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An inhibitor of mitochondrial respiration in venom of the Australian bull dog ant, Myrmecia gulosa
J. InsectPhysiol.,1970,Vol. 16,pp. 1531to 1542.Pergamon
Press. P&ted
in Great Britain
AN INHIBITOR OF MITOCHONDRIAL RESPIRATION IN VENOM OF THE AUSTRALIAN BULL DOG ANT,
MYRMECIA LILIAN
M. EWEN*
GULOSA and D. ILSET
School of Biochemistry, University of New South Wales, Kensington, New South Wales, Australia (Received 16 February 1970) Abstract-Venom of the Australian bull dog ant, lkfyrmecia gulosa, is shown to inhibit the respiration of insect mitochondria. An effect of mammalian cytochrome-c in alleviating the inhibition in the systems used is described. A position of action for the inhibitor between cytochromes-b and -c in the terminal electron transfer system is indicated. The stability of the inhibitor and its electrophoretic behaviour are consistent with that of a small basic protein. Isolation of the inhibitor molecule is achieved by chromatography of the venom on Sephadex G-75. INTRODUCTION IN RECENTyears proteins and peptides have been shown to contribute a significant proportion of the biogenic effects of many proteinaceous venoms. Kinins and kinin-like materials have been found in hymenopterous species such as the wasp (JAQUESand SCHACHTER,1954) and hornet (BHOOLAet al., 1960, 1961), and kininlike effects of scorpion venoms have been associated with proteinaceous constituents of these venoms (DINIZ and GONCALVES,1956; ADAM and WEISS, 1959). The neurotoxic action of the venom of North African scorpions has been attributed to constituent basic proteins (MIRANDAand LISSITZKY, 1961), and gastrointestinal stimulatory action caused by the secretion from the posterior salivary glands of cephalopods (ANASTASIand ERSPAMER,1962; ERSPAMERand ANASTASI,1962) is caused by the endecapeptide, eledoisin. Cephalotoxin, a paralytic component of octopus venom, appears to be a protein associated with hexosamines (GHIRETTI, 1959, 1960) and the peptides crotamine and apamin from rattlesnake and bee venoms, respectively, possess spasmogenic activity (GONCAL~, 1956; HABERMANN and REIZ, 1964). Melittin, shown to be the component of bee venom most toxic by intravenous injection to mice (HABERMANN,1965), is a peptide which contracts smooth muscle, increases capillary permeability (HABE-, 1955), releases histamine and serotonin from mast cells and thrombocytes, alters mitochondrial structure, inhibits constituent respiratory systems, and uncouples oxidative
* Present address: Department of Chemical Pathology, Vancouver General Hospital, Vancouver, B.C., Canada. t Present address: Institute for Medical Research, Johannesburg, South Africa. 1531
1532
LILIAN
M.E~EN AND D. ILSE
phosphorylation (HABEFUUNN, 1954). This work now reports the isolation of a small biogenic protein from venom of the Australian bull dog ant, Myrmecia gulosa, (F.). An inhibitory effect of the venom on the respiration of isolated insect mitochondria and investigation of the stability, separation, and characterization of the inhibitor molecule as a protein are described. MATERIALS
AND METHODS
Venom was obtained by dissection of individual ants. Insects were anaesthetized with carbon dioxide, secured under modelling clay with the gaster protruding, and the complete venom apparatus then withdrawn. Adhering tissues were removed, the venom reservoir was rinsed with distilled water, the wall punctured, and the pure venom drained on to a microscope slide. The material was desiccated over phosphorus pentoxide and stored at a temperature of 5°C. Individual ants yielded on the average 0.5 mg of dried material. Housefly (MUSCQdomestica) and blowfly (Lucilia cuprina) mitochondria were used in test systems to investigate the influence of venom and venom fractions on insect flight muscle mitochondrial respiration. Succinate and ar-glycerophosphate were used as substrates. Mitochondria were prepared in the cold (0-5°C). Flies were chilled, a weighed quantity (1 g) of thoraces prepared and ground for 30 set in 8 ml of ice-cold 0.25 M sucrose, using a Potter-Elvehjem homogenizer. The resulting suspension was filtered through double gauze, the debris rinsed with 2 ml of 0.25 M sucrose and the combined filtrates centrifuged for 5 min at 150 g to sediment remaining debris (SACKTOR and COCHRAN, 1958). The suspended mitochondria were then recentrifuged for 5 min at 3000g (SACKTOR, 1953), the supernatant discarded, the sedimented mitochondria washed once on the centrifuge with 8 ml of 0.25 M sucrose, and resuspended in 3 ml of 0.25 M sucrose. For quantitative estimation of oxygen consumption appropriate aliquots (0.05 to 0.2 ml) of the suspension were added to double side-arm Warburg flasks containing an incubation mixture, of 2.0 ml total volume, composed of 62.5 p-moles of trisHCl buffer, pH 7.4, 30 p-moles of phosphate buffer, pH 7.4, 15 p-moles of KCl, 2 p-moles of EDTA (d iaminoethanetetra-acetic acid, disodium salt), and 5 p-moles of MgCl,. The centre well contained O-2 ml of 20% (w/v) KOH to absorb CO, produced. After a 10 min equilibration period, 50 p-moles of substrate were added from one side-arm and oxygen consumption was measured by conventional manometry. Venom was added from the second side-arm to the respiring preparation after 15 min incubation and oxygen consumption recorded for a further 45 min. Inhibition, measured over the period succeeding venom addition, was calculated using the formula: ~1 0,
consumed by control system-$
0, consumed by inhibited system
~1 0, consumed by control system
100 x-. 1
Mitochondrial protein was estimated by the method of CLELANDand SLATER(1953). When used, 10 p-moles of 2,3,5-triphenyl tetrazolium chloride were added to 2.0 ml of control and test media prepared as before and the mixture, contained
MITOCHONDRIAL
RESPIRATION INHIBITOR
IN BULL DOG ANT VENOM
1533
in stoppered tubes, was incubated at 37°C for 30 min; 5 ml of glacial acetic acid were then added, the reduced dye extracted into 3-O ml of toluene, and the absorbance measured at 480 nm. Where thionin (3,7-diaminophenothiazonium chloride) was used, 1.6 mg o/owere added to the mixture contained in Thunberg tubes, the tubes flushed with argon to prevent reoxidation of the dye by atmospheric oxygen, and the time required for complete reduction of the dye was measured. Proteolytic digestion of venom was effected using a commercial preparation of Pronase-P.* The venom, in a O-9 ml incubate containing 8 p-moles of CaCl, and 150 p-moles of phosphate buffer, pH 7.4, was digested at 37°C for appropriate periods of time. Heat inactivation (5 min in boiling water) of the enzyme stopped digestive activity. Remaining protein was measured by the biuret reaction (GORNALL et al., 1949). Dialysis, electrophoresis, and column chromatography were used during Aqueous solutions of venom were separation of individual venom components. dialysed against distilled water for periods of up to 96 hr using seamless cellulose tubing, % in. and % in. (Visking Co., Chicago, Ill.). Paper strips (Schleicher and Shiill paper No. 2043) were used as the supporting medium in fractionation of Electropherograms were stained with Amidoschwarz venom by electrophoresis. 10B and venom fractions from parallel strips were eluted with distilled water. Columns of Sephadex G-25 (1 x 110 cm) and G-75 (2.5 x 40 cm and 2.5 x 90 cm) were used during separation of venom fractions by column chromatography and O-067 M phosphate buffer, pH 7.4 or 70% formic acid were used as eluting media. The protein content of 5 ml fractions was measured by absorbance at 280 nm. After separation by each technique respiratory inhibitory activity of lyophilized fractions was measured by manometry as before. RESULTS
Inhibition
of insect mitochondrial
respiration
by M. gulosa venom
Inhibition of housefly mitochondrial respiration by increasing levels of M. gulosa venom is shown in Fig. 1. The form of inhibition showed a straight-line relationship with lower concentrations of venom followed by a non-linear increase as venom concentration per mg of mitochondrial protein was increased. Housefly and blowfly mitochondrial preparations were similarly inhibited by the venom when measured under identical conditions (Fig. 2). Results shown in Figs. 1 and 2 were derived using succinate as substrate. Inhibition of mitochondrial respiration occurred also when Lu-glycerophosphate was used as substrate. Reduction of the dye 2,3,5-triphenyltetrazolium chloride by respiring mitochondria was decreased in test systems containing venom compared with control media lacking venom. Time for complete reduction of thionin was increased when venom was present in the incubation medium. Addition of 0.01 p-moles of FAD to the usual incubation medium containing succinate as substrate did not influence the inhibitory effects of * Pronase-P, from culture medium of Streptomyces griseus, enforced protein disintegrate. Kaken Chemical Company Ltd., Japan.
