ra , voL 17, pv . s9t-s99. O Paraamon Pieu Ltd. 1979. Printed ia areat Hritain.
ao4~-otott~h iot-os9tsoz.ooro
PHOSPHOLIPASE A AND B ACTIVITTES OF REPTILE AND HYM'ENOPTERA VENOMS J>~1tsY
E. FLSfCH13R, * Wu .L~1tD B. ELL1oTr,t JecoH Is1I~Y~ and PHILIP Rossxsr~tc*
'Section of Pharmacology and Toxiwlogy, University of Connecticut, School of Pharmacy, Slams, Connecticut, U.S .A . tDepartment of Biochemistry, State University of New York at Buffalo, Buffalo, N.Y ., U.S .A. $Department of Pharmacology and Physiology, Tel-Aviv University, Raurat-Aviv, Israel (Acceptedjoypublication 13 March 1979) J. E. PkBrt~t, W. B. ELUOrr, J. ISHAY and P. RosexseRa. Phospholipase A and B activities of reptile and Hymenoptera venons. Toxtcon 17, 591-599, 1979.-Phospholipase B (PLB) activity was found in a variety of snake, Gila monster and Hymenoptt:ra venons . Hytnenoptera venons, except bee, are an especially rich source of PLB. This was shown by incubation of txude venons with lysophosphatidylcholine and subsequent titration of liberated fatty acids. PLB activity was not found in pure a- or ß-bungarotoxin, a-cobrotoxin, or one crude snake venom (Crotalus horrtdrrs horrtdus). Snake, Gila monster and bce venons exhibit higher PLB activity at an alkaline pH, while hornet venom PLB activity is greater at a neutral pH . Phospholipase A, (PLAN activity was determined using phosphatidylcholine and egg yolk as substrates . In reptile and bee venons PLA, activity is much higher than PLB activity. In contrast, hornet venom PLA, and PLB activities are nearly equal. In reph7e venons PLA, activity is optimal at neutral pH, in contrast to PLB activity, suggesting that separate proteins or active sites are responsible for PLA, and PLB activities . The effect of boiling or hosting venonns in either an acidic or alkaline milieu upon subsequent PLA, and PLB activity, measured at 37°C, was examined . The PLA, and PLB activities of all vcnoms tested were lost upon boiling at pH 9~4, except for the PLA, activity of Notechis scutatus venom which retained about 30~ of its activity . Boiling at pH 5~5 results in greatly varying extents of retention of PLA, and PLB activities, dependent upon the venom examined . Therefore, the heat stability characteristics for each venom must be expeaimentally determined, not assured. Boiling destroys PLA, and PLB activities of oriental hornet venom at about the sane rate . No conclusive results were obtained from the heating studies as to whether PLA, and PLB activities reside upon the same molecule . However, PLA, and PLB activities of oriental hornet venom wore separated using triple tandem column gel permeation chromatography, demonstrating the existence of two separate proteins for these activities . INTRODUCTION
PxosrxotiPnst3s catalyze the hydrolysis of phospholipids at specific sites (ANSELL et al., 1973), and have been extensively employed as tools to elucidate the role of phospholipids in the structure and function of biological membranes (R06BNBERG, 1976 ; ZWML et al., 1973 ; Coxnl:.an and Da VR1es, 1965). Phospholipase A$ (PLA,; EC 3.1 .1 .4) catalyzes the removal of the fatty acid from diacyl phosphoglycerides yielding the 1-monoacyl phosphatide, or lyso-compound. Although snake venons are generally believed to possess only PLA, activity (Axsat,L et al., 1973 ; COrmRE~ and DE V1tIHS, 1965) there have been reports of phospholipase B (PLB) activity in some snake venoms (DoE1tY and P>3nltsox, 1964 ; Mox~a~ et al., 1969 ; Sxrt.onx et a1., 1973a, 1973b; ELLIOTT, 1978). PLB (EC 3.1 .1 .5) catalyzes the removal of the remaining fatty acid from monoacyl phosphoglycerides. The PLB activity detected in crude snake venoms is consistently lower than the PLA, activity in the same venom (DOSRY and Ps~xsox, 1964; Mox~~n et al., 1969). PLB activity in 591
592
J. E. FLETCHER, W. B. ELLIOTT, J. iSHAY and P. ROSENBERG
several of the venoms examined was manifested only at a pH greater than 8; that is, above the pH range normally employed in PLA$ assays . This is in contrast to other animal and plant sources of PLB which exhibit optimal activity in an acidic milieu (KATFS, 1960). Of special interest is the finding of SHILOAH et al. (1973a, 1973b) that PLB activity can possibly reside upon the same molecule possessing PLAs activity, however, the specific activity of PLB was considerably less than that of PLA $. This study was undertaken to test for the existence of PLB activity across a wide variety of snake venoms, as well as to determine if this activity resides upon the same molecule as does the PLA $ activity. The recent discovery of a rich source of PLB in the oriental hornet (Vespa orientalis) (ROSENBERG et al., 1977) led us to also investigate venoms of other Hymenoptera as possible sources of PLB activity. MATERIALS AND METHODS Part A Lysophosphatidylcholine was obtained from Pierce Chemical Co. (Illinois, U.S .A.) and Sigma Chemical Co . (Missouri, U.S.A .). Phosphatidylcholine was obtained from Sigma Chemical Co . (Missouri, U.S.A .) . Both substrates were > 98 ~ pure as measured by phosphorus analysis after thin-layer chromatographic separation . Acanthophis antarcticus venom was purchased from L. Light & Co. Ltd. (Colnbrook, England) . NaJa raja, VVpera palaestinae, Vipers russelll, Ophiophagus haunah, Crotalus adamartteus, Agklstrodon piscivorus, Crotalus atrox, Bothrops atrox and Agkistrodon contortrix mokeson venoms were purchased from Ross Allen's Reptile Institute (Florida, U.S.A .). Heloderma suspectum and Heloderma horrldum venoms and pure a-bungarotoxin, ß-bungarotoxin and a~obrotoxin were purchased from Miami Serpentarium (Florida, U.S .A .) . Hemachatus haemachatus venom was purchased from Pierce Chemical Co . (Illinois, U.S .A.) . Crotalus horridus horridus and Vespa orientalis venoms were respectively gifts of D. C. R. Hackenbrock (Department of Anatomy, University of North Carolina School of Medicine) and one of the co-authors (J . L) . Vespulo arenaria, Vespa maculata and Apis mellihca venoms were provided by one of the co-authors (W .B .E.) . Notechis scutatus, Burrgarus caeruleus and Dendroaspis polyltpis venoms were obtained from both L. Light & Co., Ltd. (Colnbrook, England) and Miami Serpentarium (Florida, U.S .A .) . Results were similar using venom from either source. PLA, and PLB activities were measured by titration of free fatty acids using the method of Dow (1956). Tris buffer (0~1 M) containing 0~5 mM CaCI, and either 3 mg lysophosphatidylcholine or 4~5 mg phosphatidykholine, adjusted to the appropriate pH, was incubated with appropriate concentrations of the venom in a final volume of 1 ml at 37 °C for 20 min. The reaction was terminated by the addition of extraction mixture. Blanks without enzyme or without substrate were run in each assay and were in close agreement with zero time samples which were also routinely run. Tris has been shown to be an adequate buffer under these conditions (RassrrHEtec et al., 1977). When the venoms were boiled for 15 min at either pH 9~4 or 5~5 they were in 0~1 M Tris buffer containing 0~5 mM CaCI,. The enzymatic assay was then run at pH 7.4 and 9~4 as described above. Homogenized Vespa orientalis venom sacs were boiled for either one or three min rather than the 15 min used with the other venoms . in all experiments using phosphatidylcholine as the substrate, sonication preceded incubation with the venom . Protein determination for Vespa orientalis venom sacs was performed by the method of Lowav tt al. (1951) using bovine serum albumin as the standard . Part B Lysophosphatidylcholine was obtained from Applied Science Laboratories (Pennsylvania, U.S .A.). The crude venoms of Apis mellihca, Vespa maculata, Vespa vulgaris, Vespulo macullfrons and Vtspula arenaria were provided by one of the co-authors (W .B .E.) and Vesper orientalis by another co-author (J .L). PLA, and PLB activities were measured by titration of free fatty acids using the method of DE Hays et al. (1968) as described by None et al. (1976). The substrate, either 5 ml of egg yolk homogenatt (1 yolk homogenized in 200 ml with distilled water) or 3 mg egg lysophosphatidylcholine, is incubated with crude venom (having an activity of approximately 0~3751unole/min) in a fina18 or 10 ml volume containing 1~3 x 10-' M Na desoxycholate, 0~5 ~ bovine serum albumin and 3 mM CaCI,. Enzyme activity at 40°C was followed by automatic titration of free fatty acids at pH 8~0 with 001 N NaOH using a Radiometer (Copenhagen) pH slat model TTl l/SRB2/ABU12/TTA31 . In the heating experiments, V. vulgaris and V. maculata venoms (2~8 mg/ml) were heated at pH 6~2 and 5~8 respectively for 30 min. After heating, the PLA, and PLB activities of aliquots (5-25 ul) of the heated samples were determined at 40 °C and pH 8 using the procedure described above. The PLA, and PLB activities of V. orientalis venom sacs were separated using three columns in tandem . P100 (100-200 mesh ; 88 x 2~5 cm), P10 (91 x 2~5 cm) and G25 (95 x 2~5 ctn) columns were used in the descending, ascending and descending modes, respectively . The eluant was ammonium formate (0~1 M) at
Phospholipases A and B
593
pH 4~5. V. orientalls venom saa (1 "25 g) were suspended in 100 ml ammonium formate (0~1 M), adjusted to pH 4"5, and centrifuged at 10,000 rev/min in a Sorvall centrifuge. The supernatant (1 g) was applied to the column . The flow rate was 9 ml/hr (approximately 5 ml/tube). A total of 500 fractions were collected yielding 748 mg total recovery . Protein was measured at both 280 and 230 nm in a Hitachi-Perkin Elmer 139 UV-Vas spectrophotometer, using distilled water as a blank. PLA, and PLB activities of aliquots (1-10 ul) of the fractions were determined at 40°C and pH 8 using the procedure described above. RESULTS
Part A Table 1 shows for various snake, Gila monster and Hymenoptera venoms, as well as purified toxins, their PLA $ and PLB activities using respectively purified phosphatidylcholine and lysophosphatidylcholine as substrates . The analysis was run both at pH 7"4 and 9"4, since maximal enzymatic activity may either be at neutral or alkaline pH (see TABLE 1. PHOSPHOLiPASE
A, (PLAN AND PHOSPHOLIPASE B (PLB) ACIIViTn~ OF
Venom Hymenoptera Apls melltfica Yespamaculata Yespaorientalis'(venomsae) Vespulaarenaria Snake ~lcanthophisantarcticus Agkistrodon contortrix mokeson Agkistrodon pisclvorus Bitas arietans Bothrops atrox Bu~garuscaeruleus Crotalesadamanteus Crotales atrox Crotales horridus horridus Dendroaspispolylepis Hemachatushaemachatus NaJa naja Notechlsscutatus Ophiophagushannah Yiperapalaestinae t?pera russelli Gila monster Helodermahorridum Heloderma suspectum Toxins a-bungarotoxin§ ß-bungarotoxin§ a-cobrotoxin II
PLA, ~-Equiv FFA pH 7"4 pH 9"4 1 "73 f0 "08 2"88f0"04 5"35f0 "19 1"74f0~14
(2) (2) (4) (2)
-
VARIOUS VENOMS AND TOXIIJS
PLB u-Equiv FFA pH 9"4 pH 7"4
1 " 71 f0~04 (2) 3"48f0"13 (4) -
0~14f0~02 4"28f0~41 4"43f0" 11 4~32f0~23
(2) (2)$ (4) (2)t
0"76 (1) 4"06~0 "Ol$ (2) 2"75f0~07 (4) 3"87f0 "04 (2)t
0"65f0" 11 (2)
123f0"10 (2)
0 (2) 0 (2) 0 (2) 0 (2) 0"97f0"03 0" lOfO"00 0~11 f0~06 0 (2) 0"39~0"OS 0~33f0"OS 0~33 f0~09 2"83f0~14 0"24~0 "00 0~26 0~23 f0"00
020f0"06 0~51 f0~10 0"32~0~05 0~25 f0~02 1"49~0 "04 0"54f0~13 0~25 f0"02 0 (2) 1~37f0~02 1 "13f0~03 0"91 f0 "00 2"67f0"08 0~54f0~01 0"79f0 "Ol 0~72 f0~01
(2) (2) (2) (2) (3) (2) (2)
2~33f0"08 (2) 2"13f0"03 (2) 2~32f0~14 (4) 2~20~0"03 (2) 3"08f0"07 (4) 1"83~0"08 (2) -
2~03f0"Ol 2"18f0" 10 1~73f0~02 1"62 f0"04 2"19~0"OS 1"13f0"04 -
2"13f0"09 (2) -
1"56f0"OS (2) -
0"28f0~03 (2) 0 (2)
0"56f0 "OS (2) 0~57 f0 "02 (2)
-
-
0 (2) 0 (1) 0 (2)
0 (2) 0 (2) 0 (2)
(2) (2) (4) (2) (4) (2)
(3) (2) (2) (2) (4) (2) (3) (2) (1) (2)
(2) (4) (2) (3) (2) (2) (2)
'0'02 mg protein. t0~5 mg crude venom. $1 "0 mg crude venom ß0"2 mg bungarotoxin . Ilo-S mg cobrotoxin . One ml solutions of substrate [either 4"5 mg phosphatidylcholine or 3~0 mg lysophosphatidykholine (approximately 6 lunoles of each)] were incubated with venom or toxin at 37°C for 20 min at either pH 7~4 or pH 9"4. Number of samples is given in parentheses. Values are recorded as means < S.E., with values below 0"09 k~quiv free fatty acids (FFA) being recorded as zero (limit of reliability for method used) . Except as noted in the Table 0"5 mg and 3"0 mg of crude venoms were used respectively in the PLA, and the PLB assays.
