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A bioassay for cobra cardiotoxin activity using semi-isolated cockroach heart
Tozicar VoL 30. No. 3, pp. 295-301, 1992. Printed in Great Britain.
0041-0101/92 $5 .00 + .00 ® 1992 Perpmon Pres plc
A BIOASSAY FOR COBRA CARDIOTOXIN ACTIVITY USING SEMI-ISOLATED COCKROACH HEART MARC J. KLOMMEN,' ANGELO J. VITALE,' MAYME J. TRUMBLE, 2 CARLA RUDER WESSON and WILLIAM R. TRUMBLE2*
'Division of Entomology, and Department of Bacteriology and Biochemistry, University of Idaho, Moscow, ID 83843, U.S .A . (Received 30 July 1991 ; accepted 30 October 1991)
M. J. KLowDEN, A. J. VITALE, M. J. TRumBLE, C. R. WESSON and W. R. TRuMBLE. A bioassay for cobra cardiotoxin activity using semi-isolated cockroach heart. Toeicon 30, 295-301, 1992.-A semi-isolated cockroach heart preparation was used to rapidly determine the activity of cobra cardiotoxin, monitored as a direct response on heart rate . This preparation produced a dose-response curve in the presence of active cardiotoxin and demonstrated that cardiotoxin retained its biological activity after boiling, although Cardiotoxnn activity was destroyed by heating in the presence of dithiothreitol. Experiments that cross-linked radiolabeled cardiotoxin to solubilized cockroach heart membranes suggested that cardiotoxin bound specifically to a 59,000 mob. wt membrane protein in this tissue . INTRODUCTION
venom is a remarkable source of diverse biologically active basic peptides. Although the chemical structures of many of these peptides are quite similar, they exhibit different types of pharmacological actions including potent neurotoxic, cytotoxic and cardiotoxic activities (Tu, 1977). Cardiotoxins (CTX) are 60 amino acid peptides that constitute the major peptide component of cobra venom (TAN, 1982). More than 40 cardiotoxins have been isolated, and their amino acid sequences suggest that they belong to a large family of similar molecules. The effect of purified CTX on heart muscle was first observed by SARKAR (1947) who reported that CTX arrested the heartbeat of cats and stopped perfused toad hearts . The amino acid sequence of CTX from the Taiwan cobra, Naja naja atra, was subsequently determined by NARITA and LEE (1970) . While the physiological effects of cardiotoxin have been well studied (reviewed by HARvEY, 1985), the molecular mechanisms by which CTX induces muscle contraction are not known. A variety of techniques have been employed to evaluate the activity of CTX. Many of these involve expensive and time-consuming preparations, including the use of live animals (SARKAR, 1947; SuN and WALKER, 1986), whole organs (SARKAR, 1947; WOLF et al., 1968), cardiac and skeletal tissue preparations (Ho et al., 1975; SmAU et al., 1978; HARvEY et al., 1982; SUN and WALKER, 1986; BODGES et al., 1987), cultured cells COBRA
" To whom correspondence should be addressed. 295
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(CONDREA et al., 1964; Ho et al., 1975; TONSING et al., 1983; HODGES et al., 1987; JIANG et al., 1989) and membrane vesicles (NAYLER et al., 1976; SHIAu et al., 1978; FouRiE et al ., 1983; HUANG and TRu1BLE, 1991). Our studies of CTX required a rapid and inexpensive assay for the biological activity of cardiotoxins and analog peptides . This paper describes the development of a semi-isolated cockroach heart preparation that can be used as a bioassay for cardiotoxin activity . MATERIALS AND METHODS Cardiotoxin The Naja naja atra GTX used in these experiments was purchased from Ventoxin, Inc . (Frederick, MD,
U .S .A .) or purified in our laboratory from whole lyophilized venom by a combination of cation exchange chromatography (Mono-S FPLC column ; Pharmacia, Piscataway, NJ, U .S .A .) and reverse phase chromatography (RPC-10 FPLC column ; Pharmacia) .