LILIAN M.EWEN AND D. ILSE
1534 added
venom.
periods
of up to
reaction
mixture
Measures
such
as sonication
of mitochondrial preparations for Addition of ADP to the
3 min did not alter inhibition by venom. did
not
influence
mitochondrial
Mitoch.
respiration
suggesting
that
protein
2 - 1.5mg. 3 - 2.3 mg. 2.0
4.0
Venom
6.0
8.0
(mg.)
1. Inhibition of mitochondrial oxygen consumption by M. gulosa venom. The composition of tlie reaction mixture, of 2-O ml total volume, was as detailed in the text. Succlnate was used as substrate. Results show the inhibition caused by increasing concentrations of venom, using three levels of mitochondrial protein. FIG.
100 90
*-a
Housefly
A-A
Blowfly
mitoch. mitoch.
ex
80 ‘;; k ; z N
0
70
. ..
/
60 50
.J
40 30 20
: ‘OO
.
/ A
” 1.0
.
Mitochondrial
protein
(mg.)
Effect of M. gulosa venom on the oxygen consumption of housefly and blowfly mltochondrial preparations. The composition of the reaction mixture, of 2-O ml total volume, was as detailed in the text. Succinate was used as substrate. Oxygen consumption of inhibited mitochondrial systems containing 2.0 mg of venom/2*0 ml incubation medium is expressed as a percentage of normal control respiration in similar systems lacking venom. FIG.
2.
oxidative phosphorylation was uncoupled from electron transport in the mitochondrial preparations used and the venom acted to inhibit electron transport. Bovine plasma albumin used by some workers (WOJTCZAK and WOJTCZAK, 1959, 1960) to prevent uncoupling of oxidative phosphorylation did not affect inhibition caused by venom when 30 mg of albumin were added to control and test media.
MITOCHONDRIAL
RESPIRATION
INHIBITOR
IN
BULL
DOG
ANT
VENOM
1.535
Mammalian cytochrome-c (O-04 p-moles/2 ml of reaction medium) did, however, alleviate inhibition of mitochondrial respiration by venom. With higher levels of mitochondrial protein or lower levels of venom, resulting in low percentage inhibition, complete prevention of inhibition was achieved (Fig. 3). With lower
5 IO 15
20 25 30 35
40
45
50 55 60
Time (mins.) FIG. 3. Effect of mammalian cytochrome-c in alleviating the inhibition of housefly flight muscle mitochondrial respiration caused by M. gulosa venom. The composition of the reaction mixture was as described in the text and contained 2.4 mg lof mitochondrial protein/2*0 ml; succinate was used as substrate. l, 0, + 0.04 p-moles Control preparation; 0 - - - - l , +2-O mg of venom; Oof mammalian cytochrome-c; 0 - - - - 0, $-2-O mg of venom+0*04 p-moles of mammalian cytochrome-c.
concentrations of mitochondrial protein or higher concentrations of venom, thus greater inhibition, partial alleviation was achieved with added cytochrome-c (Table 1). In these cases alleviation was not further increased by higher concentrations (up to 0.16 p-males/2.0 ml incubate) of mammalian cytochrome-c (Table 2). Stability of the respiratory inhibitor of M. gulosa venom In dry venom the inhibitor was stable at room temperature for at least 1 month. The molecule was non-volatile and could be held under low pressure (7 x lo-$ mm Hg) without loss of activity. Lyophilization from aqueous solution did not impair its inhibitory activity. The inhibitor was stable to heat. Aqueous solutions of venom heated at 100°C for up to 30 min retained > 80 per cent of the original inhibitory potency. After such treatment the molecule remained uncoagulated in aqueous solution together with 75 per cent of the total biuret reactive material. The inhibitor was not extracted into lipid solvents, except butanol. These solvents did cause varying losses in inhibitory activity, however. On treatment of venom at room temperature with ether, acetone, ethanol, and methanol the activity which remained was confined to the coagulated protein residue and could subsequently be extracted into aqueous solution.
1536
LILIAN M. EWEN AND D. ILSE
Venom solutions shaken with 98% (v / v ) acetone showed no loss in potency, but 85% (v/v) acetone caused an 18 per cent loss. Ether similarly slightly reduced inhibitory activity. With 67% (v/v) ethanol, 89 per cent of total activity was lost. TABLE
I-EFFECT
OF MAMMALIAN CYTOCHROME-c ON INHIBITION RESPIRATION BY M. @llOSa VENOM* Inhibition
Mitochondrial protein (mg)
(mg)
Exp. 1
1.0
Exp. 2
1.1
Exp. 3
3.1
Exp. 4
3.0
0.5 1.0 2.0 3.0 2.0 3.0 4.0
text.
TABLE
o-00 0.04 0.08 0.12 0.16 * text. t $
39 32 25 30 30 29 30
was as described
in the
OF INCREASING CONCENTRATIONS OF CYTOCHROME-C ON INHIBITION MITOCHONDRIAL RESPIRATION BY M. @dOSa VENOM * Inhibition
Mitochondrial protein (mg) Venom (mg): Cytochrome-c (r_l moles)
Decrease
0 19 36 45 8 29 38
39 51 61 75 38 58 68
* Volume of incubate, 2.0 ml. The reaction mixture Succinate (50 p-moles) was used as substrate.
2-EFFECT
(per cent)
Test ( + 0.02 p-moles cytochrome-c)
Control (no added cytochrome-c)
Venom
OF MITOCHON~RIAL
1.0
OF
(per cent)
1.1
1.8
3.1
3.0
: 0.5 1.0 2.0 ATDSADADADADADADAD
-
39 0 0
75 49 42
39 39
51 9 0
42 51
61 34 35
27 27
3.0
2.0
2.0
-
26 33
78 46
-
32
Volume of incubate, 2.0 ml. The reaction mixture Succinate (50 p-moles) was used as substrate. Actual. Decrease.