594
J. E. FLETCHER, W, B. ELLIOTT, J. ISHAY and P. ROSENBERG
~. O
ô~9â \'s S
ô.
Q
000
M
O
~~O
~
O
000
h
O
M~O
~
O~
.. vv
., v
_~ -{-I +I O
-}{
r,
,. O~
~ et .~r, N__d' O OO
. 0
â
ô.
°x
vvv 000
O
N_ O
-}{
u
H N
_N O
ô
N
~d N w
~
aO_~ H oH ô al
N
b
Ô irO
M
G
00
â ô . 0
O
a~~o
â~x a
Ô~ ~
G
~-I h ~
.. v
~
n ô
.°.° b
W i
v
~
O
N
0
v
v
~s .. OOv
s O
bd
ô
-i i -~ O
. 0
v o
oH
° û
v a
.. h qy ~
~~
0~~0
a .,
_ N v N O
v ~ N
n b
vvv
û
v
bbb -Ii +I 'i'i
O 'i i
O -Ii
s~
d
$~ô
N~b
h
..
.., h
â°~,~,
~~ a~
N
s
N F"
c â~ ô
b T .~-~
e~1
~ â eO n .~r,ô
O
h
Ô
N
~ ~oli°HpH
,. N,
~
N
vT
vvv
h ô
vv
N v
'r .-~
Ôv~ Ô -H o +I ~
o, ô.
° x
M
~~w, ODa ~~~â
a, ~° > ,~
W n
ä
v
~w
~
,.,
ag
w.
O . .+ ,> ~+ V ~ ~A .~ O ~r
ô
â o eo >'w° ~ ~~~~o~ ~â
b
w ~0 .
.9 â ô ,n ô w °° p+ n x
â .~
~g~~~> °' ~ J3 ~ .~
bh p~ a~NÔ~ a~ Pr"w~ ~~"~ ° Q °' a a . ., '~ ô8v ~ eo°oaô~
~ ~C ~ M~ y ~ ~ V ~ M ~~ô'~âô y ~ g ~ vv ~ $'~ vvl~v w c~BM~°~ * o ,, o a
a o(~ 0 ~~r > M
1?hospholipases A and B
595
Discussion). Also, carrying out the experiments at two pH values may provide an indication as to whether a single enzyme is responsible for both PLA, and PLB activities or if two separate enzymes exist. As can be seen in Table 1, PLA, activity was usually highest at pH 7~4 while PLB activity was usually highest at pH 9~4, except with the hornet venoms. This difference, between PLA, and PLB, in optimal pH suggests that two separate protein molecules may be responsible for the two enzymatic activities . In addition, there is no consistent relationship between the relative PLA, and PLB activities ofthe different venoms. For example, at pH 7~4 H. haemachatus has about 75 ~ of the PLA, activity of N. scutatus ; however, the PLB activity of H. haemachatus is only about 15 ~ that ofN. scutatus. Furthermore, H. horridum has about the same PLA, activity as N. naja at pH 9~4, yet the PLB activity is only 62 ~ of that demonstrated by N. naja venom. These results show that in addition to PLA, activity, many reptile and Hymenoptera venoms exhibit PLB activity. Although not all of the venoms in Table 1 were tested for PLA, activity, all are known from previous results to have PLA, activity. a-Bungarotoxin and a-cobrotoxin have no PLA, activity while ß-bungarotoxin under certain experimental conditions has PLA, activity ('I~r, 1977). Notice that a- and ß-bungarotoxin and a-cobrotoxin are devoid of PLB activity as is one of the crude snake venoms, Crotalus h. horridus, which interestingly is the one snake venom reported to have little or no PLA, activity (KocxolaTx et al., 1971). In Table 2, the effect of boiling the venoms, at pH 9~4 or 5~5, upon subsequent PLA, and PLB activities at pH 7~4 or 9~4 was examined . With the exception of N. scutatus which retained about 30 ~ of its PLA, activity, boiling at pH 9~4 destroyed almost all PLA, and PLB activities . Boiling at pH 5~5, however, resulted in varying degrees of PLA, and PLB activities being retained, with the loss of one not always associated with the loss of the other. The percent loss of PLA, and PLB activities in a single venom were in several cases dissimilar, suggesting two separate proteins . Boiling V. orientalis for 15 min abolished all activity at either pH (results not shown). Part B
Using somewhat different methods than in Part A, the PLA, and PLB activities of several Hymenoptera venoms were determined (Table 3). Bee venom (A . mellifrca) and mixed Bombes sp. venom sac extract had no PLB activity, but high PLA, activities, whereas all of the hornet venoms had both PLA, and PLB activities . TaHr.E
3.