Cockroach bioassay
The Madagascar hissing cockroach, Gromphadorhina portentosa (Blattaria: Blaberidae), was lightly anesthetized with ether until immobile and placed dorsal surface down under a dissecting microscope. The lateral margins of the abdomen were cut along each side and the ventral abdominal body wall peeled up to expose the viscera . The viscera were then carefully moved aside to expose the heart underneath, still contracting while attached to the dorsal body wall (Fig . 1) . Control preparations, in which the heart was treated with insect saline (Ertnetssr and BEADLE, 1936), continued to beat regularly at between 60 to 90 contractions per min for over 30 min (Fig . 2). Experimental preparations were treated with 25 p1 of 0 .1, 0 .5 or 1 mM CTX diluted in insect saline, applied in 5-pl drops spaced evenly along the tubular heart . The hearts were observed through a dissecting microscope and recorded on videotape for the subsequent analysis of heart rate . Using probit analysis (Statistical Analysis System, SAS Institute, Cary, NC, U.S .A .), we determined the time it took for a concentration of CTX to reduce the heart rate to one-half of its initial rate (Erso).
Cockroach heart membrane binding assay
Cockroach heart microscmes were prepared as described by AutEV et al . (1990) . CTX was radiolabeled with '2`1 by the procedure of HurnFrt and GREetrwooD (1962), purified by HPLC, and 250,000Cpm of '25I_= was incubated with the heart microscmes for 30 min on ice . The CTX was then cross-linked to binding proteins using the homobifunctional cross-linking agent, disuccinimidyl suberate (DSS, 0 .2mM ; Pierce, Rockford, IL, U .S.A .) as previously described (SAwYER et al., 1987) . The CTX-cross-linked microscme proteins were visualized on an 8-16% gradient sodium dodecyl sulfate (SDS) polyacrylamide gel following the procedure of LAEmmu (1970). The gel, stained with Coomassie brilliant blue R250, was dried prior to autoradiography .
RESULTS
Increasing concentrations of CTX dissolved in insect saline applied directly to the cockroach heart caused a dose-dependent reduction in the heartbeat (Fig. 2). Hearts that received concentrations of 0.5 mM and 1 .0 mM CTX completely ceased beating and were never observed to recover their contractions during the 30-min observation period. One millimolar CTX reduced the cockroach heartbeat by one-half (Fr o) in 1 .8 min ; 100 pM of CTX produced an ET50 in 8.2 min (Fig. 2). Heating the CTX to 100 °C for up to 40 min had no effect on its activity, but when heated 40 min in the presence of 1 mM dithiothreitol (DTI), biological activity of CTX was completely destroyed (Fig. 3). Twenty minutes of heating in the presence of DTT significantly reduced CTX activity (Fig. 3). Figure 4, lanes 1 and 2, illustrates the total protein profile of '2.I-CTX-cross-linked cockroach heart microsomal membrane preparations. No difference in the pattern of
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FIG. 1. COCKROACH HEART BIOAS&AY PREPARATION.
The ventral body wall has been cut away and viscera moved to expose the intact tubular heart (H) attached to the dorsal body wall. The scale bar represents 2.0 mm.
proteins was noted between lane 1 (containing DSS and labeled CTX only) and lane 2, which was prepared identically except that 100 ,UM unlabeled CTX was included in the cross-linking reaction . Lanes 3 and 4 show the corresponding autoradiograph from the Coomassie stained gel. 125 1-CTX that had been incubated with cardiac microsomal membranes was cross-linked to at least one cockroach heart protein such that the crosslinked product showed an apparent mol. wt of 66,000 (a 59,000 receptor protein plus a 7000 CTX molecule; Fig. 4, lane 3). Effective competition for binding and cross-linking of '2'I-CTX occurred with the addition of 100pM unlabeled CTX (Fig. 4, lane 4), indicating that CTX bound specifically to a 59,000 mol. wt membrane protein of the cockroach heart.
M. J. KLOWDEN et al.
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0
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U
2. THE RATE OF CONTRACTION OF SEM-69OLATED COCKROACH HEART PREPARATIONS TREATED wrrH SALINE (CIRCLES) OR INCREASING AMOUNTS OF CTX (SQUARES = 0.I MM ; DIAMONDS = 0.5 MM ; TRIANGLES = 1 mM). FIG .
