Toxeeon, 1976, VoL 14, pp . 221-233 . Per~amon Pree. Printed In Great Britain.
ENZYMATIC CHARACTERISTICS OF CROTALUS PHOSPHOLIPASE A~ AND TIDE CROTOXIN COMPLEX H~xxlxa BREITIiALJPT Institute of Pharmacology, Justus-Liebig-University, D-63 Gietsen, Frankfurter Str. 107, West Germany (Accepted for publlcntion
9
December
197
H. BnelrawPr. Enzymatic oharaoteristics of crotales phospholipase A, and the crotoxin complex. Toxlcon 14, 221-233, 1976.-Crotalus durlssus terrlficus phospholipase A, a component of the crotoxin complex, is of the A, type. The enzyme hydrolyzes aqueous dispersions of egg yolk lipoproteins with zero order kinetics . Optimum activity occurs at 53-57°C and pH 7~0-8~0. The rate of enzymatic attack on monomeric substrates is negligible. Rapid hydrolysis is observed when the substrate is above the critical micelle concentration. The enzymatic phospholipase activity is 1100 itmoles per min and mg enzyme using aqueous solutions of diheptanoyllecithin, containing 50 mM Ca'+, or egg yolk emulsions, containing400 mM Ca'+ as substrate. Crotalus phospholipase A, requires Ca'+ for catalytic activity. The activity of the enzyme is also enhanced by Na+, K+, Mg'+, Mn'+, and deoxycholate . Zn'+, Cu'+, Na,-EDTA, phosphate, oxalate, sulfide, cyanide, citrate, fatty acids, and lysolecithin inhibit the onzyme . Low concentrations of Ba'+ increase the enzymatic activity, whereas high concentrations inhibit the enzyme. Crotalus phospholipase A, is an extremely stable enzyme, resisting heating (85°C), lyophilization, repeated thawing, urea (6 M), and acidic solvents such as 10~ formic acid. The enzyme is inactivated by exposure to pH values higher than 9~0 or bycontact with cellophane membranes . Crotapotin, the second component of the crotoxin complex, has no phospholipase activity, but inhibits the crotales phospholipase A,. Three moles of crotapotin decrease the activity of 1 mole of phospholipase A, to a residual activity of 10~, independent of the pH range or preincubation time used. The inhibition can be prevented by deoxycholate or by Ca'+, but not by high concentrations of NaCI or KCI. lá egg yollc suspension, hydrolysis by the phospholipase-crotapotin complex occurs after a lag period . The duration of latency depends on the amounts ofcrotapotin added to the enzyme. INTRODUCTION PHOSPHOLIPASE A, (EC 3.1 .1.4) catalyzes the hydrolysis of fatty acyl esters at the 2-position of 1,2-diacyl-sn-phosphoglycerides. This enzyme occurs in the venoms of snakes, scorpions, bees and in several mammalian tissues (CONDRSA and nE VRIFS, 1965) . The isolation and biochemical characterization of the phospholipases from the Crotales durissus terrifices (Brazilian rattlesnake) venom are reported elsewhere (HABERMANN and RüBSA~rr, 1971 ; RiYBSA1~x et al., 1971 ; HsrrDON and FRASrtxE1.-COxRAT, 1971 ; Hoxsr et al., 1972 ; BREI1'HAUPT et al., 1974,1975 ; OMORI-SATOx et al., 1975) . Other studies deal with the neurotoxic and myotoxic activities of the basic crotalus phospholipase Ace (BREITHAUPT and HAS>~tANx,1973 ; BRazu, et x1.,1973 ; BRSr1z-1AUPT,197~ . The toxic crotalus phospholipase A$ is considered a protein homologous to other phospholipases as well as to several presynaptic neurotoxins (BREITHAUPT et al., 1975) . The basic crotalus phospholipase binds to the acidic polypeptide crotapotin. The resulting phospholipase~;rotapotin complex resembles the naturally occurring crotoxin which is the main toxic fraction of the Crotales TOXICON l976 Yol. !~
ul
222
HENNING BRETTHAUPT
venom (SLOTTA and FRAENKEIrCONRAT, 1938). Crotapotin itself lacks the toxicity and enzymatic activity of crotoxin, but potentiates the toxicity and inhibits the enzymatic activity of the crotales phospholipase A$ (RÜasAan;N et al., 1971). This report* describes the enzymatic properties of the basic crotales phospholipase A$. durissus terrifrcus
MATERIALS AND METHODS
Crotalus durissus terriJicus venom was obtained from Dr. Hücherl, Instituto Butantan, Säo Paulo, Brazil . The crude venom was dissolved in 0016 N HCI, and crotoxin was prepared by precipitation at its iscelectric point 4~8according to the method of SLOZ-rn and Fte.~xx>:r,-CoxxwT (1938) . The basic crotalus phospholipase A, and the acidic polypeptide crotapotin were prepared by chromatography of crotoxin on carboxymethyl cellulose (CM 32, Whatman) using a linear gradient from 0~1 to 2~S M ammonium formate buffer, pH 3~S (RthunMEx et al., 1971). The crotalus phospholipase A, and crotapotin were homogeneous with respect to polyacrylamide gel electrophoresis, iscelectric focusing, immuncelectrophoresis, sedimentation equilibrium studies, and N-terminal amino acid analyses (Bxerrxnurr et al., 1974). Hee venom phospholipase A, was prepared from crude venom by gel filtration of Sephadex G 50 according to the method of Hassu~fnxrr and RErrz (1965) . Botluops ruuwiedilphospholipase A, was prepared from crude venom by gel filtration on Sephadex G 50 and Sephadex G 25, followed by chromatography on DEAE~ellulose according to the method of Vro v. and S~rorraxc (1971a). Crotalus adamanteus phospholipase activity was tested using crude venom from the Miami Serpentarium, Florida. Phospholipase A, from porcine pancreas was a gift from Prof. G. H. de Haas, Utrecht. All other substances used in this study were of analytical grade, and commercially obtained . For determination of the positional specificity, 0~1 mg of the crotalus phospholipase A was incubated at 25° C with S mg of 1-pahnitoyl-2-oleoyl-sn-glycero-3-phosphorylcholinet dispersed in 1 ml of 0~1 M sodium-borate buffer, pH 7~2, (Ca'+ = S mM) with the aid of a few drops of ether. The degree of breakdown of the mixed-acid lecithin was followed by thin-layer chromatography on silica gel using chloroformmethanol (1 :1, v/v) as solvent. After 2 hr the hydrolysis was complete . The fractions of fatty acids and the lysolecithin fraction were extracted from the thin-layer plate by the use of chloroform-methanol (2 :1, v/v), and then analyzed by gas chromatography according to the method of ns Haas et al. (1963). Phospholipase A, activity was followed by continuous titration of the released fatty acids at constant pH, using a Combititrator 3-D (Metrohm, Herisau) . Aqueous solutions of pure synthetic short chain lecithins$ and aqueous emulsions of egg yolk were used as substrates . The egg yolk was diluted 1 :4 in physiological saline and centrifuged 6 min at 3000 g. Portions (20 ml) of the supernatants were stored at -17 °C, and thawed egg yolk emulsions were stable for 1 day. Egg yolk, diluted 1 :12 in saline without addition of Ca'* or deoxycholate was used as standard substrata. Titrations were carried out, in 3 ml of the rapidly stirred emulsion, with 0025 N NaOH at SO°C and pH 800 under a stream of nitrogen . No spontaneous hydrolysis oa-urred in this test system. The enzymatic activity was calculated from the initial hydrolysis rate, and expressed as Eunoles fatty acids liberated per min and mg enzyme . RFCULTS
Positional specificity of the crotales phospholipase When incubated with the basic crotales phospholipase A, 1-palmitoyl-2-oleoyl-snglycero-3-phosphorylcholine yielded the fatty acid esterified at the 2-position, while the 1-bound fatty acid remained within the corresponding lysocompound . The evidence that the crotales phospholipase A acts as a phospholipase A, was supported by the selective hydrolysis at position 2 of a 1-sH-pahnitoyl-2-l 4C-linoleoyl-sn-glycero-3-phosphatidylethanolamine . No phospholipase A1 or B activity was observed upon incubation of 0~1 mg of the enzyme in 025 ml of 2 mM Cas+-20 mM Tris-HCI, pH 7~4, containing 0~ 1 mM of the labeled substrate (Kunze, personal communication) . *Some of the data reported here have boen presented at the Third International Symposium on Animal and Plant Toxins 1972, Darmstadt (Germany). fThis mixed lecithin was synthetized in the laboratory of Prof. G. H. de Haas, Utrecht. $1,2-d heptanoyl-sn-glycero-3-phosphorylcholine was obtained from Prof. G. H. de Haas, Utrecht and 1,?rdíhexanoyl~n-glyoero-3-phosphorylcholine was a gift from Prof. Ch . A. Vernon, London . TOXJCON J976 Yot. 14
Crotalus Phospholipase A, and Crotoxin
223
Substrate requirements of the crotalus phospholipase A, The time course of enzymatic hydrolysis of native ovolecithin follows zeroorderkinetics . However, the rate of hydrolysis decreased shortly after starting the reaction. The phospholipase A, activity was calculated from the initial hydrolysis rate, and the activity was expressed as ~tmoles fatty acids liberated per min and mg enzyme . The reproducibility of titration data was within f 5 ~. The hydrolysis by crotalus phospholipase A, of the egg yolk lipoproteins was not complete within 15 min in comparison to bee venom phospholipaseAz (Fig.1) . Figure lA demonstrates that the addition of a second aliquot of substrate in-
to
m
A
addition of egg yolk
5
FIO. 1 . TIAffi COURSE OF HYDROLYSIS OF E00 YOLK LIPOPR0IEINS BY CROTALUS PHOSPHOLiPASH A, . (A) :
Titration curves recorded after addition of 100IIg of crotalus phospholipase ( ) and 100 Rg of bee vonom phospholipaso (---), respectively, to 3 ml of e88 y°~~ diluted 1 :12 in 015 M NaCI. Thirtcen min after starti the h drol emulsion was added. (B) : Thirtcen mm~after start' ythe h adroltl~of Iml of e~ yolk yob diluted 1:12 in 015 M NaCI~ 6 y 100 Wg of cxotalus phospholipase, an additional aliquot ~ of phospholipass was added ( ). For comparison, hydrolysis by 100 Ng or bee venom phospholipase is shown (---) . Each curve represents one of three titration experiments (end point pH 800; 50°C) with resulting pmoles of libeaated fatty acids at 1, S, 10, and 15 min within a standard error of f 1 ~.