Paosrxocrnesa A, (PLA ~Nn rxosrsoLiFese OF 9EVERAL HYMBNOFTERA VENOMs
Venom Apis mellifrca Bambus sp . venom sac extract Ytspa maculata Vespa vuigaris Ytspuia maculj/rons
PLA, 559 50 25 125
B (PLB) ~crmrrns PLB 0 0 62 19 18
Activity determined at pH 8 by automatic titration of fatty acid release from either egg yolk (PLAN or lysophatidylcholine (PLH). Results expressed as pM/min/mg .
The effect of heating upon the PLA, and PLB activities of two Hymenoptera venoms using egg yolk and purified lysophosphatidylcholine respectively as substrates is shown in Fig. 1 . The two activities decrease with increasing temperature at a similar rate for both venoms . These results could be explained by either a single molecule having both enzymatic activities or two separate proteins with PLA, and PLB activities respectively having the same heat stability characteristics.
59 6
J. E. FLETCHER, W. B. ELLIOTT, J. ISHAY and P. ROSENBERG so
a c É i
t 8
u a c n ä r!S 30
10
Temperature,
~C
FIG. 1. EFFECT OF HEATING UPON THE PHOSPHOLIPASE A, (PLAN AND PH03PHOLIPASE B (PLB) ACTIVTIIE4 OF V. völgarls AND V maCGlatO HORNET VENOM3.
Following 30 min heating at pH 6~2 (V. vulgaris) or 5~8 (V. maculata), enzymatic activity was measured at 40 °C and pH 8 by automatic titration of free fatty acids. Egg yolk (PLAN or lysophosphatidylcholine (PLB) were used as substrates : f- " PLA V. vuJgarts ; ~-~ PLH, V. vulgaris ; !:: O PLA,, V. maculata ; p-p PLB, V. maculata.
The PLB activities of yellow hornet (Y. arenaria) and baldface hornet (V. maculata) were determined at pH 7~4 and 9~2. The activities were 5-10 ~ greater at pH 7~4 than 9~2, in excellent agreement with results shown in Table 1. In Fig . 2 the PLA a and PLB activities of V. orientalis venom sacs are shown to be associated with different fractions collected on a tandem column . Therefore, it appears that separate proteins are responsible for the PLA s and PLB activities of the oriental hornet venom, even though these two proteins have similar heat stability characteristics (Table 2). DISCUSSION
We have found PLB activity in a variety of snake, Gila monster and Hymenoptera venoms. The richest source of PLB in snake venom (N. scutatus) was much less active than any of the Hymenoptera venoms with the exception of bee and mixed Bombes sp. venoms, which had little or no activity depending upon method used. The use of relatively large amounts of venom for many of our studies was necessary in order to detect the relatively low levels of PLB activity. Greater PLA Q activity was detected using one-sixth the amount of venom used in the PLB assay ; therefore, at least in the reptiles tested, the discovery of PLB activity is more of phylogenetic interest than for use as a potential source of the
Phospholipases A and B
597
c ô â
Fraction FiO . 2. SEPARATION OP FHOSPHOLIPASE A~ (PLAN AND PH03PHOLn'ASE B (PLB) ACrIVITIE4 OF V Or~ClJtaUs VENOM SAC EXTRACT. Y. orientalts venom sacs (1 ~25 g) were suspended and applied to a tandem column . Protein eluted was followed by absorption at 230 nm and 280 nm . Enzymatic activity was determined at pH 8 by automatic titration of fatty acid release from either egg yollc (PLAN or lysophosphatidalcholine (PLB) .
enzyme. In contrast, the hornets V. orientales, V. arenaria, Y. rnaculata and V. vulgaris are rich sources of PLB. The purity of the substrates was verified by thin layer chromatography to ensure that tha activity observed was due to PLA$ and PLB activities . It might be argued that some of the activity recorded as being due to PLAs is actually due to PLB activity since the product of PLA$ action (lysophospholipid) is the substrate for the PLB enzyme . This is not likely to be a significant source of error in the case of snakes, Gila monster or bee venoms, since with the low amounts of venoms used for the PLAs assay, little or no PLB activity would be observed. In order to assay PLB activity in these venoms, it was necessary to use 3~0 mg ofvenom rather than 0"5 mg used in the PLA, assay. However, for the hornet venoms, which have much greater PLB activity, a significant portion (but not all) of the activity listed as being due to PLAs may actually represent PLB activity. Since PLB activity in snakes is maximal in the range ofpH 8 "5-10"0 (DoStY and PBAxsox, 1964) and PLAß activity is optimal around pH 6"5-8~0 (CormxBA and DeVx~s, 1965), the crude venoms were incubated with substrate at both pH 7~4 and pH 9~4. Our experiments confirmed the earlier reports showing somewhat higher snake venom PLAs activity at pH 7"4 and slightly higher PLB activity at pH 9"4. In contrast, the hornet venoms show higher PLB activity at pH 7"4. There was no direct proportional relationship between PLA s and PLB activities in the various venoms.