Each point represents the mean of four different preparations; vertical lines represent standard errors . The ET,os for 0.1, 0.5 and 1.0 mM doses were 8.2, 3.6 and 1.8 min, respectively. CTX was added at 0 min. DISCUSSION
Insect hearts have been used frequently in bioassays of pharmacological agents (MILLER and METCALF, 1968 ; SCHAEFER and MILES, 1970; MAJORi et al., 1972; COLLINS and MILLER, 1977). We have found that the semi-isolated cockroach heart can also serve as the basis for a rapid and effective bioassay for cardiotoxin activity . Heart preparations treated with saline alone showed no change in the heart rates during a 30-min observation period, but saline containing concentrations of 0.1 mM CTX or greater affected the heart in a dose-dependent manner (Fig. 2). Because others (SUN and WALKER, 1986 ; HUANG and TRUIBLE, 1991) have measured biological activity with CTX concentrations as low as 10 juM, we examined the possible reasons for the apparently lower sensitivity of the
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Time (min) FIG. 3 . THE EFFECT OF HEAT AND DTT ON CTX ACTIVITY . Circles : 1.0 mM CTX (no DTT) heated for 30-40min at 100°C, ETm = 1 .7 min; diamonds : 1.0 mM CTX + 1 mM DTT (no heat treatment), ET50 = 1 .6 min; squares: 1 .0mM CTX + 1 mM DTT heated 20 min at 100°C, ET,s = 4.2 min ; triangles: 1.0 mM GTX + I mM DTT heated 40 min at 100°C . Vertical lines represent standard errors .
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FIG . 4 . ' 25I-CTX BINDING To COCKROACH HEART MEMINaANs. Microsomal membrane was incubated with "I-CTX and cross-linked with 0.2 mM disuccinimidyl suberate (lanes 1 and 3) . Specific binding was assessed by including 100 AM unlabeled CTX with the ' 1 I-= during the cross-linking reactions (lanes 2 and 4). Lane 1 shows 30-50 jug of membrane reacted with the cross-linking reagent DSS and 250,000 counts of HPLC-purified 1251-CI7{, lane 2 was prepared similarly except that 100ÁM unlabeled CTX was included in the cross-linking reaction. Lanes 3 and 4 show the corresponding sutoradiograph from the 8-16% SDS-polyacrylamide gel . Mol. wts are shown in kilodaltons.
cockroach bioassay . ToNsiNG et al. (1983) reported that CTX adsorbs to glass and plastic, reducing the amount of CTX that can be recovered from a container, and in one experiment we included 250mM mannitol to CTX solutions to stabilize the protein and help prevent adsorption . Under these conditions, 10 pM CTX significantly reduced the cockroach heartbeat (data not shown), indicating a comparable sensitivity of cockroach heart preparations to other assays . Huxa and CI-liiv (1977) reported that CTX could be denatured by heating above 88°C . In an attempt to demonstrate the ability of the bioassay to distinguish between active and inactive CTX, we boiled the peptide for as long as 40 min without observing reduced biological activity (Fig. 3). Because the CTX molecule contains four disulfide bridges (FRYKLurD and EAxlïzt, 1975) that are essential for CTX activity (TONSIIdG et al., 1983), we added 1 mM DTT to reduce the disulfide bonds. When CTX was heated for 40 min at 100°C with DTT, its biological activity was completely destroyed (Fig. 3). A shorter heating for 20min reduced, but did not eliminate, its activity (Fig. 3).