3
creased the activity of crotalus phospholipase indicating that the enzyme was still active in the egg yolk suspension. Figure 1B indicates that the addition of a second aliquot ofcrotalus phospholipase A, did not significantly increase the hydrolysis rate of the egg yolk lipoproteins. Even after addition of high amounts of crotalus phospholipase (1 mg) the hydrolysis of the egg yolk lipoproteins was not complete within 15 min. Similar incomplete hydrolyses were observed using egg yolk suspensions as substrate which contained up to 100 mM Ca'+ or up to 10 mM sodium deoxycholate. Strong inhibition occurred when 1-pahnitoyl-sn-glycero-3-phosphorylcholine or palmitoic acid were added to the egg yolk suspension. The lysocompound (0~ 1 mM) decreased TOPICON 1976 YoI. I4
224
HSNNINß
BRETTHAUPT
the hydrolysis by the phospholipase (20 gg) of egg yolk, diluted 1 :12 in 0~ 15 M NaCI (3 ml) by about 10 ~, 1 mM by 30 ~, and 2 mM by 50 %. The free fatty acid (1 mM) inhibited the phospholipase activity by about 30 ~. Using egg yolk diluted 1 :12 in 015 mM NaCI (3 ml, pH 800, 50°C) the velocity of hydrolysis increased linearly with the enzyme concentration of 1-50 lIg per 3 ml of egg yolk suspension . However, at higher concentrations (> 50 lIg of phospholipase per 3 ml) the increase in the reaction velocity slowed down . After addition of 20 Ftg of crotalus phospholipase Ae to 3 ml of egg yolk, diluted 1 :50, 1 :40, 1 :30, 1 :20, and 1 :10, respectively, in 015 M NaCI (pH 800, 50°C) the enzymatic activity increased linearly with the substrate concentration and slowed down to a maximal activity of 60 itmoles per min and mg enzymes, using egg yolk diluted 1 :10 in saline. The enzymatic activities of phospholipases from bee venom and porcine pancreas were 10 times higher, using egg yolk emulsions as substrate. With aqueous dispersions of diheptanoyllecithin in 100 mM NaCI-100 mM CaCI,1 mM Tris-HCI, pH 800 (3 mn, the enzymatic activity of the crotalus phospholipase increased up to 1100 llmoles per min and mg enzyme . As shown in Fig. 2, regression line zoo oo E
FIQ.
2. ENZYMATIC
mM Diheptanoyllecithin
ACTIVrrY OF CROTALUS PHOSPHOLn'ASE A, : DEPENDENCE ON THE CONCENTRATION OF DIIiEPTANOYLLECITHIN . Two lIg of thecrotalus phospholipase A, were added to 3 ml of 100 mM NaCI-100 mM CaCI,-1 mM Tris-HCI, containing various concentrations of diheptanoyllecithin (abscissa) . The ordinate represents the enzymatic activity in lunoles per min and mg phospholipase. All reactions at pH 800 and 30°C. (~) : ~ f s (n = 3) . Regression line was calculated by the method of least squares .
intercepts the abscissa at 0~7 mM diheptanoyllecithin . This finding may indicate the preference of the crotalus phospholipase Ae for micellar solutions. The critical micelle concentration of diheptanoyllecithin in aqueous solution was reported to be 1~5-2~0 mM (Boxsaty et al., 1972). The preference of the crotalus phospholipase for micellar substrates was clearly demonstrated with aqueous solutions of dihexanoyllecithin . The enzymatic hydrolysis of dihezanoyllecithin in concentrations up to the critical micelle concentration TOXICON 1976 Vol . II
Crotalus IPhospholipase A, and Crotoxin E c É ;,, 20o-
225
A
_~ eoo T
~ 400 U N
200
400
600
800
mM
Ca2 `
a E C_
E
N
_m
B
600~
O
z cooT
~ 200{
,r
.Cl 20
~
40
~
60
~
mM SDC
3. ENZYMATIC ACI'MIY OP CROTALUS PHOSPHOLIPASE A, : DEPENDENCE ON THE CONCENTRATION OF CALCIUM (A) OR SODIUM DEOXYCHOLATE (B) . (A) : 20 iIg of phospholipase A, wero added to 3 ml of egg yolk, diluted 1 :12 in 015 M NaCI (") and 1 M NaCI (p), respectively. Moreover, 2 Rg of the enzyme wero added to 3 ml of 3 mM diheptanoyllecithin-100 mM NaCI-1 mM Tris-HCt ( "). The ordinate represents the FIa.
enzymatic activity calculated from the initial hydrolysis rate. (B) : 20 pg of phospholipase A, wire added to 3 ml of egg yolk, diluted 1 :12 in 015 M NaCI. All reactions at pH 8'00 and SO°C. SDC = sodium dooxycholate.
Of 11 mM [ROHOLT and SCHLAMOWITZ (1961) ; BONSEN et al. (1972) OOCilrred Very Slowly . The reaction rate increased rapidly using diheptanoyllecithin in concentrations above the critical micelle concentration . Ca'+ was an absolute requirement for crotalus phospholipase A, activity (Fig. 3A). Maximal activation occurred with 50 mM Ca'+ using diheptanoyllecithin, and with 400 mM Ca$+ using egg yolk emulsion as substrate. In both systems, the specific activities increased up to 1100 Ftmoles per min and mg enzyme . As shown in Fig. 3B, addition of the surfactant sodium deoxycholate to the egg yolk emulsion increased the phospholipase activity from 60 up to 550 Etmoles per min and mg enzyme . With egg yolk suspensions containing 8 mM deoxycholate and 30 mM Ca2+ the enzymatic activity increased up to 12001tmoles per min and mg phospholipase. The enzymatic activity of the crotalus phospholipase A, varied with the concentration of NaCI or KCl in the egg yolk suspension (Fig. 4). No hydrolysis was measured observed in egg yolk, diluted 1 :12 in distilled water. The hydrolysis rate was 50 ltmoles per min and mg enzyme in egg yolk, diluted 1 :12 in 015 M NaCI or KCI, and 150ltmoles per min and mg enzyme in egg yolk, diluted 1 :12 in 1 M NaCI or KCI. With egg yolk suspensions, containing more than 1 M NaCI or KCI, the velocity of hydrolysis decreased to about 100 moles per min and mg enzyme . The enzymatic activity of the phospholipase-crotapotin complex crotoxin also varied with the concentration of NaCI or KCI . As shown in Fig. 4, high concentrations of NaCI or KCl did not dissociate the crotoxin complex . The enzymatic activity of 16 ltg of the phospholipase (10 ltg}-crotapotin (6 ltg) complex was TOXICON 1976 Yal. II
22 6
HENNING BREITHAUPT
0.15
1.0
4
M NaCI
FIO. 4. ENZYMATIC ACTMTY OF THE CROTALUS PHOSPHOLIPASE A, AND CROTO7QN : DEPENDENCE ON THS CONCENTRATION OF SODIUM CHLORIDE .
Ten ~g of crotalus phospholipase A, (PhA) and 16 ug of crotoxin, respectively, were added to 3 ml of egg yolk, diluted 1 :12 in solutions of varying NaCI content. The abscissa indicates the final salt concentration, the ordinate represents the enzymatic activity at pH 800 and SO°C. Sixteen hg of the enzyme-inhibitor complex crotoxin contained 10 lIg of phospholipase and 6 pg of crotapotin (molar ratio 1 :1). (~) : ~ f s (n = 4).
within the range of 0~1 ~5 M NaCI or KCl about 3 times lower than the enzymatic activity of 10 ltg phospholipase alone. When Ca'+ (0~1 M) was added to egg yolk suspensions containing 0~ 15 and 1 M NaCI, respectively, the phospholipase Ae activity increased to 1100 ltmoles per min and mg enzyme. Sucrose (0~3 M) did not influence the enzymatic activity in egg yolk, diluted 1 :12 in 0~ 15 M NaCI. With diheptanoyllecithin as substrate the optimal pH for the activity of the basic crotalus phospholipase A, was between 7 and 8 (Fig. SA). The pH-activity curve obtained for egg yolk suspension without addition of Ca$+ or deoxycholate yielded an optimum at pH 7-8, and a smaller second optimum at pH 8~8 (Fig. 7B) . This `shoulder' in the pHactivity curve of the crotalus phospholipase A$ was reproducible in all experiments made with different amounts of substrate and phospholipase As. Using egg yolk suspension which contained 100 mM Ca$+, this second pH optimum drifted to pH 9~5 (Fig. SB) . The pH-optimum of the phospholipase~rotapotin complex (molar ratio 1 :1) was symmetrical with a broad maximal region, centering around pH 8~5 (Fig. SB) . In egg yolk, diluted 1 :12 in 0~15 M NaCI without addition ofCaE+,the highest enzymatic activity was found at 53-57°C. The energy of activation calculated from the Arrhenius plot was 11,000 (25-58°C) and 23,800 (15-25°C) cal per mole for the hydrolysis of native egg yolk phospholipads . Figure 6 demonstrates the remarkable heat stability of the crotalus phospholipase Aa, especially at pH values below 7. The enzyme activity did not decrease after exposure to 50°C and pH 7~5 for 1 week. At pH 9~0 slow inactivation occurred at 50°C within 12 hr. The enryme, 8 times frozen (-17°C) and thawed, was fully active. Repeated lyophilization (5 times) did not reduce its activity. The crotalus phospholipase Az exposed 24 hr to 6 M urea-10 mM Tris-HCI, pH 6~0, still had 80~ of its activity after removing the urea by gel filtration. The enryme was stable in distilled water for several weeks. The crotalus phospholipase was fully active after l 2-hr exposure to 10 ~ formic acid . On the other hand the enryme lost its activity by contact with dialysis membranes. A 24-hr exposure of 10 mg of the enzyme to cellophane membrane (20 cm') reduced the enrymatic activity to about 50 ~. TOXICON1976 Y°l. It
22 7
Crotalus Phospholipase A, and Crotoxin
A
E c_ ~E á~ m
0 i
FIa .