59 8
J . E. FLETCHER, W. B . ELLIOTT, J . ISHAY and P . ROSENBERG
Several of the snake venom PLB values are not in agreement with the results of Do>r1tY and Plinxsoty (1964) . Most notably, higher relative activity was observed for N. scutatus venom and lower activity was observed for A. piscivorus venom. However, variations of enzyme activity within species as well as within individual snakes have been reported ($ARKBR and Dl:vl, 1968). Differences in experimental conditions could also explain differences between this and other reports. The heating experiments do not clearly indicate whether the PLAQ and PLB activities are associated with separate proteins or the same protein. With N. scutatus venom a clear difference in the heat sensitivity of the two enzymatic activities was observed . This suggests separate proteins as being responsible for the two enzymatic activities. A less likely possibility would be two active sites on a single protein. In contrast the PLA$ and PLB activities of several other venoms were affected similarly by heat . PLA$ activity is thought to be stable to boiling in an acidic medium and destroyed by boiling at an alkaline pH (COND1tEA and D>äVx»rs, 1965). In our experiments, however, the PLA$ activity was not always stable to boiling at pH 5~5 or to heating above 75°C for 30 min, at pH 5'8 or 6~2. D. polylepis and V. orientalis venoms lost greater than 80 ~ of their PLAp activities when boiled for 15 and 3 min, respectively, at pH 5~5. In contrast, B. caeruleus venom retained 95 ~ of its PLA$ activity after boiling at pH 5~5 for 15 min. The heat stability of PLAs at acidic pH cannot therefore be assumed, but must be determined for each venom. While the PLAP activity of several venoms was destroyed by boiling at pH 9~4, it is interesting that under these conditions N. scutatus venom retains about 30 ~ of its activity. In addition to varying stabilities of PLA$ to heating or boiling. PLB activity also demonstrated varying degrees of stability. PLB from Pseudechis porphyriacus venom was previously shown to be relatively stable to boiling for 2 hr at pH 7, retaining 21 ~ of its activity (D01?RY' and PSAxsoN, 1964). In our experiments the PLB activities were abolished in all venoms tested after 15 min of boiling at pH 9~4. After boiling or heating (80°C) at an acidic pH the venoms exhibited varying retention of activity, from greater than 80 ~ (B. caeruleus venom) to 0 ~ (D . polylepis and hornet venoms). We have found that in Y. orientalis venom the PLAs and PLB activities reside upon different molecules as determined by column separation and subsequent analysis of enzyme activity . The next logical step is to purify the enzymes of other Hymenoptera and snake venoms and conduct further analysis of the PLA, and PLB activities to determine conclusively whether these activities reside on the same protein. The results of this paper indicate that specific phospholipases from different venoms vary considerably in their properties, despite a similar catalytic site of action. Therefore, as mentioned by SHILOAH et al. (1973b), the classification of an enzyme as a PLA% may be far to restrictive in defining its properties . Acknowledgements-The authors acknowledge the skilled technical assistance of Mr . RoHexT Sam . This research was supported in part by grants from the University of Connecticut Research Foundation (P .R .) and the National Institutes of Health, NINCDS 1IR01 NS 1478801 (W.B .E .)] . REFERENCES i, G . B ., Hwwrxoxxe, J. N . and D~wsox, R . M . C. (Eds .) (1973) Form and Function ofPhospholipids AxsFi BBA Library, Vol . 3, second edn ., New York : Elsevier. Cormxen, E . and DEVt~s, A. (1965) Venom phospholipase A : a review. Toxlcon 2, 261 . DE Havas, G . H ., POSTEMA, N . M ., NIEUWENHUIZEN, W . and vnx DeElvex, L . L. M . (1968) Purification and properties of phospholipase A from porcine pancreas . Biochim. biophys. Acta 159, 103.
Phospholipases A and B
59 9
Do~eY, H. M. and Peaesox, J. E. (1964) Phospholipase B in snake venoms and bee venom. Biochem. J. 92, 599. Dow, V. P. (1956) A relation between non~sterified fatty acid in plasma and the metabolism of glucose. J. clln. Invest. 3S, 150. Eutoz-r, W. B. (1978) The chemistry and immunology of reptilian venom. In : Biology of the Reptilia, Vol. 8B, p. 163, (Gnxs, C. and Gnxs, R. A., Eds.). London : Academic Press. Kn~s, M. (1960) Lipolytic enzymes. In : LipidMetabolism, p. 165, (BLOCii, K., Ed .). New York : Wiley. Kocxou.~n, W. F., LEDFORD, E. B., Dnr,v, J. G. and BILLINGS, T. A. (1971) Toxicity and some enzymatic properties and activities in the venoms of Crotalidae, Elapidae and Viperidae. Toxicon 9, 131 . Lowxv, O. H., RoseHxoucx, N. J., Fnltx, O. L. and RnrrnnL.r.., R. J. (1951) Protein measurement with the Folin phenol reagent . J. biol. Chem . 193, 265. MOIiaMEn, A. H., Ka~L, A. and AYOBB, M. H. (1969) Studies of Phospholipase A and B activities of Egyptian snake venoms and a scorpion toxin. Toxin 6, 293. Nf+ue, B. C., NaIIe, C., DENNE, S., WYPYCH, J., AIeHesnfnx, C. E. and ELLIOrr, W. B. (1976) Immunologic comparison of phospholipases A present in Hymenoptera insect venoms. J. Allergy clip . Imn:un. 58, 101. RosExsexa, P. (1976) Bacterial and snake venom phospholipases : enzymatic proves in the study of structure and function in bicelectrically excitable tissues. ln : Animal, Plant acrd Microbial Tozins, Vol. 2, p. 229, (Oxsaxn, A., Ed.). New York : Plenum . RosexHSxG, P., IsIInY, J. and GrrrER, S. (1977) Phospholipases A and B activities of the oriental hornet (Vespa orientalis) venom and venom apparatus. Toxicon 15, 141 . Snlex.a,a, N. K. and D$vI, A. (1968) Enzymes in snake venoms. In : Venomous Animals and their Venoms, Vol. 1, p. 167, (BUCHERL, W., BucICLev, E. E. and DouLOFeu, V., Eds.). New York : Academic Press. SxILOnII, J., Kt>etxsxv, C., DsVxiFS, A. and B~GeR, A. (1973a) Phospholipase B activity of a purified Phospholipase from Vipera palaestinae venom. J. Lipid Res. 14, 267. Sxrtoax, J., KLIHANSKY, C. and DEVxIFS, A. (1973b) Phospholipase iscenzymes from Naja naja venom. II Phospholipase A and B activities . Toxicon 11, 491. Tu, A. (1977) Venoms : Chemistry and Molecular Biology. New York : Wiley. ZwaaL, R F. A., RoeLOrsex, B. and Cou.sY, C. M. (1973) Localization of red cell membrane constituents . Biochim, biophys. Acta 300, 159.