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Because CTX binds specifically to rat heart cells (TONSING et al., 1983), we wanted to determine whether the toxin also bound to the cockroach heart membrane . When 'III-CTX was incubated with cockroach heart microsomal membranes and cross-linked with DSS, specific binding was shown to occur. The autoradiograph of the SDSpolyacrylamide gel showed 2 bands (Fig. 4, lane 3), each of which was effectively abolished by the addition of 100,uM unlabeled CTX to the cross-linking reaction mixture (Fig. 4, lane 4). We have not determined whether the upper band at the top of the gel represents a high mol. wt CTX-binding protein or simply an aggregation of protein that could not enter the gel. However, we have concluded that CTX binds specifically to at least one cockroach heart membrane protein with a mol. wt of approximately 59,000. The existence of a CTX binding protein in the cockroach heart indicates a potential for using this bioassay to address questions concerning the mechanism of CTX action. We have found that the analysis of the cockroach heart rate, as described by this bioassay, provides a sensitive indication of biological activity for CTX which can be performed without the need for electrophysiological monitoring . It is likely, however, that future measurements of electrophysiological activity will allow us to record more subtle changes that might occur in the heart muscle, such as variations in amplitude or electrical potential, and to increase the sensitivity of the bioassay . The utility of the described bioassay is based primarily on its ease of preparation, rapid evaluation and low cost. Our laboratories use this bioassay principally for two applications: to assess the activity of CTX which we purify from whole venom and to screen for CTX activity of recombinant gene products expressed from a synthetic gene encoding CTX that we have recently constructed (unpublished data). Moreover, we have used this assay to determine that the "I-CTX prepared for cross-linking experiments retained its biological activity after the iodination process (data not shown) . Since this preparation provides an intact stable beating heart to which pharmacological agents may be directly added, it may be applicable to the bioassay of many cardioactive compounds. Acknowledgements-This research has been supported in part by National Science Foundation Grant # RII8902065, and the Idaho State Board of Education Specific Research Grant Program #91-055. This is publication number 9159 of the Idaho Agricultural Experiment Station .
REFERENCES A'REY, J. A ., BECK, C . F ., MURAKAett, K ., TANKSLEY, S . J ., DEERINCK, T. J ., ELLtsm N, M . H . and SuTKo, J . L . (1990) Identification and localization of two triad junctional foot protein isoforms in mature avian fast twitch skeletal muscle. J. biol. Chem. 265, 14187-14194. CoLLnvs, C . and Mil LER, T . (1977) Studies on the action of biogenic amines on cockroach heart . J. exp . Biol. 67, 1-15 . CoNDREA, E ., DE VRIEs A. and MAGER, J. (1964) Hemolysis and splitting of human erythrocyte phospholipids by snake venoms . Biochlm. biophys. Acta 84, 60-73 . EPHRuss', B . and BEADLE, G . W . (1936) A technique of transplantation for Drosophila . Am . Nat . 70, 218-225. FouRiE, A . M ., MELTZER, S ., BERMAN, M . C . and Louw, A . I . (1983) The effect of cardiotoxin on (Ca +Mg)ATPase of the erythrocyte and sarcoplasmic reticulum. Biochem . Int . 6, 581-591 . FRYKLUND, L . and EAKER, D . (1975) The complete covalent structure of a cardiotoxin from the venom of Naja nigricollis (African black-necked spitting cobra) . Biochemistry 14, 2865-2871 . HARVEY, A . L . (1985) Cardiotoxins from cobra venoms : possible mechanisms of action . J . Toxicol. Toxin Rev . 4, 41-69 . HARVEY, A. L., MARSHALL, R . J . and KARLssoN, E. (1982) Effects of purified cardiotoxins from the Thailand cobra (Naja naja siamensis) on isolated skeletal and cardiac muscle preparations . Toxicon 20, 379-396. Ho, C. L ., LEE, C. Y. and Lu, H . H . (1975) Electrophysiological effects of cobra cardiotoxin on rabbit heart cells . Toxicon 13, 437-446 .