S. ENZYMATIC ACTIVITY OF CROTALUS PHOSPHOLiPA3E A, AND CROTOXIN : DEPENDENCE ON THE pH OF AQUEOUS SOLUTIONS OF DIHEPTANOYLLECITHIN (A) OR E00 YOLK SUSPENSIONS (B) . (A) : Two EIg of phospholipase A, were added to 3 ml of 3 mM diheptanoyllecithin-100 mM
NaCI-10 mM CaCI,-1 mM sodium citrate-NaOH and Tris-HCI, respectively. All reactions at SO°C. ("): ~ f s (n = 3). (13) : ("-") : Sixteen ug of Crotoxin, containing 10 ug of phospholipase and 6 Wg of crotapotin, were added to 3 ml of egg yolk, diluted 1 :12 in O~1S M NaCI (for hydrolysis rate, see left ordinate). ("-") : Ten ug of phospholipase A, were added to 3 ml of egg yolk, diluted 1 :12 in 0" 1S M NaCI (for hydrolysis rate see 1át ordinate). (O-O): Ten u8 of phospholipase A, were added to 3 ml of egg yolk, diluted 1:12 in 0"15 M NaCI-100 mM CaCI, (for hydrolysis rate see inserted ordinate). All reactions at 50°C .
However, only 10~ of the phospholipase was adsorbed by this membrane within 1 day, and up to 20 ~ within 5 days. Solutions of phospholipase which had lost their activity by a 5-day~xposure to cellophane were fully reactiviated by the addition of NaCI (> 0"3 Nn
60
°
20_ `H : . 3Ó l0Ó
220 min
Fla. tÍ. EFFECT OF HEAT ON THE PH03PHOLIPASE A, ACITVITY. Crotalus phospholipase A, dissolved in 0" 1 M glycine-NaCI/NaOH (1 mgJml) at pH 6, 7, and 8, respectively, was heated to 8S°C for times indicated by the abscissa . The time course of inactivation by the heat-treatment was recorded for aliquots of the enzyme (SO pg), which were added to 3 ml of an egg yolk suspension, diluted 1 :12 in 0" 1S M NaCI-0 " 1 M CaCI,. Phospholipase activity was measured at pH 8"00 and SO°C. TOXICON 1976 Vol. !~
22 8
HENNING BREITHAUPT
TARIE I. THB HFFECl' OP VARIOUS IONS ON THE ENZYMATIC ACTIVITY OF THE CROTALUS PHOSPHOLIPASE
Cation added" Enzymatic activity (5 mM) (lunoles/min m~ Na 60 Ca 200 Mn 180 Ha 140 Mg 140 Al 100 Fe'+ 70 Fe'+ 60 Cu'+ 12 Zn 0 Ba (300 mM) 0 "Chloride as anion. tSodium as cation.
Anion addedt (5 mM) Chloride Nitrite Nitrate Sulfate Iodide Thiosulfate Phosphate Oxalate Sulfide Cyanide Phosphate (100 mM)
Enzymatic activity (llmoles/min m~ 60 60 60 60 60 60 30 30 30 30 5
A~
or Ca'+ (10 mM). The phospholipase A, could not be desorbed by NaCI or CaCI, from the cellophane membranes. In egg yolk, diluted 1 :12 in 015 M NaCI without addition of Ca'+, the activity of 50 ltg of the enzyme was influenced by various ions (Table 1). The complete inhibition of 50 wg enzyme by 5 mM EDTA was reversed by calcium, but not by Mn'+, Mga+ or deoxy cholate. The strong inhibition by CuB+ (5 mM) was reversed by Cas+ (25 mM) or Mgs+ (50 mM); the inhibition by Ba'+ (300 mM) was reversed by Caß+ (5 mM), but not by Mg'+ (5-300 mM). Aflter an incubation time of 30 min, SO ltg of heparin decreased the activity of 50 pg enzyme to 60 ~ residual activity. Synthetic polyglutamic acids, with molecular weights of 7000 or 30,000, did not influence the enzymatic activity. Enzymatic activity of the crotales phospholipase A $ as in,Jfuenced by crotapotin
The acidic polypeptide crotapotin inhibited the enrymatic activity of the basic crotales phospholipase A,. Three moles of crotapotin decreased the activity of 1 mole phospholipase A, to a residual enzymatic activity of 10 ~, as calculated from the initial hydrolysis rate of diheptanoyllecithin (Fig. 7A). As shown in Fig. 7B, a lag period of about 5 min preceded maximal hydrolysis using egg yolk as substrate for the phospholipase-~rotapotin complex (molar ration 1 :3). Crotoxin complexes, containing phospholipase A$ and crotapotin in the molar ratio of 1 :1, caused no latent period. The development of latency was independent of incubation time . However, the duration of the lag period increased with the amount of crotapotin added to the enyeme . The `natural' crotoxin complex, which can be isolated from the crude venom (SLOTTA and FRAENKEL-CONRAT, 1938) produced a lag period not longer than 5 sec. The enzymatic activity of crotoxin decreased upon the addition of additional crotapotin. Phospholipase A$ alone did not show this latency. The activity of phospholipase AE, inhibited by crotapotin, was calculated from that part of the time curve where maximal hydrolysis occurred . Reproducibility was within ~ 10 ~. The inhibition of the crotales phospholipase As activity by crotapotin could be prevented by deoxycholate, using egg yolk emulsion as substrate (Fig. 8). The inhibition of phospholipase As activity by crotapotin decreased in aqueous solutions of diheptanoyllecithin containing high concentrations of Cas+ (Fig. 9A). The inhibition, however, was not influenced by Ca'+, when egg yolk emulsions were used as substrate (Fig. 9B). As shown in Fig. 4, the inhibition of phospholipase activity by crotapotin was not reversed by NaCI. Crotapotin did not inhibit the activities of other phospholipases, e.g. those from the venoms of Crotales adamanteus, Bothrops neuwiedü, bee, or porcine pancreas . TOXICON 1976 Yol. l~
IFYO .
Î. TIME COURSE OF HYDROLYSIS OF DII~EPTANOYLI~CITHIN (A) OR EGG YOLK LiPOPROTEIN3 (B) HY CROTALUS PHOSPHOLIPASB A, ALONE OR IN COMBINATION WrrH CROTAPOTIN . (A): Titration curves recorded after addition of 10 fig of phospholipase alone or together with 20 pg of crotapotin to 3 ml of 3 mM diheptanoyllecithin-100 mM NaCI-10 mM CaCI,1 mM Tris-HCI, pH 800. (B): Time course recorded after addition of 1001Ig of phospholipase alone or together with 2001Ig of crotapotin to 3 ml of egg yolk, diluted 1 :12 in O~1S M NaCI . Each curve represents one of three titration experiments showing kmoles of fatty acids liberated at 1, S, and 10 min within a standard error of f 1 ~. All titrations at 50°C and pH 800 (end point titration) .
Ng Crotapotin FiO. S. EFPEGT OF DEOXYCHOLATE ON THE ENZYMATIC ACI'IYfIY OF PHOSPHOLIPASB A, Ai.ONE OR TOGETHER WITH CROTAPO'Ißd. Twenty pg of phospholipaso A, together with 0, 20, 50 or 100 IIg of crotapotin wero added to 3 ml of egg yolk, diluted 1 :12 in 015 M NaCI . All reactions at pH 800, SO°C. The activities were related to the hydrolysis rates of phospholipase A, without inhibitor added. The reaction mixtures contained 0 and 10 mM sodium deoxycholate (S1JC), respectively. The mean values ( ") and standard deviation ranges are demonstrated graphically (n = S) . TOXICON 1976 Vol.