38 18 15 25 8
3.0
4.0
-
30 23 13 30
58 35 32 39 33
-
23 26 19 25
was as described
68 47 38 36 34
21 30 32 34
in the
Hot 95% (v / v ) eth anol caused a 37 per cent loss in 15 min and hot 60% (v/v) methanol an 84 per cent loss over the same period. With 85% (v/v) butanol, inhibitory activity equalling 22 per cent of the total was extracted into the liquid
MITOCHONDRIAL RESPIRATION INHIBITORIN BULL DOGANTVENOM
1537
phase. Inhibition caused by an aqueous extract of the remaining coagulated protein amounted to 45 per cent of the total inhibition of control samples. With 92% butanol no activity was detected in the liquid phase, but inhibition by an aqueous extract of the remaining coagulated protein was 13 per cent less than that of control samples. Treatment of venom with 10% (w/v) perchloric acid for 30’min at room temperature did not destroy inhibitory activity. Similarly when venom was allowed to stand at room temperature in 70% (w/v) formic acid or 0.1 N ammonia for 3 days, no decrease in activity occurred when compared with control samples. Hydrolysis of venom protein with 6 N HCl at 110°C in a sealed tube reduced inhibitory potency of the venom by 55 per cent in 1 hr. Proteolytic digestion of venom with Pronase-P also produced a progressive decrease in inhibitory activity as venom protein decreased over increasing periods of time (Table 3). After 45 hr no inhibitory activity was detected in these preparations. TABLE 3-EFFECT
OF PROTROLYTICDIGESTIONON THE POTENCYOF THE RESPIRATORY INHIBITOROF M. &OSa VENOM* Time of digestion (hr)
Venom protein (% of total)
Inhibition (%)
Remaining activity (% of total)
0.00 0.25 0.50 1.00 2.00 4.00 8.00 20.00 45.00
100 78 74 72 68 61 42 40 39
67 49 40 36 27 22 11 10 0
74 60 54 40 32 17 15 0
* In testing for inhibitory activity, 1.7 mg of mitochondrial protein were used per 2.0 ml of incubation medium. The reaction mixture was as described in the text. Succinate (50 pmoles) was used as substrate.
Separation of inhibitory fraction from whole venom The inhibitory molecule of M. gulosa venom did not dialyse through $ in. Visking tubing but could pass through @ in. tubing (Table 4). CRAIG and KING (1962) found that pepsin of MW 35,500 had a 50 per cent escape time of 80 hr using $$ in. Visking tubing. Ovalbumin of MW 45,000 diffused more slowly than pepsin. Results in this work were consistent therefore with a MW greater than 5000 but less than 50,000 for the inhibitor. The molecule was eluted in the exclusion volume of a column of Sephadex G-25 confirming that the MW exceeded 5000. The exclusion limit of Sephadex G-75 is approximately 50,000. Chromatography of venom on this grade of Sephadex was therefore used to separate venom
LILIAN M. EWEN AND D. ILSE
1538
TABLE ~-EFFECT OF DIALYSISON M. gulosa VENOM Inhibition (per cent)
Inhibition (per cent)
Exp. No.*
Mitochondrial protein (mg) t
1 2 3 4 5 6 7 8 9
2.0 2.0 2.0 1.6 1.6 1.7 1,s 2.2 1.8
Dialysed venom (8 in. tubing) :. Nondialysed venom
Retentate
Dialysate
Exp. No.*
Mitochondrial protein (mg) t
80 66 71 63
71 69 64 80 84 75 62 72 56
0 0 0 0 0 0 0 0 0
1A 2A 3A 4A 5A 6A
1.3 1.3 2.8 2.8 2.1 2.2
Dialysed venom (# in. tubing): Nondialysed venom 93 86
93 69
Retentate
Dialysate
57 25 17 0 55 0
48 41 33 25 57 61
* Venom was treated as follows: 1-5. 4.0 mg of venom in 1.2 ml of distilled water dialysed for 67 hr against an equal volume of distilled water. 6. 4.0 mg of venom in 0.75 ml of distilled water dialysed for 92 hr against three changes of 1.0 ml of distilled water. 7. 4.0 mg of venom in 0.5 ml of distilled water dialysed for 72 hr against three changes of 1.0 ml of distilled water. 8. 5-O mg of venom in 1.0 ml of distilled water dialysed for 88 hr against four changes of 2.0 ml of distilled water. 9. Supernatant from 4.0 mg of heat-treated venom in 0.5 ml of distilled water, dialysed for 72 hr against three changes of 1.0 ml of distilled water. 1A. 4.0 mg of venom in 0.8 ml of distilled water dialysed for 60 hr against 1.0 ml of distilled water. 2A. Supernatant from 4 mg of heat-treated venom in 0.8 ml of distilled water dialysed for 60 hr against 1-O ml of distilled water. 3A. 5.0 mg of venom in 1.0 ml of distilled water dialysed for 92 hr against four changes of 1.0 ml of distilled water. 4A. Supematant from 5.0 mg of heat-treated venom in 1.0 ml of distilled water dialysed for 92 hr against four changes of 1.0 ml of distilled water. 5A. 6.0 mg of venom in 1.0 ml of distilled water dialysed for 92 hr against three changes of 1.0 ml of distilled water. 6A. 5.0 mg of venom in 1.0 ml of distilled water dialysed for 92 hr against four changes of 2.0 ml of distilled water. t Per 2-O ml of reaction mixture; composition as described in the text. $ Visking Co., Chicago, Illinois. fractions. eluting
Phosphate media.
The
buffer,
O-067 M, pH 7.4 or 70%
respiratory
inhibitor
overlapping peaks with the first smaller peak (Fig. 4).
was eluted peak forming
formic from
acid were used as
the columns
a shoulder
in two
to the second
MITOCHONDRIAL RESPIRATION INHIBITOR IN BULLDOGANTVENOM
1539
On electrophoresis of whole or heat-treated venom in 0.067 M phosphate buffer, pH 7.4, the inhibitor molecule migrated towards the cathode with the basic proteinaceous fractions of the venom. Inhibitory fractions eluted from a column of Sephadex G-75 migrated similarly on electrophoresis. 0.350-
790
.-.
0.300-
-80 -70
/i
0.250-
-60 :
f/
0.200.
z ;
O.lSO-
;;;j :,j
0.050-
-50
-
'
I\
1 _j
1 \
: I j;___,j/
: s ._
-40 f
I\ /
0.100-
v
I \
-30
I\
x
i;...
;,
i
l,,..J,
2o
-10
‘, .P/ 0.~00 :: ’ , , , , , , , ; , , , ’ 0 20 25 30 35 40 45 50 55 60 65 70 Fraction FIG. 4. Chromatography of M. gulosa venom on Sephadex G-75 (bead form) using 70% (W/V) formic acid as eluting agent; 25 mg of venom applied to a 2-5 x 90 cm column. Elution rate 30 ml/hr a-----0, E 280 nm of eluted fractions; A - - - A, percentage inhibition of housefly flight muscle mitochondrial respiration. In estimating inhibitory activity, 1.8 mg of mitochondrial protein were used/ 2-o ml incubation medium; composition as described in the text. Succinate was used as substrate. DISCUSSION
That bull dog ant venom can inhibit the oxygen consumption of respiring insect mitochondria is clear. The position and mechanism of inhibition of the electron transport system are less apparent. The terminal respiratory system of insects has been examined in some detail by several workers (CHANCEand PAPPENHEIMER,1954; PAPPENHEIMER and WILLIAMS, 1954; SHAPPIRIO and WILLIAMS, 1957, CHANCEand SACKTOR,1957) and CHANCEand SACKTOR(1958) have studied in particular the respiratory chain in housefly flight muscle mitochondria. There the scheme of electron transport is visualized as corresponding to the sequence shown overleaf. 2,3,5-Triphenyltetrazolium chloride accepts electrons from reduced cytochrome oxidase (NACHLASet al., 1960). Lessened reduction of this dye by respiring mitochondria in the presence of venom thus indicates a position of action for venom on the electron transport chain at any position preceding this. Thionin with an E,’ of + O-063would be expected to accept electrons from reduced cytochrome-b in the respiratory chain. Thus if the point of inhibition by venom succeeded the point at which thionin accepts reducing equivalents dye reduction 48
LILIAN M. EWENANDD. ILSE
1540
would be increased. Decreased action preceding cytochrome-c.
