonoma de Chihuahua, Mexico, and 2Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968, U.S.A.).
A PROTEINASEwas isolated from Crotalus basiliscus venom by DEAE-Sephadex A-S0 column chromatography and HPLC with a Rainin Dynamax-300 AX column. The proteinase (132) is a metalloproteinase, which has a tool. wt of 27,500 and a pl of 5.2. B2 causes hemorrhage in mice, is fibrinolytic, fibriogenolytic, hydrolyses casein and hide powder azure. It also inactivates complement in a dose and time-dependent fashion. The lyric activity of guinea pig and human complement disappears when incubated with B2 for 30 min at 37°C. The protease was preincuhated at different temperatures for 45 min prior to testing its effect on complement. There was an increase in the inhibitory activity at 50°C. The effect of the protease on Clq, C3 and CA were examined. B2 degraded the chain of C4 within 5 min after incubation at 37°C. The p and gamma chains of CA were unaffected. Clq and C3 were also not affected. This indicates that the classical pathway was affected by the protease, though it may likewise affect other fractions not yet tested in the alternate pathway. Acknowledgement- Supported by NIH Grant RR08012. A simple and rapid affinity column for the purification ofphospholipases A, and A2from animal venoms. JAVlER VARGAS=VILLARREAL, ' JEsus MARTIN-POLO,'MANUEL J. VARELA2 and ALEJANDROC. ALAGON'('Centro de Investigacion sobre Ingenieria y Biotecnologia, Universidad Nacional Autonoma de Mexico, A.P. 510-3, Cuernavaca, Mor. 62260, Mexico, and ~La Nauyaca, S.C., E. Carranza 487, Mexico, D.F. 09440, Mexico).
A NEW AFFINITYadsorbent for A,- and A2-type phospholipases was prepared. Dimethyl-DL-2,3,-distearoyloxypropyl-2'-hydroxyethylammonium (Rosenthal inhibitor) was coupled to CH-Sepharose 4B, through carbodiimide chemistry. Rosenthars inhibitor is a synthetic phosphatidylcholine analogue that acts as a competitive inhibitor and has been shown to inhibit phospholipase A activity of venoms (ROSENTHALand GF.YER, 1960) and cells (FRYE and FRIOU, 1975; LONG-KRuG et al., 1985). Phospholipase A 2 from Heloderma horridum horridum and Crotalus basiliscus venoms bind to the immobilized ligand in the presence of Ca 2+ and can be easily eluted under acidic conditions. The enzyme from Crotalus was suitable for N-terminal sequence analysis. The homologous protein from Heloderma venom can now be purified with 75% recovery in one single column which contrasts with the 10% yield obtained after four chromatography steps (SOSAet al., 1986). This affinity media proved also very effective in the purification of Ca2+-independent phospholipase A, from vespid venoms. REFERENCES FRYE, L. D. and FRIou, G. J. (1975) Nature 258, 333. LONG-KRUG, S. A., FISHER, K. J., HYSMITH,R. M. and RAVDIN,J. I. (1985) J. Infect. Dis. 152, 536. ROSENTI-IAL,A. F. and GEYER, R. P. (1960) J. Biol. Chem. 235, 2202. SOSA, B. P., ALAGON,A. C., MARTIN, B. M. and POSSANI,L. D. (1986) Biochemistry 25, 2927. A ricin variant [RCAr0-(-Tyr)] which lacks tyrosine residues, but retains in vivo and in vitro biological activity. ROaERT W. WAN~MACHERJR, WILLIAML. THOMPSONand RICHARDE. DINTEgMAN(Pathophysiology Division, USAMRIID, Fort Detrick, Frederick, MD 21701-5011, U.S.A.).