l~
crotapotin iNgl
crotapotin lNgl
FIG. 9. EFFECT OF CALCIUM ON THE ENZYMATIC ACIMIY OF PHO3PHOLIPASE A, ALONE OR TOGETHER wITH CROTAPOTIN t)SINO DIIiEPTANOYLLECITHIN (A) OR EGG YOLK (B) AS SiJHSIRATE . (A) : 2 P.g of phospholipase A, together with 0, 10 or 1001Ig of crotapotin were added to 3 ml
of 3 mM diheptanoyllecithin-100 mM NaCI-1 mM Tris-HCI, pH 800 . The substrates contained 10, 100, and S00 mM Ca'+, respectively. The activities were related to the hydrolysis rates of phospholipase A, without inhibitor added . The mean values (") and standard deviation ranges are demonstrated graphically (n = 4). B) : 20 pg of phospholipase A, together with 0, 10, 50 or 100 t.Ig of crotapotin were added to 3 ml of egg yolk, diluted 1 :12 in 015 M NaCI with 0, 10, 100, and 500 mM Ca'+, respectively. The enzymatic activities were related to the hydrolysis rates of the phospholipase A, without inhibitor added. Since these relative activities were not dependent on the concentrations of Ca'+ in the substrates, the mean values ( ")and standard deviation ranges are summarized in onegraphic presentation (n =12). All reactions at 50°C and pH 800. DISCUSSION
As with most other snake venom phospholipases (nE HAAS et al., 1968) the crotalus phospholipase is of the As type . By studying the velocity of breakdown of egg yolk lipoproteins and of synthetic short chain lecithins, the toxic enzyme yielded a maximal enzymatic activity of 11001tmoles per min and mg enzyme for both substrates . This is in the activity range of most other snake venom phospholipases (Vlnnl, et al., 1972). The crotalus phospholipase As fails to hydrolyze the substrate completely . Similar incomplete hydrolysis was reported for the phospholipase A from Agkistrodon piscivorus venom (AuGUS~rvx and ELLIOT, 1970). This is probably due to inhibition by the hydrolytic products. Similar to other phospholipases (BONSSx et al., 1972) the crotalus enzyme prefers substrates in the form of micelles or lipoproteins . Bee venom phospholipase A8, however, seems to hydrolyze the monomer substrate more rapidly (Si-~ot,Ixl et al., 1971). The activating effect of deoxycholate, shown for the crotalus phospholipase A$, can perhaps be explained by the requirement of phosphatide micelles which have a certain minimum zeta-potential (BANGI-IAM and DAWSON, 1959 ; DAWSON, 1963). High concentrations of NaCI or KCI, but not of saccharose, activate the crotalus phospholipase As, possibly by a change in the physicochemical state of the substrate (Boxsstr et al., 1972). $AITO and Hnx~lrnx (1962) reported a 3 times increment of the hydrolysis of ovolecithin by the Crotalus adamanteus phospholipase A when the salt concentration was increased to 0~3 M NaCI. The strict requirement for calcium is well known for many phospholipases (HABERMANN, 1957 ; Loxc and PexxY, 1957; SHIPOLINI et al., 1971 ; P1ErERSOx et al., 1974) . RoxoLT and SCHLAMOWITZ (1961) obtained with 5 mM Caß+ maximal activation of the phospholipase AQ activity of Crotalus durissus terrificus crude venom, using dihexanoyllecithin as substrate. In the present study the concentration of calcium for maximal activation of the roxrcoN r9~6 v°r. r4
Crotales Phospholipase A, and Crotoxin
23 1
pure crotales phospholipase is ten times higher, using diheptanoyllecithin as substrate. In egg yolk suspensions, maximal activation occurs at very high concentrations probably caused by adsorption to proteins . The crotales enzyme as well as phospholipases A from other sources are activated by some divalent rations. Mga+, Nia+, Coa+, and Cda+ activated Crotahtr atrox and Latlcauda semifasciata phospholipase A (Wu and Tixx>~t, 1969 ; Tu et al., 1970). The activity of the Crotalus terrif/cus enzyme increases due to exposure to Mga+ and Mna+, but, in contrast to Ca$+, these rations do not reverse the inhibition by Na,-EDTA . Calcium; moreover, abolishes the inhibition provoked by Cua+ or Baa+ . These findings support the unique role of calcium for the activity of the Crotalus terrificus phospholipase A$. Similar to this, Uz'xi: and MAGHE (1971) reported for the Crotalus adamanteus phospholipases Ae that Caa+ could not be replaced by other divalent rations. The strong inhibiting effect of phosphate buffer should be emphasized. The pH-optima of the crotales phospholipase and of crotoxin are comparable to the values, found for phospholipases from other sources (CotanxEa and DE Vxnss,1965 ; DELORI, 1973). The pH-activity curve of the crotales phospholipase A, shows an unusual shape not observed for other phospholipases . The second pH-optimum in the highly alkaline range occurs in egg yolk suspensions only, thus indicating that the egg yolk substrate may be responsible for this effect . Using aqueous solutions of the synthetic diheptanoyllecithin as substrate, the crotales phospholipase Aa shows a broad pH-activity curve with an optimum around pH 7~0-8~0. The pH-activity curve of crotoxin is similar to that of the Crotales durissus terrificus venom (ROHOLT and SCHLAMOWITZ, 1961). The crotales phospholipase Aa shows several properties classically attributed to phospholipases A. The enzyme is highly resistant to heat, acidic solvents and urea, possibly due to its large number of disulfide bridges (BxFtTxauP'r et al., 1974). In contrast to the Crotalus atrox phospholipase Aa (Wu and TINKER, 1969), the Crotalus durissus enzyme remains active upon lyophilization . However, simple contact with cellophane partially inactivates the enzyme . Reactivation occurs by calcium or by exposure to high concentration of sodium chloride . Further physical and chemical data are needed for interpretation of this phenomenon . Crotoxin, the main toxic fraction of the Crotalus durissus terr~cus venom, shows high toxicity and low phospholipase Aa activity. Crotoxin is a complex of the basic crotales phospholipase Aa and the acidic polypeptide Crotapotin (RÜssnhlerr et al., 1971). The stoichiometry of this complex varies over a wide range, but with a preference for a molar ratio of 1 :1 . Crotoxin can be considered as a toxin-potentiator complex as well as an enzymeinhibitor complex. Other naturally occurring polypeptides, also inhibiting the enzymatic action of phospholipases A, have been isolated from Naja raja (BRncnxcn et al., 1970) and Bothrops neuwiedü venom (VIDAL and STOPPAHI, 1971b) . The type and extent of inhibition, however, are not yet clarified. Moreover, these enzymes were far less toxic than the basic phospholipase Aa from Crotalus durissus terrificus venom. Crotapotin inhibits the enzymatic activity of the basic crotales phospholipase A$ to a considerable extent in all test systems used . `Natural' crotoxin, consisting mainly of 1 :1 complexes (molar ratio), possesses about 60 ~ of the enzymatic activity of the pure phospholipase Ag. Enzyme-inhibitor complexes with a molar ratio of 1 :3 show only 10 ~ of the activity of the pure enzyme . High concentrations of NaCI or KCl cannot prevent the inhibition of the enzyme by Crotapotin . Cae+, however, as well as deoxycholate antagonize the inhibiting effect of Crotapotin. T~DXICON 1976 Vol. l~
232
HENNING BREITHAUPT
Using egg yolk lipoproteins as substrate the reaction kinetics are complicated by an initial lag period, which is proportional to the inhibitor concentration used . On the other hand, this latency and the maximal velocity of hydrolysis are not influenced by the incuba tion time ofthe enzyme with crotapotin. Similar lag periods were reported for the inhibitor of Bothrops phospholipases ~VIDAL and STOPPANI, 1971b) . The polypeptide crotapotin may interact with groups which are involved in the catalytic activity of the phospholipase A8. On the other hand, its interaction with the weakly toxic enzyme provokes a strong increase of the neurotoxic and myotoxic activities . Therefore, crotapotin might become a useful tool for the study of the active centres of phospholipases A$ as well as presynaptic neurotoxins. Acknowledgements-The
author thanks Professor G. H. nE Haws for help with the studies on positional specificity and for the supply of diheptanoyllecithin. He also thanks Mrs. R. DIETRICFI for skilful technical assistance. REFERENCES
Auausrnv, J. M. and Eu.IOT, W. B. (1970) Isolation of a phospholipase A from Agktstrodon ptsctvorus venom. Biochtm. biophys. Acts 206, 98 . BwNGHwM, A. D. and DwwsoN, R. M. C. (1959) The relation between the activity of a lecithinase and the electrophoretic charge of the substrate. Bfochem. J. ?2, 486. BONSEN, P. P. M., nE Hwws, G. H., PIETERSON, W. A. and vwN DEENEN, L. L. M. (1972) Studies on phospholipase A and its zymogen from porcine pancreas . IV. The influence of chemical modification of the lecithin structure on substrate properties. Btochtm. biophys. Acts 270, 364. BRwawxcw, B. M., SwMBRwv, Y. M. and $AMHRAY, R. Y. (1970) Isolation of polypeptide inhibitior of phospholipase A from cobra venom. Eur. J. Btochem. 13, 410. BRAZIL, O. V., ExcEU., B. J. and DE Sw, S. S. (1973) The importance of phospholipase A in the action of the sotoxin complex at the frog neuromuscular junction . J. Phystol. 234, 63 P. BRSrrHwIJPT, H. (1976) Neurotoxic and myotoxic activities of the sotalus phospholipase A and its complex with sotapotin. Naunyn-Schmiedebergs Arch . Pharmak. in press. BREITHwuPT, H. and HABERMANN, E. (1973) Biochemistry and pharmacology of phospholipase A from Crotales terrificus venom as influenced by sotapotin. In : Animal andPlant Toxins, Vol. 1, p. 83, (KwISEx, E., Ed.) . Mùnchen: Goldmann . BRErrHwuPT, H., OMORI-SATOH, T. and LaNa, J. (1975) Isolation andcharacterization of three phospholipases A from the sotoxin complex. Biochim. biophys. Acts 403, 355. BREITHAUPT, H., RÜBSAMEN, K. and HAHERbIANN, E. (1974) Biochemistryy and pharmacology of the ctotoxin complex. Biochemical analysis of crotapotin and the basic sotalus phospholipase A. Eur. J. Btochem. 49, 333. CONDREA, E. and DE VRIES, A. (1965) Venom phospholipase A: a review Toxicon 2, 261 . DwwsoN, R. M. C. (1963) On the mechanism of action of phospholipase A. Btochem. J. 88, 414. DE Hwas, G . H., HEEMSKERK, C. H. T., vwN DHENEN, L. L. M., BASER, R. W. R., Gwtul-HATCHARD, J., MAGES, W. L. and THOMPSON, R. H. S. (1963) The mode of action of human pancreatic phospholipase A. Btochem. Probl. Lipids 1, 244. DE Hwws, G. H., PosTEMw, N. M., NIEUWENHUIZEN, W. and vwN DBENEN, L. L. M. (1968) Purification and properties of phospholipase A from porcine pancreas. Btochtm. biophys. Acts 159,103. DEIARI, P. J. (1973) Purification et propriétés physicochimiques, chimiques et biologiques d'une phospholipase A, toxique isolée d'un venin de serpent Viperidae : Vtpera berus. Biochtmte 55,1031 . HABERMANN, E. (1957) Manometrische Bestimmung von ~hospholipase A. Btochem. Z. 328, 474 . HAHE1tMANN, E. and RErrz, K. G. (1965) Ein neues Verfahren zur Gewinnung der Komponenten von Bienengift, insbesondere des zentralwirksamen Peptide Apamin . Btochem. Z. 341, 451 . HABI?RMANN, E. and ROBSwMEN, K. (1971) Biochemical and pharmacological analysis of the so-called sotoxin. In : Toxins of Animal and Plant Origins, Vol. 1, p. 333, (UE VRIES, A. and KOCHVA, E., Eds.) . New York : Gordon & Breach . HENDON, R. A. 8nd FttAENSEIrCONRAT, H. (1971) Biological roles of the two components of sotoxin. Proc. nain. Acad. Sct., U.S .A . 68, 1560 . HORST, J., HENDON, R. A. and FRweNxEt-CoNRwT, H. (1972) The active components of sotoxin. Biochem. biophys. Res. Commun. 46, 1042. Lorra, C. and PENNY, I. F. (1957) The structure of the naturally occurring phosphoglyoerides . 3. Action of moccasin-venom phospholipase A on ovolecithin and related substances . Biochem. J. 65, 382. TOXJCON J976 Yot. J~
Crotalus Phospholipase A, and Crotoxin
233
Oatonz-Sw~rox, T., LwNa, J., HxFtzxwurr, H. and HwHExMwNx, E. (197 Partial amino acid sequence of tha basic crotalus phospholipase A. Toxlcon 13, 69. Ptsrsnsox, W. A., Vorwsxx, J. J. and ns Hwws, G. H. (1974) Interaction of phospholipase A, and its zymogen with divalent metal ions. Biochemistry 13, 1439. RoxoLr, O. A. and SCFII.AMOVVITZ, M. (1961) Studies of the use of díëexanoyllecithin and other lecithins as substrates for phospholipase A. With addendum on aspects of micelle properties of díhexanoyllecithin . Archs Biochem . Blophys . 94, 364. RüsswMSN, K., Basrrxwurr, H. and HABERMANN, E. (1971) Biochemistry and pharmacology of the crotoxin complex. I. Subfractionation and recombination of the crotoxin complex. Naunyn-Schmiedebergs Arch. Pharmak . 270, 274. Swrro, K. and Hwxw~wx, D. J. (1962) A study of the purification and properties of the phospholipase A of Crotolus adamanteus venom. Biochemistry 1, 521. Stirroi uvt, R. A., CwuFwwsRT, G. L., Coz-ixeu., R. C., Dooxwx, S., Vsxrrox, CH. A. and Bwr~s, B. E. C. (1971) Phospholipase A from bee venom. Eur. J. Blochem. 20, 459. SLO~rrw, K. H. and FxwsxxstrCoivnwT, H. L. (1938) Schlangengifte, III. Mitteilung : Reinigung and Krystallisation des Klapperschlangen-Giftes. Ber . dt . them. Ges.71, 1076 . Tu, A. T., Pwsssr, R. B. and Toots, P. M. (1970) Isolation and characterization of phospholipase A from sea snake, Laticauda semljasciata venom. Archs Biochem . Blophys. 140, 96 . Urns, J. F. and Mwcss, W. L. (1971) Phospholipase A, : action on purified phospholipids as affected by deoxycholate and divalent cations. Can. J. Blochem . 49, 776. Vmwt., J. C., CAITANEO, P. and STOPPAM, A. O. M. (1972) Some characteristic properties of phospholipases A, from Bothrops neuwiedil venom. Archs Blochem. Blophys. 151, 168. V~nwL, J. C. and STOPPAM, A. O. M. (1971a) Isolation and purification of two phospholipases A from Bothrops venoms. Archs Blochem . Blophys . 145, 543. VtnwL, J. C. and Srorrwrn, A. O. M. (1971b) Isolation and properties of an inhibitor of phospholipase A from Bothrops neuwledli venom. Archs Biochem . Blophys. 147, 66. Wu, T:W. and TtrrxsR, D. O. (1969) Phospholipase A, from Crotalus atrox venom. I. Purification and some properties. Biochemistry S, 1958 .