reduction of this dye is consistent with a point of
a-glycero-
M. gulosa venom inhibited mitochondrial oxygen consumption using either succinate or or-glycerophosphate as substrate. The point of inhibition would appear, therefore, to be at a position on the electron transport pathway common to both succinate and a-glycerophosphate, i.e. in the region of the electron transport chain between cytochrome-b and -c, although an action as a general flavoprotein poison has not been excluded. The addition of FAD, which forms the prosthetic group of succinate dehydrogenase was, however, found not to decrease inhibition of the succinoxidase system by venom. The effect of cytochrome-c in alleviating inhibition of oxygen consumption is unexplained. Basic proteins constitute at least part of the structure of both the inhibitory molecule of M. gdosa venom and of cytochrome-c. The effect of the cytochrome might thus be as a competitor of the inhibitory molecule. Alternatively, the action of added mammalian cytochrome-c in alleviating the inhibition of oxygen consumption caused by venom could possibly be in the provision of a bypass from substrate to unaffected parts of the respiratory chain. In uncoupled mitochondria electrons can be passed directly from the flavoprotein to cytochrome-c, or to cytochrome-c, bypassing cytochrome-b (GILMOUR, 1961). Under these conditions, mitochondrial electron transport is still inhibited by antimycin A which acts at a position between cytochromes-b and -c. If venom acted at a similar locus, inhibited mitochondrial electron transport might be substituted in uncoupled mitochondria by direct electron transport from substrate, through mammalian cytochrome-c, to succeeding sections of the mitochondrial electron transfer system. Migration of the respiratory inhibitor molecule towards the cathode on electrophoresis at pH 74 and separation from other venom fractions on Sephadex G-75, together with the stability of the inhibitor in 70% formic acid and O-1 N ammonia and its susceptibility to proteolytic digestion techniques, are consistent with the structure of a small basic protein. The elution spectrum of this venom on Sephadex G-75 shows considerable similarity to that of bee venom separated on Sephadex G-50 (HABERMANN,1965) with the respiratory inhibitor, like meiittin in the elution pattern of bee venom, forming the most predominant peak.
MITOCHONDRIAL RESPIRATION INHIBITORIN BULLDOGANT VENOM
1541
In this study the molecule has been characterized as an inhibitor of mitochondrial respiration. It may possess, however, like melittin in bee venom, a much wider activity spectrum. Estimation of the toxicity of the purified material to laboratory animals compared with that of whole venom may give an indication of the importance of the molecule as a biologically active constituent of bull dog ant venom. REFERENCES K. R. and WEISS C. (1959) Actions of scorpion venom on skeletal muscle. Br. J. Pharmac. 14, 334-339. ANASTASIA. and ERSPAMERV. (1962) Occurrence and some properties of eledoisin in extracts of posterior salivary glands of l&done. Br.J. Pharmac. 19, 326-336. BHOOLAK. D., CALLEJ. D., and SCHACHTER M. (1960) The identification of acetylcholine, 5-hydroxytryptamine and other substances in hornet venom. J. Physiol., Lond. 151, 35-36P. BHOOLA K. D., CALLE J. D., and SCHACHTERM. (1961) Identification of acetylcholine, S-hydroxytryptamine, histamine and a new kinin in hornet venom (V. crabro). J. Physiol., Lond. 159, 167-182. CHANCEB. and PAPPENHEIMER A. M., JR. (1954) Kinetic and spectrophotometric studies of cytochrome b, in midgut homogenates of Cecropia. J. biol. Chem. 209, 931-943. CHANCEB. and SACKTORB. (1958) Respiratory metabolism of insect flight muscie-XI. Kinetics of respiratory enzymes in flight muscle sarcomes. Archs Biochem. Biophys. 76, 509-531. CLELANDK. W. and SLATERE. C. (1953) Respiratory granules of heart muscle. Bi0chem.J. 53, 547-556. CRAIG I. C. and KING T. P. (1962) Dialysis. In Methods of Biochemical Analysis (Ed. by GLICK D.) 10, 175-199. Interscience, New York. DINIZ C. R. and GONCALVE~ J. M. (1956) Some chemical and pharmacological properties of Brazilian scorpion venoms. In Venoms (Ed. by BUCKLEYE. E. and PORGESN.), pp. 131-139. AAAS, Washington. ERSPAMER V. and ANASTASIA. (1962) Structure and pharmacological actions of eledoisin, the active endecapeptide of the posterior salivary glands of Eledone. Experientia 18, 58-59. GHIRETTIF. (1959) Cephalotoxin: the crab-paralysing agent of the posterior salivary glands of cephalopods. Nature, Lond. 183, 1192-1193. GHIRETTIF. (1960) Toxicity of octopus saliva against Crustacea. Ann. N. Y. Acad. Sci. 90, 726-741. GILMOURD. (1961) Biochemistry of Insects. Academic Press, New York. GONCALVESJ. M. (1956) Purification and properties of Crotamine. In Venoms (Ed. by BUCKLEYE. R. and PORGESN.), pp. 261-273. AAAS, Washington. GORNALL A. G., BARDAWILLC. J., and DAVID M. M. (1949) Determination of serum proteins by means of biuret reaction. J. biol. Chem. 177, 751-766. HABERMANNE. (1954) Hemmung der oxydativen Phosphorylierung durch tierische Gifte. Naturwissenschaften 41, 429430. HABERMANNE. (1955) uber Permeabilitatsiinderungen durch tierische Gifte. Arch. e@. Path. Pharmak. 225, 158-160. HABERMANN E. (1965) Recent studies on Hymenoptera venoms. In PYOC.2nd int. Pharmacol. Meeting, Prague (1963) (Ed. by RAUDONATH. W. and VANECEKJ.) 9, 53-61. Pergamon Press, Oxford. HABERMANNE. and REIZ K. G. (1964) Apamin: Ein vasisches, zentral erregendes Polypeptide aus Beinengift. Naturwissenschaften 51, 61. ADAM
1542
LILIAN M. EVEN ANDD. ILSE
JAQV~S R. and SCHACHTERM. (1954) Presence of histamine, S-hydroxytryptamine and potent, slow contracting substances in wasp venom. Br. J. Pharmac. 9, 53-58. MIRANDAF. and LISSITZKYS. (1961) Scorpamins: The toxic proteins of scorpion venoms. Nature, Land. 190,443-444. NACHLA~M. M., MARGULIESS. I., and SELIGMANA. M. (1960) Sites of electron transfer to tetrazolium salts in succinoxidase systems. r. biol. Chem. 235, 2739-2743. PAPPENHEIMERA. M. and WILLIAMS C. M. (1954) Cytochrome b, and the dihydrocoenzyme l-oxidase system in the Cecropia silkworm. J. biol. Chem. 209, 915-929. SACKTORB. (1953) Investigation on the mitochondria of the house fly, Musca domestica L. J. gen. Physiol. 36, 371-387. SACKTORB. and COCHRAND. G. (1958) The respiratory metabolism of insect flight muscle -1. Manometric studies of oxidation and concomitant phosphorylation with sarcosomes. Archs Biochem. Biophys. 74, 266-276. SHAPPIRIOD. G. and WILLIAMS C. M. (1957) The cytochrome system of the Cecropia silkworm-11. Spectrophotometric studies of oxidative enzyme systems in the wing epithelium. Proc. R. Sot. Lond. (B) 147, 233-246. WOJTCZAKL. and WOJTCZAKA. B. (1959) The action of serum albumin on oxidative phosphorylation in insect mitochondria. Biochim. biophys. Acta 31, 297-299. WOJTCZAKL. and WOJTCZAKA. B. (1960) Uncoupling of oxidative phosphorylation and inhibition of ATP-P, exchange by a substance from insect mitochondria. Biochim. biophys. Acta 39, 277-286.