ao4~-otott~h iot-os9tsoz.ooro
PHOSPHOLIPASE A AND B ACTIVITTES OF REPTILE AND HYM'ENOPTERA VENOMS J>~1tsY
E. FLSfCH13R, * Wu .L~1tD B. ELL1oTr,t JecoH Is1I~Y~ and PHILIP Rossxsr~tc*
'Section of Pharmacology and Toxiwlogy, University of Connecticut, School of Pharmacy, Slams, Connecticut, U.S .A . tDepartment of Biochemistry, State University of New York at Buffalo, Buffalo, N.Y ., U.S .A. $Department of Pharmacology and Physiology, Tel-Aviv University, Raurat-Aviv, Israel (Acceptedjoypublication 13 March 1979) J. E. PkBrt~t, W. B. ELUOrr, J. ISHAY and P. RosexseRa. Phospholipase A and B activities of reptile and Hymenoptera venons. Toxtcon 17, 591-599, 1979.-Phospholipase B (PLB) activity was found in a variety of snake, Gila monster and Hymenoptt:ra venons . Hytnenoptera venons, except bee, are an especially rich source of PLB. This was shown by incubation of txude venons with lysophosphatidylcholine and subsequent titration of liberated fatty acids. PLB activity was not found in pure a- or ß-bungarotoxin, a-cobrotoxin, or one crude snake venom (Crotalus horrtdrrs horrtdus). Snake, Gila monster and bce venons exhibit higher PLB activity at an alkaline pH, while hornet venom PLB activity is greater at a neutral pH . Phospholipase A, (PLAN activity was determined using phosphatidylcholine and egg yolk as substrates . In reptile and bee venons PLA, activity is much higher than PLB activity. In contrast, hornet venom PLA, and PLB activities are nearly equal. In reph7e venons PLA, activity is optimal at neutral pH, in contrast to PLB activity, suggesting that separate proteins or active sites are responsible for PLA, and PLB activities . The effect of boiling or hosting venonns in either an acidic or alkaline milieu upon subsequent PLA, and PLB activity, measured at 37°C, was examined . The PLA, and PLB activities of all vcnoms tested were lost upon boiling at pH 9~4, except for the PLA, activity of Notechis scutatus venom which retained about 30~ of its activity . Boiling at pH 5~5 results in greatly varying extents of retention of PLA, and PLB activities, dependent upon the venom examined . Therefore, the heat stability characteristics for each venom must be expeaimentally determined, not assured. Boiling destroys PLA, and PLB activities of oriental hornet venom at about the sane rate . No conclusive results were obtained from the heating studies as to whether PLA, and PLB activities reside upon the same molecule . However, PLA, and PLB activities of oriental hornet venom wore separated using triple tandem column gel permeation chromatography, demonstrating the existence of two separate proteins for these activities . INTRODUCTION
PxosrxotiPnst3s catalyze the hydrolysis of phospholipids at specific sites (ANSELL et al., 1973), and have been extensively employed as tools to elucidate the role of phospholipids in the structure and function of biological membranes (R06BNBERG, 1976 ; ZWML et al., 1973 ; Coxnl:.an and Da VR1es, 1965). Phospholipase A$ (PLA,; EC 3.1 .1 .4) catalyzes the removal of the fatty acid from diacyl phosphoglycerides yielding the 1-monoacyl phosphatide, or lyso-compound. Although snake venons are generally believed to possess only PLA, activity (Axsat,L et al., 1973 ; COrmRE~ and DE V1tIHS, 1965) there have been reports of phospholipase B (PLB) activity in some snake venoms (DoE1tY and P>3nltsox, 1964 ; Mox~a~ et al., 1969 ; Sxrt.onx et a1., 1973a, 1973b; ELLIOTT, 1978). PLB (EC 3.1 .1 .5) catalyzes the removal of the remaining fatty acid from monoacyl phosphoglycerides. The PLB activity detected in crude snake venoms is consistently lower than the PLA, activity in the same venom (DOSRY and Ps~xsox, 1964; Mox~~n et al., 1969). PLB activity in 591
592
J. E. FLETCHER, W. B. ELLIOTT, J. iSHAY and P. ROSENBERG
several of the venoms examined was manifested only at a pH greater than 8; that is, above the pH range normally employed in PLA$ assays . This is in contrast to other animal and plant sources of PLB which exhibit optimal activity in an acidic milieu (KATFS, 1960). Of special interest is the finding of SHILOAH et al. (1973a, 1973b) that PLB activity can possibly reside upon the same molecule possessing PLAs activity, however, the specific activity of PLB was considerably less than that of PLA $. This study was undertaken to test for the existence of PLB activity across a wide variety of snake venoms, as well as to determine if this activity resides upon the same molecule as does the PLA $ activity. The recent discovery of a rich source of PLB in the oriental hornet (Vespa orientalis) (ROSENBERG et al., 1977) led us to also investigate venoms of other Hymenoptera as possible sources of PLB activity. MATERIALS AND METHODS Part A Lysophosphatidylcholine was obtained from Pierce Chemical Co. (Illinois, U.S .A.) and Sigma Chemical Co . (Missouri, U.S.A .). Phosphatidylcholine was obtained from Sigma Chemical Co . (Missouri, U.S.A .) . Both substrates were > 98 ~ pure as measured by phosphorus analysis after thin-layer chromatographic separation . Acanthophis antarcticus venom was purchased from L. Light & Co. Ltd. (Colnbrook, England) . NaJa raja, VVpera palaestinae, Vipers russelll, Ophiophagus haunah, Crotalus adamartteus, Agklstrodon piscivorus, Crotalus atrox, Bothrops atrox and Agkistrodon contortrix mokeson venoms were purchased from Ross Allen's Reptile Institute (Florida, U.S.A .). Heloderma suspectum and Heloderma horrldum venoms and pure a-bungarotoxin, ß-bungarotoxin and a~obrotoxin were purchased from Miami Serpentarium (Florida, U.S .A .) . Hemachatus haemachatus venom was purchased from Pierce Chemical Co . (Illinois, U.S .A.) . Crotalus horridus horridus and Vespa orientalis venoms were respectively gifts of D. C. R. Hackenbrock (Department of Anatomy, University of North Carolina School of Medicine) and one of the co-authors (J . L) . Vespulo arenaria, Vespa maculata and Apis mellihca venoms were provided by one of the co-authors (W .B .E.) . Notechis scutatus, Burrgarus caeruleus and Dendroaspis polyltpis venoms were obtained from both L. Light & Co., Ltd. (Colnbrook, England) and