rorrco~ lsró vot. t~
ENZYMATIC CHARACTERISTICS OF CROTALUS PHOSPHOLIPASE A~ AND TIDE CROTOXIN COMPLEX H~xxlxa BREITIiALJPT Institute of Pharmacology, Justus-Liebig-University, D-63 Gietsen, Frankfurter Str. 107, West Germany (Accepted for publlcntion
9
December
197
H. BnelrawPr. Enzymatic oharaoteristics of crotales phospholipase A, and the crotoxin complex. Toxlcon 14, 221-233, 1976.-Crotalus durlssus terrlficus phospholipase A, a component of the crotoxin complex, is of the A, type. The enzyme hydrolyzes aqueous dispersions of egg yolk lipoproteins with zero order kinetics . Optimum activity occurs at 53-57°C and pH 7~0-8~0. The rate of enzymatic attack on monomeric substrates is negligible. Rapid hydrolysis is observed when the substrate is above the critical micelle concentration. The enzymatic phospholipase activity is 1100 itmoles per min and mg enzyme using aqueous solutions of diheptanoyllecithin, containing 50 mM Ca'+, or egg yolk emulsions, containing400 mM Ca'+ as substrate. Crotalus phospholipase A, requires Ca'+ for catalytic activity. The activity of the enzyme is also enhanced by Na+, K+, Mg'+, Mn'+, and deoxycholate . Zn'+, Cu'+, Na,-EDTA, phosphate, oxalate, sulfide, cyanide, citrate, fatty acids, and lysolecithin inhibit the onzyme . Low concentrations of Ba'+ increase the enzymatic activity, whereas high concentrations inhibit the enzyme. Crotalus phospholipase A, is an extremely stable enzyme, resisting heating (85°C), lyophilization, repeated thawing, urea (6 M), and acidic solvents such as 10~ formic acid. The enzyme is inactivated by exposure to pH values higher than 9~0 or bycontact with cellophane membranes . Crotapotin, the second component of the crotoxin complex, has no phospholipase activity, but inhibits the crotales phospholipase A,. Three moles of crotapotin decrease the activity of 1 mole of phospholipase A, to a residual activity of 10~, independent of the pH range or preincubation time used. The inhibition can be prevented by deoxycholate or by Ca'+, but not by high concentrations of NaCI or KCI. lá egg yollc suspension, hydrolysis by the phospholipase-crotapotin complex occurs after a lag period . The duration of latency depends on the amounts ofcrotapotin added to the enzyme. INTRODUCTION PHOSPHOLIPASE A, (EC 3.1 .1.4) catalyzes the hydrolysis of fatty acyl esters at the 2-position of 1,2-diacyl-sn-phosphoglycerides. This enzyme occurs in the venoms of snakes, scorpions, bees and in several mammalian tissues (CONDRSA and nE VRIFS, 1965) . The isolation and biochemical characterization of the phospholipases from the Crotales durissus terrifices (Brazilian rattlesnake) venom are reported elsewhere (HABERMANN and RüBSA~rr, 1971 ; RiYBSA1~x et al., 1971 ; HsrrDON and FRASrtxE1.-COxRAT, 1971 ; Hoxsr et al., 1972 ; BREI1'HAUPT et al., 1974,1975 ; OMORI-SATOx et al., 1975) . Other studies deal with the neurotoxic and myotoxic activities of the basic crotalus phospholipase Ace (BREITHAUPT and HAS>~tANx,1973 ; BRazu, et x1.,1973 ; BRSr1z-1AUPT,197~ . The toxic crotalus phospholipase A$ is considered a protein homologous to other phospholipases as well as to several presynaptic neurotoxins (BREITHAUPT et al., 1975) . The basic crotalus phospholipase binds to the acidic polypeptide crotapotin. The resulting phospholipase~;rotapotin complex resembles the naturally occurring crotoxin which is the main toxic fraction of the Crotales TOXICON l976 Yol. !~
ul
222
HENNING BRETTHAUPT
venom (SLOTTA and FRAENKEIrCONRAT, 1938). Crotapotin itself lacks the toxicity and enzymatic activity of crotoxin, but potentiates the toxicity and inhibits the enzymatic activity of the crotales phospholipase A$ (RÜasAan;N et al., 1971). This report* describes the enzymatic properties of the basic crotales phospholipase A$. durissus terrifrcus
MATERIALS AND METHODS
Crotalus durissus terriJicus venom was obtained from Dr. Hücherl, Instituto Butantan, Säo Paulo, Brazil . The crude venom was dissolved in 0016 N HCI, and crotoxin was prepared by precipitation at its iscelectric point 4~8according to the method of SLOZ-rn and Fte.~xx>:r,-CoxxwT (1938) . The basic crotalus phospholipase A, and the acidic polypeptide crotapotin were prepared by chromatography of crotoxin on carboxymethyl cellulose (CM 32, Whatman) using a linear gradient from 0~1 to 2~S M ammonium formate buffer, pH 3~S (RthunMEx et al., 1971). The crotalus phospholipase A, and crotapotin were homogeneous with respect to polyacrylamide gel electrophoresis, iscelectric focusing, immuncelectrophoresis, sedimentation equilibrium studies, and N-terminal amino acid analyses (Bxerrxnurr et al., 1974). Hee venom phospholipase A, was prepared from crude venom by gel filtration of Sephadex G 50 according to the method of Hassu~fnxrr and RErrz (1965) . Botluops ruuwiedilphospholipase A, was prepared from crude venom by gel filtration on Sephadex G 50 and Sephadex G 25, followed by chromatography on DEAE~ellulose according to the method of Vro v. and S~rorraxc (1971a). Crotalus adamanteus phospholipase activity was tested using crude venom from the Miami Serpentarium, Florida. Phospholipase A, from porcine pancreas was a gift from Prof. G. H. de Haas, Utrecht. All other substances used in this study were of analytical grade, and commercially obtained . For determination of the positional specificity, 0~1 mg of the crotalus phospholipase A was incubated at 25° C with S mg of 1-pahnitoyl-2-oleoyl-sn-glycero-3-phosphorylcholinet dispersed in 1 ml of 0~1 M sodium-borate buffer, pH 7~2, (Ca'+ = S mM) with the aid of a few drops of ether. The degree of breakdown of the mixed-acid lecithin was followed by thin-layer chromatography on silica gel using chloroformmethanol (1 :1, v/v) as solvent. After 2 hr the hydrolysis was complete . The fractions of fatty acids and the lysolecithin fraction were extracted from the thin-layer plate by the use of chloroform-methanol (2 :1, v/v), and then analyzed by gas chromatography according to the method of ns Haas et al. (1963). Phospholipase A, activity was followed by continuous titration of the released fatty acids at constant pH, using a Combititrator 3-D (Metrohm, Herisau) . Aqueous solutions of pure synthetic short chain lecithins$ and aqueous emulsions of egg yolk were used as substrates . The egg yolk was diluted 1 :4 in physiological saline and centrifuged 6 min at 3000 g. Portions (20 ml) of the supernatants were stored at -17 °C, and thawed egg yolk emulsions were stable for 1 day. Egg yolk, diluted 1 :12 in saline without addition of Ca'* or deoxycholate was used as standard substrata. Titrations were carried out, in 3 ml of the rapidly stirred emulsion, with 0025 N NaOH at SO°C and pH 800 under a stream of nitrogen . No spontaneous hydrolysis oa-urred in this test system. The enzymatic activity was calculated from the initial hydrolysis rate, and expressed as Eunoles fatty acids liberated per min and mg enzyme . RFCULTS
Positional specificity of the crotales phospholipase When incubated with the basic crotales phospholipase A, 1-palmitoyl-2-oleoyl-snglycero-3-phosphorylcholine yielded the fatty acid esterified at the 2-position, while the 1-bound fatty acid remained within the corresponding lysocompound . The evidence that the crotales phospholipase A acts as a phospholipase A, was supported by the selective hydrolysis at position 2 of a 1-sH-pahnitoyl-2-l 4C-linoleoyl-sn-glycero-3-phosphatidylethanolamine . No phospholipase A1 or B activity was observed upon incubation of 0~1 mg of the enzyme in 025 ml of 2 mM Cas+-20 mM Tris-HCI, pH 7~4, containing 0~ 1 mM of the labeled substrate (Kunze, personal communication) . *Some of the data reported here have boen presented at the Third International Symposium on Animal and Plant Toxins 1972, Darmstadt (Germany). fThis mixed lecithin was synthetized in the laboratory of Prof. G. H. de Haas, Utrecht. $1,2-d heptanoyl-sn-glycero-3-phosphorylcholine was obtained from Prof. G. H. de Haas, Utrecht and 1,?rdíhexanoyl~n-glyoero-3-phosphorylcholine was a gift from Prof. Ch . A. Vernon, London . TOXJCON J976 Yot. 14
Crotalus Phospholipase A, and Crotoxin
223
Substrate requirements of the crotalus phospholipase A, The time course of enzymatic hydrolysis of native ovolecithin follows zeroorderkinetics . However, the rate of hydrolysis decreased shortly after starting the reaction. The phospholipase A, activity was calculated from the initial hydrolysis rate, and the activity was expressed as ~tmoles fatty acids liberated per min and mg enzyme . The reproducibility of titration data was within f 5 ~. The hydrolysis by crotalus phospholipase A, of the egg yolk lipoproteins was not complete within 15 min in comparison to bee venom phospholipaseAz (Fig.1) . Figure lA demonstrates that the addition of a second aliquot of substrate in-
to
m
A
addition of egg yolk
5
FIO. 1 . TIAffi COURSE OF HYDROLYSIS OF E00 YOLK LIPOPR0IEINS BY CROTALUS PHOSPHOLiPASH A, . (A) :
Titration curves recorded after addition of 100IIg of crotalus phospholipase ( ) and 100 Rg of bee vonom phospholipaso (---), respectively, to 3 ml of e88 y°~~ diluted 1 :12 in 015 M NaCI. Thirtcen min after starti the h drol emulsion was added. (B) : Thirtcen mm~after start' ythe h adroltl~of Iml of e~ yolk yob diluted 1:12 in 015 M NaCI~ 6 y 100 Wg of cxotalus phospholipase, an additional aliquot ~ of phospholipass was added ( ). For comparison, hydrolysis by 100 Ng or bee venom phospholipase is shown (---) . Each curve represents one of three titration experiments (end point pH 800; 50°C) with resulting pmoles of libeaated fatty acids at 1, S, 10, and 15 min within a standard error of f 1 ~.