Press. P&ted
in Great Britain
AN INHIBITOR OF MITOCHONDRIAL RESPIRATION IN VENOM OF THE AUSTRALIAN BULL DOG ANT,
MYRMECIA LILIAN
M. EWEN*
GULOSA and D. ILSET
School of Biochemistry, University of New South Wales, Kensington, New South Wales, Australia (Received 16 February 1970) Abstract-Venom of the Australian bull dog ant, lkfyrmecia gulosa, is shown to inhibit the respiration of insect mitochondria. An effect of mammalian cytochrome-c in alleviating the inhibition in the systems used is described. A position of action for the inhibitor between cytochromes-b and -c in the terminal electron transfer system is indicated. The stability of the inhibitor and its electrophoretic behaviour are consistent with that of a small basic protein. Isolation of the inhibitor molecule is achieved by chromatography of the venom on Sephadex G-75. INTRODUCTION IN RECENTyears proteins and peptides have been shown to contribute a significant proportion of the biogenic effects of many proteinaceous venoms. Kinins and kinin-like materials have been found in hymenopterous species such as the wasp (JAQUESand SCHACHTER,1954) and hornet (BHOOLAet al., 1960, 1961), and kininlike effects of scorpion venoms have been associated with proteinaceous constituents of these venoms (DINIZ and GONCALVES,1956; ADAM and WEISS, 1959). The neurotoxic action of the venom of North African scorpions has been attributed to constituent basic proteins (MIRANDAand LISSITZKY, 1961), and gastrointestinal stimulatory action caused by the secretion from the posterior salivary glands of cephalopods (ANASTASIand ERSPAMER,1962; ERSPAMERand ANASTASI,1962) is caused by the endecapeptide, eledoisin. Cephalotoxin, a paralytic component of octopus venom, appears to be a protein associated with hexosamines (GHIRETTI, 1959, 1960) and the peptides crotamine and apamin from rattlesnake and bee venoms, respectively, possess spasmogenic activity (GONCAL~, 1956; HABERMANN and REIZ, 1964). Melittin, shown to be the component of bee venom most toxic by intravenous injection to mice (HABERMANN,1965), is a peptide which contracts smooth muscle, increases capillary permeability (HABE-, 1955), releases histamine and serotonin from mast cells and thrombocytes, alters mitochondrial structure, inhibits constituent respiratory systems, and uncouples oxidative
* Present address: Department of Chemical Pathology, Vancouver General Hospital, Vancouver, B.C., Canada. t Present address: Institute for Medical Research, Johannesburg, South Africa. 1531
1532
LILIAN
M.E~EN AND D. ILSE
phosphorylation (HABEFUUNN, 1954). This work now reports the isolation of a small biogenic protein from venom of the Australian bull dog ant, Myrmecia gulosa, (F.). An inhibitory effect of the venom on the respiration of isolated insect mitochondria and investigation of the stability, separation, and characterization of the inhibitor molecule as a protein are described. MATERIALS
AND METHODS
Venom was obtained by dissection of individual ants. Insects were anaesthetized with carbon dioxide, secured under modelling clay with the gaster protruding, and the complete venom apparatus then withdrawn. Adhering tissues were removed, the venom reservoir was rinsed with distilled water, the wall punctured, and the pure venom drained on to a microscope slide. The material was desiccated over phosphorus pentoxide and stored at a temperature of 5°C. Individual ants yielded on the average 0.5 mg of dried material. Housefly (MUSCQdomestica) and blowfly (Lucilia cuprina) mitochondria were used in test systems to investigate the influence of venom and venom fractions on insect flight muscle mitochondrial respiration. Succinate and ar-glycerophosphate were used as substrates. Mitochondria were prepared in the cold (0-5°C). Flies were chilled, a weighed quantity (1 g) of thoraces prepared and ground for 30 set in 8 ml of ice-cold 0.25 M sucrose, using a Potter-Elvehjem homogenizer. The resulting suspension was filtered through double gauze, the debris rinsed with 2 ml of 0.25 M sucrose and the combined filtrates centrifuged for 5 min at 150 g to sediment remaining debris (SACKTOR and COCHRAN, 1958). The suspended mitochondria were then recentrifuged for 5 min at 3000g (SACKTOR, 1953), the supernatant discarded, the sedimented mitochondria washed once on the centrifuge with 8 ml of 0.25 M sucrose, and resuspended in 3 ml of 0.25 M sucrose. For quantitative estimation of oxygen consumption appropriate aliquots (0.05 to 0.2 ml) of the suspension were added to double side-arm Warburg flasks containing an incubation mixture, of 2.0 ml total volume, composed of 62.5 p-moles of trisHCl buffer, pH 7.4, 30 p-moles of phosphate buffer, pH 7.4, 15 p-moles of KCl, 2 p-moles of EDTA (d iaminoethanetetra-acetic acid, disodium salt), and 5 p-moles of MgCl,. The centre well contained O-2 ml of 20% (w/v) KOH to absorb CO, produced. After a 10 min equilibration period, 50 p-moles of substrate were added from one side-arm and oxygen consumption was measured by conventional manometry. Venom was added from the second side-arm to the respiring preparation after 15 min incubation and oxygen consumption recorded for a further 45 min. Inhibition, measured over the period succeeding venom addition, was calculated using the formula: ~1 0,
consumed by control system-$
0, consumed by inhibited system
~1 0, consumed by control system
100 x-. 1
Mitochondrial protein was estimated by the method of CLELANDand SLATER(1953). When used, 10 p-moles of 2,3,5-triphenyl tetrazolium chloride were added to 2.0 ml of control and test media prepared as before and the mixture, contained
MITOCHONDRIAL
RESPIRATION INHIBITOR
IN BULL DOG ANT VENOM
1533
in stoppered tubes, was incubated at 37°C for 30 min; 5 ml of glacial acetic acid were then added, the reduced dye extracted into 3-O ml of toluene, and the absorbance measured at 480 nm. Where thionin (3,7-diaminophenothiazonium chloride) was used, 1.6 mg o/owere added to the mixture contained in Thunberg tubes, the tubes flushed with argon to prevent reoxidation of the dye by atmospheric oxygen, and the time required for complete reduction of the dye was measured. Proteolytic digestion of venom was effected using a commercial preparation of Pronase-P.* The venom, in a O-9 ml incubate containing 8 p-moles of CaCl, and 150 p-moles of phosphate buffer, pH 7.4, was digested at 37°C for appropriate periods of time. Heat inactivation (5 min in boiling water) of the enzyme stopped digestive activity. Remaining protein was measured by the biuret reaction (GORNALL et al., 1949). Dialysis, electrophoresis, and column chromatography were used during Aqueous solutions of venom were separation of individual venom components. dialysed against distilled water for periods of up to 96 hr using seamless cellulose tubing, % in. and % in. (Visking Co., Chicago, Ill.). Paper strips (Schleicher and Shiill paper No. 2043) were used as the supporting medium in fractionation of Electropherograms were stained with Amidoschwarz venom by electrophoresis. 10B and venom fractions from parallel strips were eluted with distilled water. Columns of Sephadex G-25 (1 x 110 cm) and G-75 (2.5 x 40 cm and 2.5 x 90 cm) were used during separation of venom fractions by column chromatography and O-067 M phosphate buffer, pH 7.4 or 70% formic acid were used as eluting media. The protein content of 5 ml fractions was measured by absorbance at 280 nm. After separation by each technique respiratory inhibitory activity of lyophilized fractions was measured by manometry as before. RESULTS
Inhibition
of insect mitochondrial
respiration
by M. gulosa venom
Inhibition of housefly mitochondrial respiration by increasing levels of M. gulosa venom is shown in Fig. 1. The form of inhibition showed a straight-line relationship with lower concentrations of venom followed by a non-linear increase as venom concentration per mg of mitochondrial protein was increased. Housefly and blowfly mitochondrial preparations were similarly inhibited by the venom when measured under identical conditions (Fig. 2). Results shown in Figs. 1 and 2 were derived using succinate as substrate. Inhibition of mitochondrial respiration occurred also when Lu-glycerophosphate was used as substrate. Reduction of the dye 2,3,5-triphenyltetrazolium chloride by respiring mitochondria was decreased in test systems containing venom compared with control media lacking venom. Time for complete reduction of thionin was increased when venom was present in the incubation medium. Addition of 0.01 p-moles of FAD to the usual incubation medium containing succinate as substrate did not influence the inhibitory effects of * Pronase-P, from culture medium of Streptomyces griseus, enforced protein disintegrate. Kaken Chemical Company Ltd., Japan.