IN THE process of characterizing ricin (RCA~0) from two different commercial sources, we observed that one lot of ricin was deficient in tyrosine (Tyr) residues, as determined by hydrolysis in 6N HC1 and analysis by ion exchange chromatography. The in vivo and in vitro biological activity of this ricin variant [RCAto-(- Tyr)] was compared with a lot of ricin [RCAt0-(+ Tyr)] that had a Tyr content close to previously observed values (LIN and Ll, 1980). The i.e. LDs0 for RCA~0-(-Tyr)] in the mouse was 5.04(3.89- 6.50) #g/kg compared to 3.72(2.92-4.68) #g/kg for RCAt0-(+ Tyr). Mean time to death was 99.2+ 14.5 and 75.5+ 12 hr, respectively. The EDs0 for inhibition of protein synthesis in Vero cells was not significantly different: 65 pg/ml for RCA~o-(-Tyr) and 80 pg/ml for RCA~0-(+Tyr). The pututative cleft, which is responsible for the enzymatic activity of ricin (+ Tyr) A-chain, contains 8 Tyr residues (RoBERTUS, 1988). In addition, chemical modification studies have been implicated Tyr 248 in the 'strong' galactose binding site of the B-chain of ricin. Despite the apparent importance
628
Third Pan-American Symposium--Abstracts
of Tyr in the active sites of ricin, the Tyr-deficient variant evaluated in our laboratory did contain full in vivo and in vitro biological activity. REFERENCES LIN, T. T. S. and L1, S. S. L. (1980) Fur. J. Biochem. 105, 227. ROaERTUS, J. D. (1988) lmmunotoxins (FRANKEL,A. E., Ed.), pp. 11-24, Kluwer Academic pub. In conducting the research described in this report, the investigators adhered to the "Guide for the Care and Use of Laboratory Animals", as prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources Commission on Life Sciences-National Research Council. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care. The views of the author(s) do not purport to reflect the positions of the Department of the Army or the Department of Defense.
PSP content of roe cannot be predicted from that in other tissues of Bay of Fundy sea scallops (Placopecten magellanicus). W. WATSON-WRIGHT,I D. RICHARD, 2 A. BELLIVEAU,3 A. McGuIRE 3 and I. MARSHALL4 (~Department of Fisheries and Oceans, Inspection Services Branch, Halifax, Nova Scotia, Canada B3J 2S7; 2Inspection Services Branch, Black's Harbour, New Brunswick, and 3Inspection Services Branch and 4Fisheries Habitat Branch, Yarmouth, Nova Scotia, Canada). THE 'ROE-ON' scallop industry is relatively new in Canada and as such the ability to predict concentrations of paralytic shellfish poisoning (PSP) in the roe of Placopecten megallanicus from tissues normally discarded (e.g. hepatopancreas, rims) is of interest. Live scallops were collected between 1978 and 1984 and again in 1988 from the Bay of Fundy in eastern Canada, an area known for its annual blooms of Alexandrium tamarensis and consequent intoxification of shellfish. Hepatopancreas, gonads, gills, rims and adductor muscles were separated, extracted and analyzed for PSP using the A.O.A.C mouse bioassay. PSP was non-detectable in all of the 63 adductor muscles analyzed. PSP was detectable in gonadal tissue in 69% of the samples (n = 41). The lowest values for the viscera at which PSP became detectable in the gonadal tissue were 1620/,g/100 g and 310 gg/100 g for hepatopancreas and rims respectively. PSP was detected in only 36% of the gills and was not correlated with that in gonads (r = 0.061; P = 0.80). Quantifiable PSP in gonadal tissue correlated significantly (P < 0.05) with that in hepatopancreas (r = 0.31; P = 0.048) but not rims (r = 0.21; P = 0.19). When only those roe PSP values close to the acceptable limit of 80 #g/100 g were included (range = 50-100), the correlation between PSP content of gonads with hepatopancreas essentially disappeared (r = 0.18; P = 0.60; n = 11) as did that with rims ( r = 0.16; P = 0 . 6 3 ; n = 12). The highest PSP value in any tissue was detected in the hepatopancreas (45,000 #g/100 g) which corresponded with a roe value of 470/zg/100g whereas the highest roe value (1700 #g/100 g) corresponded with a hepatopancreas value of 6700 #g/100 g. On the other occasion where a value of 470/,g/100 g was recorded in the roe, the hepatopancreas contained only 3100 #g/100 g. The lack of correlation between gonads and the other tissues of the Placopecten magellanicus suggests that PSP content in normally discarded tissues is not indicative of that in gonadal tissue and as such should not be used by regulatory agencies, industry or consumers to forecast roe toxicity.