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HoDGEs, S . J ., AGBAn, A . S ., HARvEY, A. L . and HIDER, R. C. (1987) Cobra cardiotoxins: purification, effects on skeletal muscle and structure/activity relationships. Eur. J. Biochem . 166, 373-383 . HUANG, J.-L . and TRumi rp, W. R . (1991) Cardiotoxin from cobra venom affects the Ca-Mg-ATPase of cardiac sarcolemmal membrane vesicles . Toxicon 29, 31-41 . HUNG, M . C . and CHEN, Y . H . (1977) Conformational stability of a snake cardiotoxin . Int . J. Pept. Protein Res. 10, 277-285 . HuNTm, W . M . and GREENwooD, F. C . (1962) Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature Lond. 194, 495-496. JUNG, M.-S ., fmcFn;R, J . E . and SMITH, L . A. (1989) Effects of divalent cations on snake venom cardiotoxininduced hemolysis and 3H-deoxyglucose-(-phosphate release from human red blood cells. Toxicon 27, 1297-1305 . LAEMMLI, U. K . (1970) Cleavage of structural proteins during the assembly of the coat of bacteriophage T4. Nature 227, 680-685 . MAJOw, G ., IlEmm, S . and CASAGLIA, O . (1972) Effect of black widow spider venom on the cockroach heart. J. Insect Physiol. 18, 913-927 . MILLER, T . and ME cALF, R. L . (1968) The cockroach heart as a bioassay organ . Entomologia exp. appl. 11, 455-463 . NARITA, K . and Lea, C. Y . (1970) The amino acid sequence of cardiotoxin from Formosan cobra (Naja naja atra) venom. Biochem . biophys. Res. Commun . 41, 339-343 . NAYLER, W. G ., SULLIVAN, A. T., DuNNErr, J ., $LADE, A . M . and TREnFrBwm, E. R . (1976) The effect of a cardiotoxic component of the venom of the Indian cobra (Naja nigricollis) on the subcellular structure and function of heart muscle. J. mot Cell Cardiol. 8, 341-360. SARKAR, N . K . (1947) Isolation of cardiotoxins from cobra venom (Naja tripudians, monocellate variety) . J. Indian them. Sac . 24, 227-232 . SAWYER, S . T., KRANTz, S . B. and LUNA, J . (1987) Identification of the receptor for erythropoietin by crosslinking to Friend virus-infected erythroid cells. Proc . nain. Acad. Sci. U.S.A . 84, 3690-3694. ScHAEFER, C . W . and MILES, J. (1970) Inhibition by acetylcholine of cockroach heart (Gromphadorhina portentosa) . Comp . gen . Pharmacol. 1, 426-430. SmAU, S .-Y . L., YANG, M . S., CHEN, K . J . and Ian, C . Y . (1978) Effects of cobra Cadotoxin on ATPase activity, uCa++ uptake and conformation of biological membranes . In Toxins: Animal, Plant and Microbial, pp. 197-210 (RosENBMG, P., Ed .) . Oxford : Pergamon Press . SUN, J . J . and WALKER, M . J. A . (1986) Actions of cardiotoxins from the southern Chinese cobra (Naja naja atra) on rat cardiac tissue . Toxicon 24, 233-'245 . TAN, N.H . (1982) Cardiotoxins from the venom of Malayan cobra (Naja naja sputatrix) . Archs Biochem. Biophys . 218, 51-58 . Tomm, L., Pommtnt, D. J. J., Louw, A. I . and Vlsmk L . (1983) The binding of snake venom cardiotoxins to heart cell membranes . Biochim. biophys. Acts 732, 282-288. Tu, A. T . (1977) Venoms: Chemistry and Molecular Biology. New York: Wiley . WoLF, J ., SALABF, H ., AmiaRosE, M . and LARSEN, P . R . (1968) The basic proteins of cobra venom . II. Mechanism of action of cobramine B on thyroid tissue. J. Not Chem. 243, 1290-1296.