Miami Serpentarium (Florida, U.S .A .) . Results were similar using venom from either source. PLA, and PLB activities were measured by titration of free fatty acids using the method of Dow (1956). Tris buffer (0~1 M) containing 0~5 mM CaCI, and either 3 mg lysophosphatidylcholine or 4~5 mg phosphatidykholine, adjusted to the appropriate pH, was incubated with appropriate concentrations of the venom in a final volume of 1 ml at 37 °C for 20 min. The reaction was terminated by the addition of extraction mixture. Blanks without enzyme or without substrate were run in each assay and were in close agreement with zero time samples which were also routinely run. Tris has been shown to be an adequate buffer under these conditions (RassrrHEtec et al., 1977). When the venoms were boiled for 15 min at either pH 9~4 or 5~5 they were in 0~1 M Tris buffer containing 0~5 mM CaCI,. The enzymatic assay was then run at pH 7.4 and 9~4 as described above. Homogenized Vespa orientalis venom sacs were boiled for either one or three min rather than the 15 min used with the other venoms . in all experiments using phosphatidylcholine as the substrate, sonication preceded incubation with the venom . Protein determination for Vespa orientalis venom sacs was performed by the method of Lowav tt al. (1951) using bovine serum albumin as the standard . Part B Lysophosphatidylcholine was obtained from Applied Science Laboratories (Pennsylvania, U.S .A.). The crude venoms of Apis mellihca, Vespa maculata, Vespa vulgaris, Vespulo macullfrons and Vtspula arenaria were provided by one of the co-authors (W .B .E.) and Vesper orientalis by another co-author (J .L). PLA, and PLB activities were measured by titration of free fatty acids using the method of DE Hays et al. (1968) as described by None et al. (1976). The substrate, either 5 ml of egg yolk homogenatt (1 yolk homogenized in 200 ml with distilled water) or 3 mg egg lysophosphatidylcholine, is incubated with crude venom (having an activity of approximately 0~3751unole/min) in a fina18 or 10 ml volume containing 1~3 x 10-' M Na desoxycholate, 0~5 ~ bovine serum albumin and 3 mM CaCI,. Enzyme activity at 40°C was followed by automatic titration of free fatty acids at pH 8~0 with 001 N NaOH using a Radiometer (Copenhagen) pH slat model TTl l/SRB2/ABU12/TTA31 . In the heating experiments, V. vulgaris and V. maculata venoms (2~8 mg/ml) were heated at pH 6~2 and 5~8 respectively for 30 min. After heating, the PLA, and PLB activities of aliquots (5-25 ul) of the heated samples were determined at 40 °C and pH 8 using the procedure described above. The PLA, and PLB activities of V. orientalis venom sacs were separated using three columns in tandem . P100 (100-200 mesh ; 88 x 2~5 cm), P10 (91 x 2~5 cm) and G25 (95 x 2~5 ctn) columns were used in the descending, ascending and descending modes, respectively . The eluant was ammonium formate (0~1 M) at
Phospholipases A and B
593
pH 4~5. V. orientalls venom saa (1 "25 g) were suspended in 100 ml ammonium formate (0~1 M), adjusted to pH 4"5, and centrifuged at 10,000 rev/min in a Sorvall centrifuge. The supernatant (1 g) was applied to the column . The flow rate was 9 ml/hr (approximately 5 ml/tube). A total of 500 fractions were collected yielding 748 mg total recovery . Protein was measured at both 280 and 230 nm in a Hitachi-Perkin Elmer 139 UV-Vas spectrophotometer, using distilled water as a blank. PLA, and PLB activities of aliquots (1-10 ul) of the fractions were determined at 40°C and pH 8 using the procedure described above. RESULTS
Part A Table 1 shows for various snake, Gila monster and Hymenoptera venoms, as well as purified toxins, their PLA $ and PLB activities using respectively purified phosphatidylcholine and lysophosphatidylcholine as substrates . The analysis was run both at pH 7"4 and 9"4, since maximal enzymatic activity may either be at neutral or alkaline pH (see TABLE 1. PHOSPHOLiPASE
A, (PLAN AND PHOSPHOLIPASE B (PLB) ACIIViTn~ OF
Venom Hymenoptera Apls melltfica Yespamaculata Yespaorientalis'(venomsae) Vespulaarenaria Snake ~lcanthophisantarcticus Agkistrodon contortrix mokeson Agkistrodon pisclvorus Bitas arietans Bothrops atrox Bu~garuscaeruleus Crotalesadamanteus Crotales atrox Crotales horridus horridus Dendroaspispolylepis Hemachatushaemachatus NaJa naja Notechlsscutatus Ophiophagushannah Yiperapalaestinae t?pera russelli Gila monster Helodermahorridum Heloderma suspectum Toxins a-bungarotoxin§ ß-bungarotoxin§ a-cobrotoxin II
PLA, ~-Equiv FFA pH 7"4 pH 9"4 1 "73 f0 "08 2"88f0"04 5"35f0 "19 1"74f0~14
(2) (2) (4) (2)
-
VARIOUS VENOMS AND TOXIIJS
PLB u-Equiv FFA pH 9"4 pH 7"4
1 " 71 f0~04 (2) 3"48f0"13 (4) -
0~14f0~02 4"28f0~41 4"43f0" 11 4~32f0~23
(2) (2)$ (4) (2)t
0"76 (1) 4"06~0 "Ol$ (2) 2"75f0~07 (4) 3"87f0 "04 (2)t
0"65f0" 11 (2)
123f0"10 (2)
0 (2) 0 (2) 0 (2) 0 (2) 0"97f0"03 0" lOfO"00 0~11 f0~06 0 (2) 0"39~0"OS 0~33f0"OS 0~33 f0~09 2"83f0~14 0"24~0 "00 0~26 0~23 f0"00
020f0"06 0~51 f0~10 0"32~0~05 0~25 f0~02 1"49~0 "04 0"54f0~13 0~25 f0"02 0 (2) 1~37f0~02 1 "13f0~03 0"91 f0 "00 2"67f0"08 0~54f0~01 0"79f0 "Ol 0~72 f0~01
(2) (2) (2) (2) (3) (2) (2)
2~33f0"08 (2) 2"13f0"03 (2) 2~32f0~14 (4) 2~20~0"03 (2) 3"08f0"07 (4) 1"83~0"08 (2) -
2~03f0"Ol 2"18f0" 10 1~73f0~02 1"62 f0"04 2"19~0"OS 1"13f0"04 -
2"13f0"09 (2) -
1"56f0"OS (2) -
0"28f0~03 (2) 0 (2)
0"56f0 "OS (2) 0~57 f0 "02 (2)
-
-
0 (2) 0 (1) 0 (2)
0 (2) 0 (2) 0 (2)
(2) (2) (4) (2) (4) (2)
(3) (2) (2) (2) (4) (2) (3) (2) (1) (2)
(2) (4) (2) (3) (2) (2) (2)
'0'02 mg protein. t0~5 mg crude venom. $1 "0 mg crude venom ß0"2 mg bungarotoxin . Ilo-S mg cobrotoxin . One ml solutions of substrate [either 4"5 mg phosphatidylcholine or 3~0 mg lysophosphatidykholine (approximately 6 lunoles of each)] were incubated with venom or toxin at 37°C for 20 min at either pH 7~4 or pH 9"4. Number of samples is given in parentheses. Values are recorded as means < S.E., with values below 0"09 k~quiv free fatty acids (FFA) being recorded as zero (limit of reliability for method used) . Except as noted in the Table 0"5 mg and 3"0 mg of crude venoms were used respectively in the PLA, and the PLB assays.