Variation in action upon smooth muscle of toxic isolates from venom ofCentruroides sculpturatus. DEAN D. WATT and MICHAEL H. DAVlDIAN (Division of Biochemistry, Department of Biomedical Sciences, Creighton University, Omaha, NE 68178, U.S.A.). VENOM from the scorpion Centruroides sculpturatus, and other scorpions as well, is a complex mixture of many protein neurotoxins in addition to other non-toxin components. There is considerable variability in biological actions among the individual toxins of the venom. When the venom from C. sculpturat~ is chromatographed on CM-Cellulose, 12 elution zones, with several different toxic components in each zone, are obtained (WATT and SIMARD, 1984). Results from studies of the effects of isolated toxic components upon smooth muscle show that the components of CMC-zone 12 best mimic the action of the crude venom. With mouse colon, the usual spontaneous, variable spiking is transformed into one major, smooth, periodic oscillatory contraction with prolonged relaxation and spikes, if any, having greatly reduced amplitude. Tetrodotoxin (TTX) blocks the major contraction and enhances low amplitude spiking, blocked by verapamil. Baseline tension may or may not be altered. A contraction, blocked by TTX, is induced by the toxic isolates in chick duodenum. Chick rectal cecum responds to CMC zone 12 by formation of a contraction oscillation, blocked by verapamil, having an irregular period. These effects on smooth muscle, and other minor effects, will be related to Na +-channel action by toxins isolated from CMC zone 12 (SIMARDet al., 1986).
627
In conducting the research described in this report, the investigators adhered to the "Guide for the Care and Use of Laboratory Animals", as prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources Commission on Life Sciences-Narional Research Council. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care. The views of the author(s) do not purport to reflect the positions of the Department of the Army or the Department of Defense. Effect o f a Crotalus basiliseus (Mexican West Coast rattlesnake) venom proteina.se (B2) on complement. ARMANVO VA~LA-R^MIm~.Z' and EpPIE D. IL~L2 (IDepartamento de Invesrigacion, Facultad de Medicina, Universidad Autonoma de Chihuahua, Mexico, and 2Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968, U.S.A.).
A PROTEINASEwas isolated from Crotalus basiliscus venom by DEAE-Sephadex A-S0 column chromatography and HPLC with a Rainin Dynamax-300 AX column. The proteinase (132) is a metalloproteinase, which has a tool. wt of 27,500 and a pl of 5.2. B2 causes hemorrhage in mice, is fibrinolytic, fibriogenolytic, hydrolyses casein and hide powder azure. It also inactivates complement in a dose and time-dependent fashion. The lyric activity of guinea pig and human complement disappears when incubated with B2 for 30 min at 37°C. The protease was preincuhated at different temperatures for 45 min prior to testing its effect on complement. There was an increase in the inhibitory activity at 50°C. The effect of the protease on Clq, C3 and CA were examined. B2 degraded the chain of C4 within 5 min after incubation at 37°C. The p and gamma chains of CA were unaffected. Clq and C3 were also not affected. This indicates that the classical pathway was affected by the protease, though it may likewise affect other fractions not yet tested in the alternate pathway. Acknowledgement- Supported by NIH Grant RR08012. A simple and rapid affinity column for the purification ofphospholipases A, and A2from animal venoms. JAVlER VARGAS=VILLARREAL, ' JEsus MARTIN-POLO,'MANUEL J. VARELA2 and ALEJANDROC. ALAGON'('Centro de Investigacion sobre Ingenieria y Biotecnologia, Universidad Nacional Autonoma de Mexico, A.P. 510-3, Cuernavaca, Mor. 62260, Mexico, and ~La Nauyaca, S.C., E. Carranza 487, Mexico, D.F. 09440, Mexico).