0041-0101/92 $5 .00 + .00 ® 1992 Perpmon Pres plc
A BIOASSAY FOR COBRA CARDIOTOXIN ACTIVITY USING SEMI-ISOLATED COCKROACH HEART MARC J. KLOMMEN,' ANGELO J. VITALE,' MAYME J. TRUMBLE, 2 CARLA RUDER WESSON and WILLIAM R. TRUMBLE2*
'Division of Entomology, and Department of Bacteriology and Biochemistry, University of Idaho, Moscow, ID 83843, U.S .A . (Received 30 July 1991 ; accepted 30 October 1991)
M. J. KLowDEN, A. J. VITALE, M. J. TRumBLE, C. R. WESSON and W. R. TRuMBLE. A bioassay for cobra cardiotoxin activity using semi-isolated cockroach heart. Toeicon 30, 295-301, 1992.-A semi-isolated cockroach heart preparation was used to rapidly determine the activity of cobra cardiotoxin, monitored as a direct response on heart rate . This preparation produced a dose-response curve in the presence of active cardiotoxin and demonstrated that cardiotoxin retained its biological activity after boiling, although Cardiotoxnn activity was destroyed by heating in the presence of dithiothreitol. Experiments that cross-linked radiolabeled cardiotoxin to solubilized cockroach heart membranes suggested that cardiotoxin bound specifically to a 59,000 mob. wt membrane protein in this tissue . INTRODUCTION
venom is a remarkable source of diverse biologically active basic peptides. Although the chemical structures of many of these peptides are quite similar, they exhibit different types of pharmacological actions including potent neurotoxic, cytotoxic and cardiotoxic activities (Tu, 1977). Cardiotoxins (CTX) are 60 amino acid peptides that constitute the major peptide component of cobra venom (TAN, 1982). More than 40 cardiotoxins have been isolated, and their amino acid sequences suggest that they belong to a large family of similar molecules. The effect of purified CTX on heart muscle was first observed by SARKAR (1947) who reported that CTX arrested the heartbeat of cats and stopped perfused toad hearts . The amino acid sequence of CTX from the Taiwan cobra, Naja naja atra, was subsequently determined by NARITA and LEE (1970) . While the physiological effects of cardiotoxin have been well studied (reviewed by HARvEY, 1985), the molecular mechanisms by which CTX induces muscle contraction are not known. A variety of techniques have been employed to evaluate the activity of CTX. Many of these involve expensive and time-consuming preparations, including the use of live animals (SARKAR, 1947; SuN and WALKER, 1986), whole organs (SARKAR, 1947; WOLF et al., 1968), cardiac and skeletal tissue preparations (Ho et al., 1975; SmAU et al., 1978; HARvEY et al., 1982; SUN and WALKER, 1986; BODGES et al., 1987), cultured cells COBRA
" To whom correspondence should be addressed. 295
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(CONDREA et al., 1964; Ho et al., 1975; TONSING et al., 1983; HODGES et al., 1987; JIANG et al., 1989) and membrane vesicles (NAYLER et al., 1976; SHIAu et al., 1978; FouRiE et al ., 1983; HUANG and TRu1BLE, 1991). Our studies of CTX required a rapid and inexpensive assay for the biological activity of cardiotoxins and analog peptides . This paper describes the development of a semi-isolated cockroach heart preparation that can be used as a bioassay for cardiotoxin activity . MATERIALS AND METHODS Cardiotoxin The Naja naja atra GTX used in these experiments was purchased from Ventoxin, Inc . (Frederick, MD,
U .S .A .) or purified in our laboratory from whole lyophilized venom by a combination of cation exchange chromatography (Mono-S FPLC column ; Pharmacia, Piscataway, NJ, U .S .A .) and reverse phase chromatography (RPC-10 FPLC column ; Pharmacia) .
Cockroach bioassay
The Madagascar hissing cockroach, Gromphadorhina portentosa (Blattaria: Blaberidae), was lightly anesthetized with ether until immobile and placed dorsal surface down under a dissecting microscope. The lateral margins of the abdomen were cut along each side and the ventral abdominal body wall peeled up to expose the viscera . The viscera were then carefully moved aside to expose the heart underneath, still contracting while attached to the dorsal body wall (Fig . 1) . Control preparations, in which the heart was treated with insect saline (Ertnetssr and BEADLE, 1936), continued to beat regularly at between 60 to 90 contractions per min for over 30 min (Fig . 2). Experimental preparations were treated with 25 p1 of 0 .1, 0 .5 or 1 mM CTX diluted in insect saline, applied in 5-pl drops spaced evenly along the tubular heart . The hearts were observed through a dissecting microscope and recorded on videotape for the subsequent analysis of heart rate . Using probit analysis (Statistical Analysis System, SAS Institute, Cary, NC, U.S .A .), we determined the time it took for a concentration of CTX to reduce the heart rate to one-half of its initial rate (Erso).