LILIAN M.EWEN AND D. ILSE
1534 added
venom.
periods
of up to
reaction
mixture
Measures
such
as sonication
of mitochondrial preparations for Addition of ADP to the
3 min did not alter inhibition by venom. did
not
influence
mitochondrial
Mitoch.
respiration
suggesting
that
protein
2 - 1.5mg. 3 - 2.3 mg. 2.0
4.0
Venom
6.0
8.0
(mg.)
1. Inhibition of mitochondrial oxygen consumption by M. gulosa venom. The composition of tlie reaction mixture, of 2-O ml total volume, was as detailed in the text. Succlnate was used as substrate. Results show the inhibition caused by increasing concentrations of venom, using three levels of mitochondrial protein. FIG.
100 90
*-a
Housefly
A-A
Blowfly
mitoch. mitoch.
ex
80 ‘;; k ; z N
0
70
. ..
/
60 50
.J
40 30 20
: ‘OO
.
/ A
” 1.0
.
Mitochondrial
protein
(mg.)
Effect of M. gulosa venom on the oxygen consumption of housefly and blowfly mltochondrial preparations. The composition of the reaction mixture, of 2-O ml total volume, was as detailed in the text. Succinate was used as substrate. Oxygen consumption of inhibited mitochondrial systems containing 2.0 mg of venom/2*0 ml incubation medium is expressed as a percentage of normal control respiration in similar systems lacking venom. FIG.
2.
oxidative phosphorylation was uncoupled from electron transport in the mitochondrial preparations used and the venom acted to inhibit electron transport. Bovine plasma albumin used by some workers (WOJTCZAK and WOJTCZAK, 1959, 1960) to prevent uncoupling of oxidative phosphorylation did not affect inhibition caused by venom when 30 mg of albumin were added to control and test media.
MITOCHONDRIAL
RESPIRATION
INHIBITOR
IN
BULL
DOG
ANT
VENOM
1.535
Mammalian cytochrome-c (O-04 p-moles/2 ml of reaction medium) did, however, alleviate inhibition of mitochondrial respiration by venom. With higher levels of mitochondrial protein or lower levels of venom, resulting in low percentage inhibition, complete prevention of inhibition was achieved (Fig. 3). With lower
5 IO 15
20 25 30 35
40
45
50 55 60
Time (mins.) FIG. 3. Effect of mammalian cytochrome-c in alleviating the inhibition of housefly flight muscle mitochondrial respiration caused by M. gulosa venom. The composition of the reaction mixture was as described in the text and contained 2.4 mg lof mitochondrial protein/2*0 ml; succinate was used as substrate. l, 0, + 0.04 p-moles Control preparation; 0 - - - - l , +2-O mg of venom; Oof mammalian cytochrome-c; 0 - - - - 0, $-2-O mg of venom+0*04 p-moles of mammalian cytochrome-c.
concentrations of mitochondrial protein or higher concentrations of venom, thus greater inhibition, partial alleviation was achieved with added cytochrome-c (Table 1). In these cases alleviation was not further increased by higher concentrations (up to 0.16 p-males/2.0 ml incubate) of mammalian cytochrome-c (Table 2). Stability of the respiratory inhibitor of M. gulosa venom In dry venom the inhibitor was stable at room temperature for at least 1 month. The molecule was non-volatile and could be held under low pressure (7 x lo-$ mm Hg) without loss of activity. Lyophilization from aqueous solution did not impair its inhibitory activity. The inhibitor was stable to heat. Aqueous solutions of venom heated at 100°C for up to 30 min retained > 80 per cent of the original inhibitory potency. After such treatment the molecule remained uncoagulated in aqueous solution together with 75 per cent of the total biuret reactive material. The inhibitor was not extracted into lipid solvents, except butanol. These solvents did cause varying losses in inhibitory activity, however. On treatment of venom at room temperature with ether, acetone, ethanol, and methanol the activity which remained was confined to the coagulated protein residue and could subsequently be extracted into aqueous solution.
1536
LILIAN M. EWEN AND D. ILSE
Venom solutions shaken with 98% (v / v ) acetone showed no loss in potency, but 85% (v/v) acetone caused an 18 per cent loss. Ether similarly slightly reduced inhibitory activity. With 67% (v/v) ethanol, 89 per cent of total activity was lost. TABLE
I-EFFECT
OF MAMMALIAN CYTOCHROME-c ON INHIBITION RESPIRATION BY M. @llOSa VENOM* Inhibition
Mitochondrial protein (mg)
(mg)
Exp. 1
1.0
Exp. 2
1.1
Exp. 3
3.1
Exp. 4
3.0
0.5 1.0 2.0 3.0 2.0 3.0 4.0
text.
TABLE
o-00 0.04 0.08 0.12 0.16 * text. t $
39 32 25 30 30 29 30
was as described
in the
OF INCREASING CONCENTRATIONS OF CYTOCHROME-C ON INHIBITION MITOCHONDRIAL RESPIRATION BY M. @dOSa VENOM * Inhibition
Mitochondrial protein (mg) Venom (mg): Cytochrome-c (r_l moles)
Decrease
0 19 36 45 8 29 38
39 51 61 75 38 58 68
* Volume of incubate, 2.0 ml. The reaction mixture Succinate (50 p-moles) was used as substrate.
2-EFFECT
(per cent)
Test ( + 0.02 p-moles cytochrome-c)
Control (no added cytochrome-c)
Venom
OF MITOCHON~RIAL
1.0
OF
(per cent)
1.1
1.8
3.1
3.0
: 0.5 1.0 2.0 ATDSADADADADADADAD
-
39 0 0
75 49 42
39 39
51 9 0
42 51
61 34 35
27 27
3.0
2.0
2.0
-
26 33
78 46
-
32
Volume of incubate, 2.0 ml. The reaction mixture Succinate (50 p-moles) was used as substrate. Actual. Decrease.