A NEW AFFINITYadsorbent for A,- and A2-type phospholipases was prepared. Dimethyl-DL-2,3,-distearoyloxypropyl-2'-hydroxyethylammonium (Rosenthal inhibitor) was coupled to CH-Sepharose 4B, through carbodiimide chemistry. Rosenthars inhibitor is a synthetic phosphatidylcholine analogue that acts as a competitive inhibitor and has been shown to inhibit phospholipase A activity of venoms (ROSENTHALand GF.YER, 1960) and cells (FRYE and FRIOU, 1975; LONG-KRuG et al., 1985). Phospholipase A 2 from Heloderma horridum horridum and Crotalus basiliscus venoms bind to the immobilized ligand in the presence of Ca 2+ and can be easily eluted under acidic conditions. The enzyme from Crotalus was suitable for N-terminal sequence analysis. The homologous protein from Heloderma venom can now be purified with 75% recovery in one single column which contrasts with the 10% yield obtained after four chromatography steps (SOSAet al., 1986). This affinity media proved also very effective in the purification of Ca2+-independent phospholipase A, from vespid venoms. REFERENCES FRYE, L. D. and FRIou, G. J. (1975) Nature 258, 333. LONG-KRUG, S. A., FISHER, K. J., HYSMITH,R. M. and RAVDIN,J. I. (1985) J. Infect. Dis. 152, 536. ROSENTI-IAL,A. F. and GEYER, R. P. (1960) J. Biol. Chem. 235, 2202. SOSA, B. P., ALAGON,A. C., MARTIN, B. M. and POSSANI,L. D. (1986) Biochemistry 25, 2927. A ricin variant [RCAr0-(-Tyr)] which lacks tyrosine residues, but retains in vivo and in vitro biological activity. ROaERT W. WAN~MACHERJR, WILLIAML. THOMPSONand RICHARDE. DINTEgMAN(Pathophysiology Division, USAMRIID, Fort Detrick, Frederick, MD 21701-5011, U.S.A.).
IN THE process of characterizing ricin (RCA~0) from two different commercial sources, we observed that one lot of ricin was deficient in tyrosine (Tyr) residues, as determined by hydrolysis in 6N HC1 and analysis by ion exchange chromatography. The in vivo and in vitro biological activity of this ricin variant [RCAto-(- Tyr)] was compared with a lot of ricin [RCAt0-(+ Tyr)] that had a Tyr content close to previously observed values (LIN and Ll, 1980). The i.e. LDs0 for RCA~0-(-Tyr)] in the mouse was 5.04(3.89- 6.50) #g/kg compared to 3.72(2.92-4.68) #g/kg for RCAt0-(+ Tyr). Mean time to death was 99.2+ 14.5 and 75.5+ 12 hr, respectively. The EDs0 for inhibition of protein synthesis in Vero cells was not significantly different: 65 pg/ml for RCA~o-(-Tyr) and 80 pg/ml for RCA~0-(+Tyr). The pututative cleft, which is responsible for the enzymatic activity of ricin (+ Tyr) A-chain, contains 8 Tyr residues (RoBERTUS, 1988). In addition, chemical modification studies have been implicated Tyr 248 in the 'strong' galactose binding site of the B-chain of ricin. Despite the apparent importance
628
Third Pan-American Symposium--Abstracts
of Tyr in the active sites of ricin, the Tyr-deficient variant evaluated in our laboratory did contain full in vivo and in vitro biological activity. REFERENCES LIN, T. T. S. and L1, S. S. L. (1980) Fur. J. Biochem. 105, 227. ROaERTUS, J. D. (1988) lmmunotoxins (FRANKEL,A. E., Ed.), pp. 11-24, Kluwer Academic pub. In conducting the research described in this report, the investigators adhered to the "Guide for the Care and Use of Laboratory Animals", as prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources Commission on Life Sciences-National Research Council. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care. The views of the author(s) do not purport to reflect the positions of the Department of the Army or the Department of Defense.