3
creased the activity of crotalus phospholipase indicating that the enzyme was still active in the egg yolk suspension. Figure 1B indicates that the addition of a second aliquot ofcrotalus phospholipase A, did not significantly increase the hydrolysis rate of the egg yolk lipoproteins. Even after addition of high amounts of crotalus phospholipase (1 mg) the hydrolysis of the egg yolk lipoproteins was not complete within 15 min. Similar incomplete hydrolyses were observed using egg yolk suspensions as substrate which contained up to 100 mM Ca'+ or up to 10 mM sodium deoxycholate. Strong inhibition occurred when 1-pahnitoyl-sn-glycero-3-phosphorylcholine or palmitoic acid were added to the egg yolk suspension. The lysocompound (0~ 1 mM) decreased TOPICON 1976 YoI. I4
224
HSNNINß
BRETTHAUPT
the hydrolysis by the phospholipase (20 gg) of egg yolk, diluted 1 :12 in 0~ 15 M NaCI (3 ml) by about 10 ~, 1 mM by 30 ~, and 2 mM by 50 %. The free fatty acid (1 mM) inhibited the phospholipase activity by about 30 ~. Using egg yolk diluted 1 :12 in 015 mM NaCI (3 ml, pH 800, 50°C) the velocity of hydrolysis increased linearly with the enzyme concentration of 1-50 lIg per 3 ml of egg yolk suspension . However, at higher concentrations (> 50 lIg of phospholipase per 3 ml) the increase in the reaction velocity slowed down . After addition of 20 Ftg of crotalus phospholipase Ae to 3 ml of egg yolk, diluted 1 :50, 1 :40, 1 :30, 1 :20, and 1 :10, respectively, in 015 M NaCI (pH 800, 50°C) the enzymatic activity increased linearly with the substrate concentration and slowed down to a maximal activity of 60 itmoles per min and mg enzymes, using egg yolk diluted 1 :10 in saline. The enzymatic activities of phospholipases from bee venom and porcine pancreas were 10 times higher, using egg yolk emulsions as substrate. With aqueous dispersions of diheptanoyllecithin in 100 mM NaCI-100 mM CaCI,1 mM Tris-HCI, pH 800 (3 mn, the enzymatic activity of the crotalus phospholipase increased up to 1100 llmoles per min and mg enzyme . As shown in Fig. 2, regression line zoo oo E
FIQ.
2. ENZYMATIC
mM Diheptanoyllecithin
ACTIVrrY OF CROTALUS PHOSPHOLn'ASE A, : DEPENDENCE ON THE CONCENTRATION OF DIIiEPTANOYLLECITHIN . Two lIg of thecrotalus phospholipase A, were added to 3 ml of 100 mM NaCI-100 mM CaCI,-1 mM Tris-HCI, containing various concentrations of diheptanoyllecithin (abscissa) . The ordinate represents the enzymatic activity in lunoles per min and mg phospholipase. All reactions at pH 800 and 30°C. (~) : ~ f s (n = 3) . Regression line was calculated by the method of least squares .
intercepts the abscissa at 0~7 mM diheptanoyllecithin . This finding may indicate the preference of the crotalus phospholipase Ae for micellar solutions. The critical micelle concentration of diheptanoyllecithin in aqueous solution was reported to be 1~5-2~0 mM (Boxsaty et al., 1972). The preference of the crotalus phospholipase for micellar substrates was clearly demonstrated with aqueous solutions of dihexanoyllecithin . The enzymatic hydrolysis of dihezanoyllecithin in concentrations up to the critical micelle concentration TOXICON 1976 Vol . II
Crotalus IPhospholipase A, and Crotoxin E c É ;,, 20o-
225
A
_~ eoo T
~ 400 U N
200
400
600
800
mM
Ca2 `
a E C_
E
N
_m
B
600~
O
z cooT
~ 200{
,r
.Cl 20
~
40
~
60
~
mM SDC
3. ENZYMATIC ACI'MIY OP CROTALUS PHOSPHOLIPASE A, : DEPENDENCE ON THE CONCENTRATION OF CALCIUM (A) OR SODIUM DEOXYCHOLATE (B) . (A) : 20 iIg of phospholipase A, wero added to 3 ml of egg yolk, diluted 1 :12 in 015 M NaCI (") and 1 M NaCI (p), respectively. Moreover, 2 Rg of the enzyme wero added to 3 ml of 3 mM diheptanoyllecithin-100 mM NaCI-1 mM Tris-HCt ( "). The ordinate represents the FIa.
enzymatic activity calculated from the initial hydrolysis rate. (B) : 20 pg of phospholipase A, wire added to 3 ml of egg yolk, diluted 1 :12 in 015 M NaCI. All reactions at pH 8'00 and SO°C. SDC = sodium dooxycholate.
Of 11 mM [ROHOLT and SCHLAMOWITZ (1961) ; BONSEN et al. (1972) OOCilrred Very Slowly . The reaction rate increased rapidly using diheptanoyllecithin in concentrations above the critical micelle concentration . Ca'+ was an absolute requirement for crotalus phospholipase A, activity (Fig. 3A). Maximal activation occurred with 50 mM Ca'+ using diheptanoyllecithin, and with 400 mM Ca$+ using egg yolk emulsion as substrate. In both systems, the specific activities increased up to 1100 Ftmoles per min and mg enzyme . As shown in Fig. 3B, addition of the surfactant sodium deoxycholate to the egg yolk emulsion increased the phospholipase activity from 60 up to 550 Etmoles per min and mg enzyme . With egg yolk suspensions containing 8 mM deoxycholate and 30 mM Ca2+ the enzymatic activity increased up to 12001tmoles per min and mg phospholipase. The enzymatic activity of the crotalus phospholipase A, varied with the concentration of NaCI or KCl in the egg yolk suspension (Fig. 4). No hydrolysis was measured observed in egg yolk, diluted 1 :12 in distilled water. The hydrolysis rate was 50 ltmoles per min and mg enzyme in egg yolk, diluted 1 :12 in 015 M NaCI or KCI, and 150ltmoles per min and mg enzyme in egg yolk, diluted 1 :12 in 1 M NaCI or KCI. With egg yolk suspensions, containing more than 1 M NaCI or KCI, the velocity of hydrolysis decreased to about 100 moles per min and mg enzyme . The enzymatic activity of the phospholipase-crotapotin complex crotoxin also varied with the concentration of NaCI or KCI . As shown in Fig. 4, high concentrations of NaCI or KCl did not dissociate the crotoxin complex . The enzymatic activity of 16 ltg of the phospholipase (10 ltg}-crotapotin (6 ltg) complex was TOXICON 1976 Yal. II
22 6
HENNING BREITHAUPT
0.15
1.0
4
M NaCI
FIO. 4. ENZYMATIC ACTMTY OF THE CROTALUS PHOSPHOLIPASE A, AND CROTO7QN : DEPENDENCE ON THS CONCENTRATION OF SODIUM CHLORIDE .