38 18 15 25 8
3.0
4.0
-
30 23 13 30
58 35 32 39 33
-
23 26 19 25
was as described
68 47 38 36 34
21 30 32 34
in the
Hot 95% (v / v ) eth anol caused a 37 per cent loss in 15 min and hot 60% (v/v) methanol an 84 per cent loss over the same period. With 85% (v/v) butanol, inhibitory activity equalling 22 per cent of the total was extracted into the liquid
MITOCHONDRIAL RESPIRATION INHIBITORIN BULL DOGANTVENOM
1537
phase. Inhibition caused by an aqueous extract of the remaining coagulated protein amounted to 45 per cent of the total inhibition of control samples. With 92% butanol no activity was detected in the liquid phase, but inhibition by an aqueous extract of the remaining coagulated protein was 13 per cent less than that of control samples. Treatment of venom with 10% (w/v) perchloric acid for 30’min at room temperature did not destroy inhibitory activity. Similarly when venom was allowed to stand at room temperature in 70% (w/v) formic acid or 0.1 N ammonia for 3 days, no decrease in activity occurred when compared with control samples. Hydrolysis of venom protein with 6 N HCl at 110°C in a sealed tube reduced inhibitory potency of the venom by 55 per cent in 1 hr. Proteolytic digestion of venom with Pronase-P also produced a progressive decrease in inhibitory activity as venom protein decreased over increasing periods of time (Table 3). After 45 hr no inhibitory activity was detected in these preparations. TABLE 3-EFFECT
OF PROTROLYTICDIGESTIONON THE POTENCYOF THE RESPIRATORY INHIBITOROF M. &OSa VENOM* Time of digestion (hr)
Venom protein (% of total)
Inhibition (%)
Remaining activity (% of total)
0.00 0.25 0.50 1.00 2.00 4.00 8.00 20.00 45.00
100 78 74 72 68 61 42 40 39
67 49 40 36 27 22 11 10 0
74 60 54 40 32 17 15 0
* In testing for inhibitory activity, 1.7 mg of mitochondrial protein were used per 2.0 ml of incubation medium. The reaction mixture was as described in the text. Succinate (50 pmoles) was used as substrate.
Separation of inhibitory fraction from whole venom The inhibitory molecule of M. gulosa venom did not dialyse through $ in. Visking tubing but could pass through @ in. tubing (Table 4). CRAIG and KING (1962) found that pepsin of MW 35,500 had a 50 per cent escape time of 80 hr using $$ in. Visking tubing. Ovalbumin of MW 45,000 diffused more slowly than pepsin. Results in this work were consistent therefore with a MW greater than 5000 but less than 50,000 for the inhibitor. The molecule was eluted in the exclusion volume of a column of Sephadex G-25 confirming that the MW exceeded 5000. The exclusion limit of Sephadex G-75 is approximately 50,000. Chromatography of venom on this grade of Sephadex was therefore used to separate venom
LILIAN M. EWEN AND D. ILSE
1538
TABLE ~-EFFECT OF DIALYSISON M. gulosa VENOM Inhibition (per cent)
Inhibition (per cent)
Exp. No.*
Mitochondrial protein (mg) t
1 2 3 4 5 6 7 8 9
2.0 2.0 2.0 1.6 1.6 1.7 1,s 2.2 1.8
Dialysed venom (8 in. tubing) :. Nondialysed venom
Retentate
Dialysate
Exp. No.*
Mitochondrial protein (mg) t
80 66 71 63
71 69 64 80 84 75 62 72 56
0 0 0 0 0 0 0 0 0
1A 2A 3A 4A 5A 6A
1.3 1.3 2.8 2.8 2.1 2.2
Dialysed venom (# in. tubing): Nondialysed venom 93 86
93 69
Retentate
Dialysate
57 25 17 0 55 0
48 41 33 25 57 61
* Venom was treated as follows: 1-5. 4.0 mg of venom in 1.2 ml of distilled water dialysed for 67 hr against an equal volume of distilled water. 6. 4.0 mg of venom in 0.75 ml of distilled water dialysed for 92 hr against three changes of 1.0 ml of distilled water. 7. 4.0 mg of venom in 0.5 ml of distilled water dialysed for 72 hr against three changes of 1.0 ml of distilled water. 8. 5-O mg of venom in 1.0 ml of distilled water dialysed for 88 hr against four changes of 2.0 ml of distilled water. 9. Supernatant from 4.0 mg of heat-treated venom in 0.5 ml of distilled water, dialysed for 72 hr against three changes of 1.0 ml of distilled water. 1A. 4.0 mg of venom in 0.8 ml of distilled water dialysed for 60 hr against 1.0 ml of distilled water. 2A. Supernatant from 4 mg of heat-treated venom in 0.8 ml of distilled water dialysed for 60 hr against 1-O ml of distilled water. 3A. 5.0 mg of venom in 1.0 ml of distilled water dialysed for 92 hr against four changes of 1.0 ml of distilled water. 4A. Supematant from 5.0 mg of heat-treated venom in 1.0 ml of distilled water dialysed for 92 hr against four changes of 1.0 ml of distilled water. 5A. 6.0 mg of venom in 1.0 ml of distilled water dialysed for 92 hr against three changes of 1.0 ml of distilled water. 6A. 5.0 mg of venom in 1.0 ml of distilled water dialysed for 92 hr against four changes of 2.0 ml of distilled water. t Per 2-O ml of reaction mixture; composition as described in the text. $ Visking Co., Chicago, Illinois. fractions. eluting
Phosphate media.
The
buffer,
O-067 M, pH 7.4 or 70%
respiratory
inhibitor
overlapping peaks with the first smaller peak (Fig. 4).
was eluted peak forming
formic from
acid were used as
the columns
a shoulder
in two
to the second
MITOCHONDRIAL RESPIRATION INHIBITOR IN BULLDOGANTVENOM
1539
On electrophoresis of whole or heat-treated venom in 0.067 M phosphate buffer, pH 7.4, the inhibitor molecule migrated towards the cathode with the basic proteinaceous fractions of the venom. Inhibitory fractions eluted from a column of Sephadex G-75 migrated similarly on electrophoresis. 0.350-
790
.-.
0.300-
-80 -70
/i
0.250-
-60 :
f/
0.200.
z ;
O.lSO-
;;;j :,j
0.050-
-50
-
'
I\
1 _j
1 \
: I j;___,j/
: s ._
-40 f
I\ /
0.100-
v
I \
-30
I\
x
i;...
;,
i
l,,..J,
2o
-10
‘, .P/ 0.~00 :: ’ , , , , , , , ; , , , ’ 0 20 25 30 35 40 45 50 55 60 65 70 Fraction FIG. 4. Chromatography of M. gulosa venom on Sephadex G-75 (bead form) using 70% (W/V) formic acid as eluting agent; 25 mg of venom applied to a 2-5 x 90 cm column. Elution rate 30 ml/hr a-----0, E 280 nm of eluted fractions; A - - - A, percentage inhibition of housefly flight muscle mitochondrial respiration. In estimating inhibitory activity, 1.8 mg of mitochondrial protein were used/ 2-o ml incubation medium; composition as described in the text. Succinate was used as substrate. DISCUSSION
That bull dog ant venom can inhibit the oxygen consumption of respiring insect mitochondria is clear. The position and mechanism of inhibition of the electron transport system are less apparent. The terminal respiratory system of insects has been examined in some detail by several workers (CHANCEand PAPPENHEIMER,1954; PAPPENHEIMER and WILLIAMS, 1954; SHAPPIRIO and WILLIAMS, 1957, CHANCEand SACKTOR,1957) and CHANCEand SACKTOR(1958) have studied in particular the respiratory chain in housefly flight muscle mitochondria. There the scheme of electron transport is visualized as corresponding to the sequence shown overleaf. 2,3,5-Triphenyltetrazolium chloride accepts electrons from reduced cytochrome oxidase (NACHLASet al., 1960). Lessened reduction of this dye by respiring mitochondria in the presence of venom thus indicates a position of action for venom on the electron transport chain at any position preceding this. Thionin with an E,’ of + O-063would be expected to accept electrons from reduced cytochrome-b in the respiratory chain. Thus if the point of inhibition by venom succeeded the point at which thionin accepts reducing equivalents dye reduction 48
LILIAN M. EWENANDD. ILSE
1540
would be increased. Decreased action preceding cytochrome-c.