PSP content of roe cannot be predicted from that in other tissues of Bay of Fundy sea scallops (Placopecten magellanicus). W. WATSON-WRIGHT,I D. RICHARD, 2 A. BELLIVEAU,3 A. McGuIRE 3 and I. MARSHALL4 (~Department of Fisheries and Oceans, Inspection Services Branch, Halifax, Nova Scotia, Canada B3J 2S7; 2Inspection Services Branch, Black's Harbour, New Brunswick, and 3Inspection Services Branch and 4Fisheries Habitat Branch, Yarmouth, Nova Scotia, Canada). THE 'ROE-ON' scallop industry is relatively new in Canada and as such the ability to predict concentrations of paralytic shellfish poisoning (PSP) in the roe of Placopecten megallanicus from tissues normally discarded (e.g. hepatopancreas, rims) is of interest. Live scallops were collected between 1978 and 1984 and again in 1988 from the Bay of Fundy in eastern Canada, an area known for its annual blooms of Alexandrium tamarensis and consequent intoxification of shellfish. Hepatopancreas, gonads, gills, rims and adductor muscles were separated, extracted and analyzed for PSP using the A.O.A.C mouse bioassay. PSP was non-detectable in all of the 63 adductor muscles analyzed. PSP was detectable in gonadal tissue in 69% of the samples (n = 41). The lowest values for the viscera at which PSP became detectable in the gonadal tissue were 1620/,g/100 g and 310 gg/100 g for hepatopancreas and rims respectively. PSP was detected in only 36% of the gills and was not correlated with that in gonads (r = 0.061; P = 0.80). Quantifiable PSP in gonadal tissue correlated significantly (P < 0.05) with that in hepatopancreas (r = 0.31; P = 0.048) but not rims (r = 0.21; P = 0.19). When only those roe PSP values close to the acceptable limit of 80 #g/100 g were included (range = 50-100), the correlation between PSP content of gonads with hepatopancreas essentially disappeared (r = 0.18; P = 0.60; n = 11) as did that with rims ( r = 0.16; P = 0 . 6 3 ; n = 12). The highest PSP value in any tissue was detected in the hepatopancreas (45,000 #g/100 g) which corresponded with a roe value of 470/zg/100g whereas the highest roe value (1700 #g/100 g) corresponded with a hepatopancreas value of 6700 #g/100 g. On the other occasion where a value of 470/,g/100 g was recorded in the roe, the hepatopancreas contained only 3100 #g/100 g. The lack of correlation between gonads and the other tissues of the Placopecten magellanicus suggests that PSP content in normally discarded tissues is not indicative of that in gonadal tissue and as such should not be used by regulatory agencies, industry or consumers to forecast roe toxicity.
Variation in action upon smooth muscle of toxic isolates from venom ofCentruroides sculpturatus. DEAN D. WATT and MICHAEL H. DAVlDIAN (Division of Biochemistry, Department of Biomedical Sciences, Creighton University, Omaha, NE 68178, U.S.A.). VENOM from the scorpion Centruroides sculpturatus, and other scorpions as well, is a complex mixture of many protein neurotoxins in addition to other non-toxin components. There is considerable variability in biological actions among the individual toxins of the venom. When the venom from C. sculpturat~ is chromatographed on CM-Cellulose, 12 elution zones, with several different toxic components in each zone, are obtained (WATT and SIMARD, 1984). Results from studies of the effects of isolated toxic components upon smooth muscle show that the components of CMC-zone 12 best mimic the action of the crude venom. With mouse colon, the usual spontaneous, variable spiking is transformed into one major, smooth, periodic oscillatory contraction with prolonged relaxation and spikes, if any, having greatly reduced amplitude. Tetrodotoxin (TTX) blocks the major contraction and enhances low amplitude spiking, blocked by verapamil. Baseline tension may or may not be altered. A contraction, blocked by TTX, is induced by the toxic isolates in chick duodenum. Chick rectal cecum responds to CMC zone 12 by formation of a contraction oscillation, blocked by verapamil, having an irregular period. These effects on smooth muscle, and other minor effects, will be related to Na +-channel action by toxins isolated from CMC zone 12 (SIMARDet al., 1986).