Ten ~g of crotalus phospholipase A, (PhA) and 16 ug of crotoxin, respectively, were added to 3 ml of egg yolk, diluted 1 :12 in solutions of varying NaCI content. The abscissa indicates the final salt concentration, the ordinate represents the enzymatic activity at pH 800 and SO°C. Sixteen hg of the enzyme-inhibitor complex crotoxin contained 10 lIg of phospholipase and 6 pg of crotapotin (molar ratio 1 :1). (~) : ~ f s (n = 4).
within the range of 0~1 ~5 M NaCI or KCl about 3 times lower than the enzymatic activity of 10 ltg phospholipase alone. When Ca'+ (0~1 M) was added to egg yolk suspensions containing 0~ 15 and 1 M NaCI, respectively, the phospholipase Ae activity increased to 1100 ltmoles per min and mg enzyme. Sucrose (0~3 M) did not influence the enzymatic activity in egg yolk, diluted 1 :12 in 0~ 15 M NaCI. With diheptanoyllecithin as substrate the optimal pH for the activity of the basic crotalus phospholipase A, was between 7 and 8 (Fig. SA). The pH-activity curve obtained for egg yolk suspension without addition of Ca$+ or deoxycholate yielded an optimum at pH 7-8, and a smaller second optimum at pH 8~8 (Fig. 7B) . This `shoulder' in the pHactivity curve of the crotalus phospholipase A$ was reproducible in all experiments made with different amounts of substrate and phospholipase As. Using egg yolk suspension which contained 100 mM Ca$+, this second pH optimum drifted to pH 9~5 (Fig. SB) . The pH-optimum of the phospholipase~rotapotin complex (molar ratio 1 :1) was symmetrical with a broad maximal region, centering around pH 8~5 (Fig. SB) . In egg yolk, diluted 1 :12 in 0~15 M NaCI without addition ofCaE+,the highest enzymatic activity was found at 53-57°C. The energy of activation calculated from the Arrhenius plot was 11,000 (25-58°C) and 23,800 (15-25°C) cal per mole for the hydrolysis of native egg yolk phospholipads . Figure 6 demonstrates the remarkable heat stability of the crotalus phospholipase Aa, especially at pH values below 7. The enzyme activity did not decrease after exposure to 50°C and pH 7~5 for 1 week. At pH 9~0 slow inactivation occurred at 50°C within 12 hr. The enryme, 8 times frozen (-17°C) and thawed, was fully active. Repeated lyophilization (5 times) did not reduce its activity. The crotalus phospholipase Az exposed 24 hr to 6 M urea-10 mM Tris-HCI, pH 6~0, still had 80~ of its activity after removing the urea by gel filtration. The enryme was stable in distilled water for several weeks. The crotalus phospholipase was fully active after l 2-hr exposure to 10 ~ formic acid . On the other hand the enryme lost its activity by contact with dialysis membranes. A 24-hr exposure of 10 mg of the enzyme to cellophane membrane (20 cm') reduced the enrymatic activity to about 50 ~. TOXICON1976 Y°l. It
22 7
Crotalus Phospholipase A, and Crotoxin
A
E c_ ~E á~ m
0 i
FIa .
S. ENZYMATIC ACTIVITY OF CROTALUS PHOSPHOLiPA3E A, AND CROTOXIN : DEPENDENCE ON THE pH OF AQUEOUS SOLUTIONS OF DIHEPTANOYLLECITHIN (A) OR E00 YOLK SUSPENSIONS (B) . (A) : Two EIg of phospholipase A, were added to 3 ml of 3 mM diheptanoyllecithin-100 mM
NaCI-10 mM CaCI,-1 mM sodium citrate-NaOH and Tris-HCI, respectively. All reactions at SO°C. ("): ~ f s (n = 3). (13) : ("-") : Sixteen ug of Crotoxin, containing 10 ug of phospholipase and 6 Wg of crotapotin, were added to 3 ml of egg yolk, diluted 1 :12 in O~1S M NaCI (for hydrolysis rate, see left ordinate). ("-") : Ten ug of phospholipase A, were added to 3 ml of egg yolk, diluted 1 :12 in 0" 1S M NaCI (for hydrolysis rate see 1át ordinate). (O-O): Ten u8 of phospholipase A, were added to 3 ml of egg yolk, diluted 1:12 in 0"15 M NaCI-100 mM CaCI, (for hydrolysis rate see inserted ordinate). All reactions at 50°C .
However, only 10~ of the phospholipase was adsorbed by this membrane within 1 day, and up to 20 ~ within 5 days. Solutions of phospholipase which had lost their activity by a 5-day~xposure to cellophane were fully reactiviated by the addition of NaCI (> 0"3 Nn
60
°
20_ `H : . 3Ó l0Ó
220 min
Fla. tÍ. EFFECT OF HEAT ON THE PH03PHOLIPASE A, ACITVITY. Crotalus phospholipase A, dissolved in 0" 1 M glycine-NaCI/NaOH (1 mgJml) at pH 6, 7, and 8, respectively, was heated to 8S°C for times indicated by the abscissa . The time course of inactivation by the heat-treatment was recorded for aliquots of the enzyme (SO pg), which were added to 3 ml of an egg yolk suspension, diluted 1 :12 in 0" 1S M NaCI-0 " 1 M CaCI,. Phospholipase activity was measured at pH 8"00 and SO°C. TOXICON 1976 Vol. !~
22 8
HENNING BREITHAUPT
TARIE I. THB HFFECl' OP VARIOUS IONS ON THE ENZYMATIC ACTIVITY OF THE CROTALUS PHOSPHOLIPASE
Cation added" Enzymatic activity (5 mM) (lunoles/min m~ Na 60 Ca 200 Mn 180 Ha 140 Mg 140 Al 100 Fe'+ 70 Fe'+ 60 Cu'+ 12 Zn 0 Ba (300 mM) 0 "Chloride as anion. tSodium as cation.
Anion addedt (5 mM) Chloride Nitrite Nitrate Sulfate Iodide Thiosulfate Phosphate Oxalate Sulfide Cyanide Phosphate (100 mM)
Enzymatic activity (llmoles/min m~ 60 60 60 60 60 60 30 30 30 30 5
A~
or Ca'+ (10 mM). The phospholipase A, could not be desorbed by NaCI or CaCI, from the cellophane membranes. In egg yolk, diluted 1 :12 in 015 M NaCI without addition of Ca'+, the activity of 50 ltg of the enzyme was influenced by various ions (Table 1). The complete inhibition of 50 wg enzyme by 5 mM EDTA was reversed by calcium, but not by Mn'+, Mga+ or deoxy cholate. The strong inhibition by CuB+ (5 mM) was reversed by Cas+ (25 mM) or Mgs+ (50 mM); the inhibition by Ba'+ (300 mM) was reversed by Caß+ (5 mM), but not by Mg'+ (5-300 mM). Aflter an incubation time of 30 min, SO ltg of heparin decreased the activity of 50 pg enzyme to 60 ~ residual activity. Synthetic polyglutamic acids, with molecular weights of 7000 or 30,000, did not influence the enzymatic activity. Enzymatic activity of the crotales phospholipase A $ as in,Jfuenced by crotapotin
The acidic polypeptide crotapotin inhibited the enrymatic activity of the basic crotales phospholipase A,. Three moles of crotapotin decreased the activity of 1 mole phospholipase A, to a residual enzymatic activity of 10 ~, as calculated from the initial hydrolysis rate of diheptanoyllecithin (Fig. 7A). As shown in Fig. 7B, a lag period of about 5 min preceded maximal hydrolysis using egg yolk as substrate for the phospholipase-~rotapotin complex (molar ration 1 :3). Crotoxin complexes, containing phospholipase A$ and crotapotin in the molar ratio of 1 :1, caused no latent period. The development of latency was independent of incubation time . However, the duration of the lag period increased with the amount of crotapotin added to the enyeme . The `natural' crotoxin complex, which can be isolated from the crude venom (SLOTTA and FRAENKEL-CONRAT, 1938) produced a lag period not longer than 5 sec. The enzymatic activity of crotoxin decreased upon the addition of additional crotapotin. Phospholipase A$ alone did not show this latency. The activity of phospholipase AE, inhibited by crotapotin, was calculated from that part of the time curve where maximal hydrolysis occurred . Reproducibility was within ~ 10 ~. The inhibition of the crotales phospholipase As activity by crotapotin could be prevented by deoxycholate, using egg yolk emulsion as substrate (Fig. 8). The inhibition of phospholipase As activity by crotapotin decreased in aqueous solutions of diheptanoyllecithin containing high concentrations of Cas+ (Fig. 9A). The inhibition, however, was not influenced by Ca'+, when egg yolk emulsions were used as substrate (Fig. 9B). As shown in Fig. 4, the inhibition of phospholipase activity by crotapotin was not reversed by NaCI. Crotapotin did not inhibit the activities of other phospholipases, e.g. those from the venoms of Crotales adamanteus, Bothrops neuwiedü, bee, or porcine pancreas . TOXICON 1976 Yol. l~
IFYO .
Î. TIME COURSE OF HYDROLYSIS OF DII~EPTANOYLI~CITHIN (A) OR EGG YOLK LiPOPROTEIN3 (B) HY CROTALUS PHOSPHOLIPASB A, ALONE OR IN COMBINATION WrrH CROTAPOTIN . (A): Titration curves recorded after addition of 10 fig of phospholipase alone or together with 20 pg of crotapotin to 3 ml of 3 mM diheptanoyllecithin-100 mM NaCI-10 mM CaCI,1 mM Tris-HCI, pH 800. (B): Time course recorded after addition of 1001Ig of phospholipase alone or together with 2001Ig of crotapotin to 3 ml of egg yolk, diluted 1 :12 in O~1S M NaCI . Each curve represents one of three titration experiments showing kmoles of fatty acids liberated at 1, S, and 10 min within a standard error of f 1 ~. All titrations at 50°C and pH 800 (end point titration) .
Ng Crotapotin FiO. S. EFPEGT OF DEOXYCHOLATE ON THE ENZYMATIC ACI'IYfIY OF PHOSPHOLIPASB A, Ai.ONE OR TOGETHER WITH CROTAPO'Ißd. Twenty pg of phospholipaso A, together with 0, 20, 50 or 100 IIg of crotapotin wero added to 3 ml of egg yolk, diluted 1 :12 in 015 M NaCI . All reactions at pH 800, SO°C. The activities were related to the hydrolysis rates of phospholipase A, without inhibitor added. The reaction mixtures contained 0 and 10 mM sodium deoxycholate (S1JC), respectively. The mean values ( ") and standard deviation ranges are demonstrated graphically (n = S) . TOXICON 1976 Vol.