Cockroach heart membrane binding assay
Cockroach heart microscmes were prepared as described by AutEV et al . (1990) . CTX was radiolabeled with '2`1 by the procedure of HurnFrt and GREetrwooD (1962), purified by HPLC, and 250,000Cpm of '25I_= was incubated with the heart microscmes for 30 min on ice . The CTX was then cross-linked to binding proteins using the homobifunctional cross-linking agent, disuccinimidyl suberate (DSS, 0 .2mM ; Pierce, Rockford, IL, U .S.A .) as previously described (SAwYER et al., 1987) . The CTX-cross-linked microscme proteins were visualized on an 8-16% gradient sodium dodecyl sulfate (SDS) polyacrylamide gel following the procedure of LAEmmu (1970). The gel, stained with Coomassie brilliant blue R250, was dried prior to autoradiography .
RESULTS
Increasing concentrations of CTX dissolved in insect saline applied directly to the cockroach heart caused a dose-dependent reduction in the heartbeat (Fig. 2). Hearts that received concentrations of 0.5 mM and 1 .0 mM CTX completely ceased beating and were never observed to recover their contractions during the 30-min observation period. One millimolar CTX reduced the cockroach heartbeat by one-half (Fr o) in 1 .8 min ; 100 pM of CTX produced an ET50 in 8.2 min (Fig. 2). Heating the CTX to 100 °C for up to 40 min had no effect on its activity, but when heated 40 min in the presence of 1 mM dithiothreitol (DTI), biological activity of CTX was completely destroyed (Fig. 3). Twenty minutes of heating in the presence of DTT significantly reduced CTX activity (Fig. 3). Figure 4, lanes 1 and 2, illustrates the total protein profile of '2.I-CTX-cross-linked cockroach heart microsomal membrane preparations. No difference in the pattern of
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FIG. 1. COCKROACH HEART BIOAS&AY PREPARATION.
The ventral body wall has been cut away and viscera moved to expose the intact tubular heart (H) attached to the dorsal body wall. The scale bar represents 2.0 mm.
proteins was noted between lane 1 (containing DSS and labeled CTX only) and lane 2, which was prepared identically except that 100 ,UM unlabeled CTX was included in the cross-linking reaction . Lanes 3 and 4 show the corresponding autoradiograph from the Coomassie stained gel. 125 1-CTX that had been incubated with cardiac microsomal membranes was cross-linked to at least one cockroach heart protein such that the crosslinked product showed an apparent mol. wt of 66,000 (a 59,000 receptor protein plus a 7000 CTX molecule; Fig. 4, lane 3). Effective competition for binding and cross-linking of '2'I-CTX occurred with the addition of 100pM unlabeled CTX (Fig. 4, lane 4), indicating that CTX bound specifically to a 59,000 mol. wt membrane protein of the cockroach heart.
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2. THE RATE OF CONTRACTION OF SEM-69OLATED COCKROACH HEART PREPARATIONS TREATED wrrH SALINE (CIRCLES) OR INCREASING AMOUNTS OF CTX (SQUARES = 0.I MM ; DIAMONDS = 0.5 MM ; TRIANGLES = 1 mM). FIG .
Each point represents the mean of four different preparations; vertical lines represent standard errors . The ET,os for 0.1, 0.5 and 1.0 mM doses were 8.2, 3.6 and 1.8 min, respectively. CTX was added at 0 min. DISCUSSION
Insect hearts have been used frequently in bioassays of pharmacological agents (MILLER and METCALF, 1968 ; SCHAEFER and MILES, 1970; MAJORi et al., 1972; COLLINS and MILLER, 1977). We have found that the semi-isolated cockroach heart can also serve as the basis for a rapid and effective bioassay for cardiotoxin activity . Heart preparations treated with saline alone showed no change in the heart rates during a 30-min observation period, but saline containing concentrations of 0.1 mM CTX or greater affected the heart in a dose-dependent manner (Fig. 2). Because others (SUN and WALKER, 1986 ; HUANG and TRUIBLE, 1991) have measured biological activity with CTX concentrations as low as 10 juM, we examined the possible reasons for the apparently lower sensitivity of the
a>
aa 0 .û 0 U
Time (min) FIG. 3 . THE EFFECT OF HEAT AND DTT ON CTX ACTIVITY . Circles : 1.0 mM CTX (no DTT) heated for 30-40min at 100°C, ETm = 1 .7 min; diamonds : 1.0 mM CTX + 1 mM DTT (no heat treatment), ET50 = 1 .6 min; squares: 1 .0mM CTX + 1 mM DTT heated 20 min at 100°C, ET,s = 4.2 min ; triangles: 1.0 mM GTX + I mM DTT heated 40 min at 100°C . Vertical lines represent standard errors .