reduction of this dye is consistent with a point of
a-glycero-
M. gulosa venom inhibited mitochondrial oxygen consumption using either succinate or or-glycerophosphate as substrate. The point of inhibition would appear, therefore, to be at a position on the electron transport pathway common to both succinate and a-glycerophosphate, i.e. in the region of the electron transport chain between cytochrome-b and -c, although an action as a general flavoprotein poison has not been excluded. The addition of FAD, which forms the prosthetic group of succinate dehydrogenase was, however, found not to decrease inhibition of the succinoxidase system by venom. The effect of cytochrome-c in alleviating inhibition of oxygen consumption is unexplained. Basic proteins constitute at least part of the structure of both the inhibitory molecule of M. gdosa venom and of cytochrome-c. The effect of the cytochrome might thus be as a competitor of the inhibitory molecule. Alternatively, the action of added mammalian cytochrome-c in alleviating the inhibition of oxygen consumption caused by venom could possibly be in the provision of a bypass from substrate to unaffected parts of the respiratory chain. In uncoupled mitochondria electrons can be passed directly from the flavoprotein to cytochrome-c, or to cytochrome-c, bypassing cytochrome-b (GILMOUR, 1961). Under these conditions, mitochondrial electron transport is still inhibited by antimycin A which acts at a position between cytochromes-b and -c. If venom acted at a similar locus, inhibited mitochondrial electron transport might be substituted in uncoupled mitochondria by direct electron transport from substrate, through mammalian cytochrome-c, to succeeding sections of the mitochondrial electron transfer system. Migration of the respiratory inhibitor molecule towards the cathode on electrophoresis at pH 74 and separation from other venom fractions on Sephadex G-75, together with the stability of the inhibitor in 70% formic acid and O-1 N ammonia and its susceptibility to proteolytic digestion techniques, are consistent with the structure of a small basic protein. The elution spectrum of this venom on Sephadex G-75 shows considerable similarity to that of bee venom separated on Sephadex G-50 (HABERMANN,1965) with the respiratory inhibitor, like meiittin in the elution pattern of bee venom, forming the most predominant peak.
MITOCHONDRIAL RESPIRATION INHIBITORIN BULLDOGANT VENOM
1541
In this study the molecule has been characterized as an inhibitor of mitochondrial respiration. It may possess, however, like melittin in bee venom, a much wider activity spectrum. Estimation of the toxicity of the purified material to laboratory animals compared with that of whole venom may give an indication of the importance of the molecule as a biologically active constituent of bull dog ant venom. REFERENCES K. R. and WEISS C. (1959) Actions of scorpion venom on skeletal muscle. Br. J. Pharmac. 14, 334-339. ANASTASIA. and ERSPAMERV. (1962) Occurrence and some properties of eledoisin in extracts of posterior salivary glands of l&done. Br.J. Pharmac. 19, 326-336. BHOOLAK. D., CALLEJ. D., and SCHACHTER M. (1960) The identification of acetylcholine, 5-hydroxytryptamine and other substances in hornet venom. J. Physiol., Lond. 151, 35-36P. BHOOLA K. D., CALLE J. D., and SCHACHTERM. (1961) Identification of acetylcholine, S-hydroxytryptamine, histamine and a new kinin in hornet venom (V. crabro). J. Physiol., Lond. 159, 167-182. CHANCEB. and PAPPENHEIMER A. M., JR. (1954) Kinetic and spectrophotometric studies of cytochrome b, in midgut homogenates of Cecropia. J. biol. Chem. 209, 931-943. CHANCEB. and SACKTORB. (1958) Respiratory metabolism of insect flight muscie-XI. Kinetics of respiratory enzymes in flight muscle sarcomes. Archs Biochem. Biophys. 76, 509-531. CLELANDK. W. and SLATERE. C. (1953) Respiratory granules of heart muscle. Bi0chem.J. 53, 547-556. CRAIG I. C. and KING T. P. (1962) Dialysis. In Methods of Biochemical Analysis (Ed. by GLICK D.) 10, 175-199. Interscience, New York. DINIZ C. R. and GONCALVE~ J. M. (1956) Some chemical and pharmacological properties of Brazilian scorpion venoms. In Venoms (Ed. by BUCKLEYE. E. and PORGESN.), pp. 131-139. AAAS, Washington. ERSPAMER V. and ANASTASIA. (1962) Structure and pharmacological actions of eledoisin, the active endecapeptide of the posterior salivary glands of Eledone. Experientia 18, 58-59. GHIRETTIF. (1959) Cephalotoxin: the crab-paralysing agent of the posterior salivary glands of cephalopods. Nature, Lond. 183, 1192-1193. GHIRETTIF. (1960) Toxicity of octopus saliva against Crustacea. Ann. N. Y. Acad. Sci. 90, 726-741. GILMOURD. (1961) Biochemistry of Insects. Academic Press, New York. GONCALVESJ. M. (1956) Purification and properties of Crotamine. In Venoms (Ed. by BUCKLEYE. R. and PORGESN.), pp. 261-273. AAAS, Washington. GORNALL A. G., BARDAWILLC. J., and DAVID M. M. (1949) Determination of serum proteins by means of biuret reaction. J. biol. Chem. 177, 751-766. HABERMANNE. (1954) Hemmung der oxydativen Phosphorylierung durch tierische Gifte. Naturwissenschaften 41, 429430. HABERMANNE. (1955) uber Permeabilitatsiinderungen durch tierische Gifte. Arch. e@. Path. Pharmak. 225, 158-160. HABERMANN E. (1965) Recent studies on Hymenoptera venoms. In PYOC.2nd int. Pharmacol. Meeting, Prague (1963) (Ed. by RAUDONATH. W. and VANECEKJ.) 9, 53-61. Pergamon Press, Oxford. HABERMANNE. and REIZ K. G. (1964) Apamin: Ein vasisches, zentral erregendes Polypeptide aus Beinengift. Naturwissenschaften 51, 61. ADAM
1542
LILIAN M. EVEN ANDD. ILSE
JAQV~S R. and SCHACHTERM. (1954) Presence of histamine, S-hydroxytryptamine and potent, slow contracting substances in wasp venom. Br. J. Pharmac. 9, 53-58. MIRANDAF. and LISSITZKYS. (1961) Scorpamins: The toxic proteins of scorpion venoms. Nature, Land. 190,443-444. NACHLA~M. M., MARGULIESS. I., and SELIGMANA. M. (1960) Sites of electron transfer to tetrazolium salts in succinoxidase systems. r. biol. Chem. 235, 2739-2743. PAPPENHEIMERA. M. and WILLIAMS C. M. (1954) Cytochrome b, and the dihydrocoenzyme l-oxidase system in the Cecropia silkworm. J. biol. Chem. 209, 915-929. SACKTORB. (1953) Investigation on the mitochondria of the house fly, Musca domestica L. J. gen. Physiol. 36, 371-387. SACKTORB. and COCHRAND. G. (1958) The respiratory metabolism of insect flight muscle -1. Manometric studies of oxidation and concomitant phosphorylation with sarcosomes. Archs Biochem. Biophys. 74, 266-276. SHAPPIRIOD. G. and WILLIAMS C. M. (1957) The cytochrome system of the Cecropia silkworm-11. Spectrophotometric studies of oxidative enzyme systems in the wing epithelium. Proc. R. Sot. Lond. (B) 147, 233-246. WOJTCZAKL. and WOJTCZAKA. B. (1959) The action of serum albumin on oxidative phosphorylation in insect mitochondria. Biochim. biophys. Acta 31, 297-299. WOJTCZAKL. and WOJTCZAKA. B. (1960) Uncoupling of oxidative phosphorylation and inhibition of ATP-P, exchange by a substance from insect mitochondria. Biochim. biophys. Acta 39, 277-286.