l~
crotapotin iNgl
crotapotin lNgl
FIG. 9. EFFECT OF CALCIUM ON THE ENZYMATIC ACIMIY OF PHO3PHOLIPASE A, ALONE OR TOGETHER wITH CROTAPOTIN t)SINO DIIiEPTANOYLLECITHIN (A) OR EGG YOLK (B) AS SiJHSIRATE . (A) : 2 P.g of phospholipase A, together with 0, 10 or 1001Ig of crotapotin were added to 3 ml
of 3 mM diheptanoyllecithin-100 mM NaCI-1 mM Tris-HCI, pH 800 . The substrates contained 10, 100, and S00 mM Ca'+, respectively. The activities were related to the hydrolysis rates of phospholipase A, without inhibitor added . The mean values (") and standard deviation ranges are demonstrated graphically (n = 4). B) : 20 pg of phospholipase A, together with 0, 10, 50 or 100 t.Ig of crotapotin were added to 3 ml of egg yolk, diluted 1 :12 in 015 M NaCI with 0, 10, 100, and 500 mM Ca'+, respectively. The enzymatic activities were related to the hydrolysis rates of the phospholipase A, without inhibitor added. Since these relative activities were not dependent on the concentrations of Ca'+ in the substrates, the mean values ( ")and standard deviation ranges are summarized in onegraphic presentation (n =12). All reactions at 50°C and pH 800. DISCUSSION
As with most other snake venom phospholipases (nE HAAS et al., 1968) the crotalus phospholipase is of the As type . By studying the velocity of breakdown of egg yolk lipoproteins and of synthetic short chain lecithins, the toxic enzyme yielded a maximal enzymatic activity of 11001tmoles per min and mg enzyme for both substrates . This is in the activity range of most other snake venom phospholipases (Vlnnl, et al., 1972). The crotalus phospholipase As fails to hydrolyze the substrate completely . Similar incomplete hydrolysis was reported for the phospholipase A from Agkistrodon piscivorus venom (AuGUS~rvx and ELLIOT, 1970). This is probably due to inhibition by the hydrolytic products. Similar to other phospholipases (BONSSx et al., 1972) the crotalus enzyme prefers substrates in the form of micelles or lipoproteins . Bee venom phospholipase A8, however, seems to hydrolyze the monomer substrate more rapidly (Si-~ot,Ixl et al., 1971). The activating effect of deoxycholate, shown for the crotalus phospholipase A$, can perhaps be explained by the requirement of phosphatide micelles which have a certain minimum zeta-potential (BANGI-IAM and DAWSON, 1959 ; DAWSON, 1963). High concentrations of NaCI or KCI, but not of saccharose, activate the crotalus phospholipase As, possibly by a change in the physicochemical state of the substrate (Boxsstr et al., 1972). $AITO and Hnx~lrnx (1962) reported a 3 times increment of the hydrolysis of ovolecithin by the Crotalus adamanteus phospholipase A when the salt concentration was increased to 0~3 M NaCI. The strict requirement for calcium is well known for many phospholipases (HABERMANN, 1957 ; Loxc and PexxY, 1957; SHIPOLINI et al., 1971 ; P1ErERSOx et al., 1974) . RoxoLT and SCHLAMOWITZ (1961) obtained with 5 mM Caß+ maximal activation of the phospholipase AQ activity of Crotalus durissus terrificus crude venom, using dihexanoyllecithin as substrate. In the present study the concentration of calcium for maximal activation of the roxrcoN r9~6 v°r. r4
Crotales Phospholipase A, and Crotoxin
23 1
pure crotales phospholipase is ten times higher, using diheptanoyllecithin as substrate. In egg yolk suspensions, maximal activation occurs at very high concentrations probably caused by adsorption to proteins . The crotales enzyme as well as phospholipases A from other sources are activated by some divalent rations. Mga+, Nia+, Coa+, and Cda+ activated Crotahtr atrox and Latlcauda semifasciata phospholipase A (Wu and Tixx>~t, 1969 ; Tu et al., 1970). The activity of the Crotalus terrif/cus enzyme increases due to exposure to Mga+ and Mna+, but, in contrast to Ca$+, these rations do not reverse the inhibition by Na,-EDTA . Calcium; moreover, abolishes the inhibition provoked by Cua+ or Baa+ . These findings support the unique role of calcium for the activity of the Crotalus terrificus phospholipase A$. Similar to this, Uz'xi: and MAGHE (1971) reported for the Crotalus adamanteus phospholipases Ae that Caa+ could not be replaced by other divalent rations. The strong inhibiting effect of phosphate buffer should be emphasized. The pH-optima of the crotales phospholipase and of crotoxin are comparable to the values, found for phospholipases from other sources (CotanxEa and DE Vxnss,1965 ; DELORI, 1973). The pH-activity curve of the crotales phospholipase A, shows an unusual shape not observed for other phospholipases . The second pH-optimum in the highly alkaline range occurs in egg yolk suspensions only, thus indicating that the egg yolk substrate may be responsible for this effect . Using aqueous solutions of the synthetic diheptanoyllecithin as substrate, the crotales phospholipase Aa shows a broad pH-activity curve with an optimum around pH 7~0-8~0. The pH-activity curve of crotoxin is similar to that of the Crotales durissus terrificus venom (ROHOLT and SCHLAMOWITZ, 1961). The crotales phospholipase Aa shows several properties classically attributed to phospholipases A. The enzyme is highly resistant to heat, acidic solvents and urea, possibly due to its large number of disulfide bridges (BxFtTxauP'r et al., 1974). In contrast to the Crotalus atrox phospholipase Aa (Wu and TINKER, 1969), the Crotalus durissus enzyme remains active upon lyophilization . However, simple contact with cellophane partially inactivates the enzyme . Reactivation occurs by calcium or by exposure to high concentration of sodium chloride . Further physical and chemical data are needed for interpretation of this phenomenon . Crotoxin, the main toxic fraction of the Crotalus durissus terr~cus venom, shows high toxicity and low phospholipase Aa activity. Crotoxin is a complex of the basic crotales phospholipase Aa and the acidic polypeptide Crotapotin (RÜssnhlerr et al., 1971). The stoichiometry of this complex varies over a wide range, but with a preference for a molar ratio of 1 :1 . Crotoxin can be considered as a toxin-potentiator complex as well as an enzymeinhibitor complex. Other naturally occurring polypeptides, also inhibiting the enzymatic action of phospholipases A, have been isolated from Naja raja (BRncnxcn et al., 1970) and Bothrops neuwiedü venom (VIDAL and STOPPAHI, 1971b) . The type and extent of inhibition, however, are not yet clarified. Moreover, these enzymes were far less toxic than the basic phospholipase Aa from Crotalus durissus terrificus venom. Crotapotin inhibits the enzymatic activity of the basic crotales phospholipase A$ to a considerable extent in all test systems used . `Natural' crotoxin, consisting mainly of 1 :1 complexes (molar ratio), possesses about 60 ~ of the enzymatic activity of the pure phospholipase Ag. Enzyme-inhibitor complexes with a molar ratio of 1 :3 show only 10 ~ of the activity of the pure enzyme . High concentrations of NaCI or KCl cannot prevent the inhibition of the enzyme by Crotapotin . Cae+, however, as well as deoxycholate antagonize the inhibiting effect of Crotapotin. T~DXICON 1976 Vol. l~
232
HENNING BREITHAUPT
Using egg yolk lipoproteins as substrate the reaction kinetics are complicated by an initial lag period, which is proportional to the inhibitor concentration used . On the other hand, this latency and the maximal velocity of hydrolysis are not influenced by the incuba tion time ofthe enzyme with crotapotin. Similar lag periods were reported for the inhibitor of Bothrops phospholipases ~VIDAL and STOPPANI, 1971b) . The polypeptide crotapotin may interact with groups which are involved in the catalytic activity of the phospholipase A8. On the other hand, its interaction with the weakly toxic enzyme provokes a strong increase of the neurotoxic and myotoxic activities . Therefore, crotapotin might become a useful tool for the study of the active centres of phospholipases A$ as well as presynaptic neurotoxins. Acknowledgements-The
author thanks Professor G. H. nE Haws for help with the studies on positional specificity and for the supply of diheptanoyllecithin. He also thanks Mrs. R. DIETRICFI for skilful technical assistance. REFERENCES
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Crotalus Phospholipase A, and Crotoxin
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Oatonz-Sw~rox, T., LwNa, J., HxFtzxwurr, H. and HwHExMwNx, E. (197 Partial amino acid sequence of tha basic crotalus phospholipase A. Toxlcon 13, 69. Ptsrsnsox, W. A., Vorwsxx, J. J. and ns Hwws, G. H. (1974) Interaction of phospholipase A, and its zymogen with divalent metal ions. Biochemistry 13, 1439. RoxoLr, O. A. and SCFII.AMOVVITZ, M. (1961) Studies of the use of díëexanoyllecithin and other lecithins as substrates for phospholipase A. With addendum on aspects of micelle properties of díhexanoyllecithin . Archs Biochem . Blophys . 94, 364. RüsswMSN, K., Basrrxwurr, H. and HABERMANN, E. (1971) Biochemistry and pharmacology of the crotoxin complex. I. Subfractionation and recombination of the crotoxin complex. Naunyn-Schmiedebergs Arch. Pharmak . 270, 274. Swrro, K. and Hwxw~wx, D. J. (1962) A study of the purification and properties of the phospholipase A of Crotolus adamanteus venom. Biochemistry 1, 521. Stirroi uvt, R. A., CwuFwwsRT, G. L., Coz-ixeu., R. C., Dooxwx, S., Vsxrrox, CH. A. and Bwr~s, B. E. C. (1971) Phospholipase A from bee venom. Eur. J. Blochem. 20, 459. SLO~rrw, K. H. and FxwsxxstrCoivnwT, H. L. (1938) Schlangengifte, III. Mitteilung : Reinigung and Krystallisation des Klapperschlangen-Giftes. Ber . dt . them. Ges.71, 1076 . Tu, A. T., Pwsssr, R. B. and Toots, P. M. (1970) Isolation and characterization of phospholipase A from sea snake, Laticauda semljasciata venom. Archs Biochem . Blophys. 140, 96 . Urns, J. F. and Mwcss, W. L. (1971) Phospholipase A, : action on purified phospholipids as affected by deoxycholate and divalent cations. Can. J. Blochem . 49, 776. Vmwt., J. C., CAITANEO, P. and STOPPAM, A. O. M. (1972) Some characteristic properties of phospholipases A, from Bothrops neuwiedil venom. Archs Blochem. Blophys. 151, 168. V~nwL, J. C. and STOPPAM, A. O. M. (1971a) Isolation and purification of two phospholipases A from Bothrops venoms. Archs Blochem . Blophys . 145, 543. VtnwL, J. C. and Srorrwrn, A. O. M. (1971b) Isolation and properties of an inhibitor of phospholipase A from Bothrops neuwledli venom. Archs Biochem . Blophys. 147, 66. Wu, T:W. and TtrrxsR, D. O. (1969) Phospholipase A, from Crotalus atrox venom. I. Purification and some properties. Biochemistry S, 1958 .
rorrco~ lsró vot. t~