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FIG . 4 . ' 25I-CTX BINDING To COCKROACH HEART MEMINaANs. Microsomal membrane was incubated with "I-CTX and cross-linked with 0.2 mM disuccinimidyl suberate (lanes 1 and 3) . Specific binding was assessed by including 100 AM unlabeled CTX with the ' 1 I-= during the cross-linking reactions (lanes 2 and 4). Lane 1 shows 30-50 jug of membrane reacted with the cross-linking reagent DSS and 250,000 counts of HPLC-purified 1251-CI7{, lane 2 was prepared similarly except that 100ÁM unlabeled CTX was included in the cross-linking reaction. Lanes 3 and 4 show the corresponding sutoradiograph from the 8-16% SDS-polyacrylamide gel . Mol. wts are shown in kilodaltons.
cockroach bioassay . ToNsiNG et al. (1983) reported that CTX adsorbs to glass and plastic, reducing the amount of CTX that can be recovered from a container, and in one experiment we included 250mM mannitol to CTX solutions to stabilize the protein and help prevent adsorption . Under these conditions, 10 pM CTX significantly reduced the cockroach heartbeat (data not shown), indicating a comparable sensitivity of cockroach heart preparations to other assays . Huxa and CI-liiv (1977) reported that CTX could be denatured by heating above 88°C . In an attempt to demonstrate the ability of the bioassay to distinguish between active and inactive CTX, we boiled the peptide for as long as 40 min without observing reduced biological activity (Fig. 3). Because the CTX molecule contains four disulfide bridges (FRYKLurD and EAxlïzt, 1975) that are essential for CTX activity (TONSIIdG et al., 1983), we added 1 mM DTT to reduce the disulfide bonds. When CTX was heated for 40 min at 100°C with DTT, its biological activity was completely destroyed (Fig. 3). A shorter heating for 20min reduced, but did not eliminate, its activity (Fig. 3).
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Because CTX binds specifically to rat heart cells (TONSING et al., 1983), we wanted to determine whether the toxin also bound to the cockroach heart membrane . When 'III-CTX was incubated with cockroach heart microsomal membranes and cross-linked with DSS, specific binding was shown to occur. The autoradiograph of the SDSpolyacrylamide gel showed 2 bands (Fig. 4, lane 3), each of which was effectively abolished by the addition of 100,uM unlabeled CTX to the cross-linking reaction mixture (Fig. 4, lane 4). We have not determined whether the upper band at the top of the gel represents a high mol. wt CTX-binding protein or simply an aggregation of protein that could not enter the gel. However, we have concluded that CTX binds specifically to at least one cockroach heart membrane protein with a mol. wt of approximately 59,000. The existence of a CTX binding protein in the cockroach heart indicates a potential for using this bioassay to address questions concerning the mechanism of CTX action. We have found that the analysis of the cockroach heart rate, as described by this bioassay, provides a sensitive indication of biological activity for CTX which can be performed without the need for electrophysiological monitoring . It is likely, however, that future measurements of electrophysiological activity will allow us to record more subtle changes that might occur in the heart muscle, such as variations in amplitude or electrical potential, and to increase the sensitivity of the bioassay . The utility of the described bioassay is based primarily on its ease of preparation, rapid evaluation and low cost. Our laboratories use this bioassay principally for two applications: to assess the activity of CTX which we purify from whole venom and to screen for CTX activity of recombinant gene products expressed from a synthetic gene encoding CTX that we have recently constructed (unpublished data). Moreover, we have used this assay to determine that the "I-CTX prepared for cross-linking experiments retained its biological activity after the iodination process (data not shown) . Since this preparation provides an intact stable beating heart to which pharmacological agents may be directly added, it may be applicable to the bioassay of many cardioactive compounds. Acknowledgements-This research has been supported in part by National Science Foundation Grant # RII8902065, and the Idaho State Board of Education Specific Research Grant Program #91-055. This is publication number 9159 of the Idaho Agricultural Experiment Station .
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