594
J. E. FLETCHER, W, B. ELLIOTT, J. ISHAY and P. ROSENBERG
~. O
ô~9â \'s S
ô.
Q
000
M
O
~~O
~
O
000
h
O
M~O
~
O~
.. vv
., v
_~ -{-I +I O
-}{
r,
,. O~
~ et .~r, N__d' O OO
. 0
â
ô.
°x
vvv 000
O
N_ O
-}{
u
H N
_N O
ô
N
~d N w
~
aO_~ H oH ô al
N
b
Ô irO
M
G
00
â ô . 0
O
a~~o
â~x a
Ô~ ~
G
~-I h ~
.. v
~
n ô
.°.° b
W i
v
~
O
N
0
v
v
~s .. OOv
s O
bd
ô
-i i -~ O
. 0
v o
oH
° û
v a
.. h qy ~
~~
0~~0
a .,
_ N v N O
v ~ N
n b
vvv
û
v
bbb -Ii +I 'i'i
O 'i i
O -Ii
s~
d
$~ô
N~b
h
..
.., h
â°~,~,
~~ a~
N
s
N F"
c â~ ô
b T .~-~
e~1
~ â eO n .~r,ô
O
h
Ô
N
~ ~oli°HpH
,. N,
~
N
vT
vvv
h ô
vv
N v
'r .-~
Ôv~ Ô -H o +I ~
o, ô.
° x
M
~~w, ODa ~~~â
a, ~° > ,~
W n
ä
v
~w
~
,.,
ag
w.
O . .+ ,> ~+ V ~ ~A .~ O ~r
ô
â o eo >'w° ~ ~~~~o~ ~â
b
w ~0 .
.9 â ô ,n ô w °° p+ n x
â .~
~g~~~> °' ~ J3 ~ .~
bh p~ a~NÔ~ a~ Pr"w~ ~~"~ ° Q °' a a . ., '~ ô8v ~ eo°oaô~
~ ~C ~ M~ y ~ ~ V ~ M ~~ô'~âô y ~ g ~ vv ~ $'~ vvl~v w c~BM~°~ * o ,, o a
a o(~ 0 ~~r > M
1?hospholipases A and B
595
Discussion). Also, carrying out the experiments at two pH values may provide an indication as to whether a single enzyme is responsible for both PLA, and PLB activities or if two separate enzymes exist. As can be seen in Table 1, PLA, activity was usually highest at pH 7~4 while PLB activity was usually highest at pH 9~4, except with the hornet venoms. This difference, between PLA, and PLB, in optimal pH suggests that two separate protein molecules may be responsible for the two enzymatic activities . In addition, there is no consistent relationship between the relative PLA, and PLB activities ofthe different venoms. For example, at pH 7~4 H. haemachatus has about 75 ~ of the PLA, activity of N. scutatus ; however, the PLB activity of H. haemachatus is only about 15 ~ that ofN. scutatus. Furthermore, H. horridum has about the same PLA, activity as N. naja at pH 9~4, yet the PLB activity is only 62 ~ of that demonstrated by N. naja venom. These results show that in addition to PLA, activity, many reptile and Hymenoptera venoms exhibit PLB activity. Although not all of the venoms in Table 1 were tested for PLA, activity, all are known from previous results to have PLA, activity. a-Bungarotoxin and a-cobrotoxin have no PLA, activity while ß-bungarotoxin under certain experimental conditions has PLA, activity ('I~r, 1977). Notice that a- and ß-bungarotoxin and a-cobrotoxin are devoid of PLB activity as is one of the crude snake venoms, Crotalus h. horridus, which interestingly is the one snake venom reported to have little or no PLA, activity (KocxolaTx et al., 1971). In Table 2, the effect of boiling the venoms, at pH 9~4 or 5~5, upon subsequent PLA, and PLB activities at pH 7~4 or 9~4 was examined . With the exception of N. scutatus which retained about 30 ~ of its PLA, activity, boiling at pH 9~4 destroyed almost all PLA, and PLB activities . Boiling at pH 5~5, however, resulted in varying degrees of PLA, and PLB activities being retained, with the loss of one not always associated with the loss of the other. The percent loss of PLA, and PLB activities in a single venom were in several cases dissimilar, suggesting two separate proteins . Boiling V. orientalis for 15 min abolished all activity at either pH (results not shown). Part B
Using somewhat different methods than in Part A, the PLA, and PLB activities of several Hymenoptera venoms were determined (Table 3). Bee venom (A . mellifrca) and mixed Bombes sp. venom sac extract had no PLB activity, but high PLA, activities, whereas all of the hornet venoms had both PLA, and PLB activities . TaHr.E
3.
Paosrxocrnesa A, (PLA ~Nn rxosrsoLiFese OF 9EVERAL HYMBNOFTERA VENOMs
Venom Apis mellifrca Bambus sp . venom sac extract Ytspa maculata Vespa vuigaris Ytspuia maculj/rons
PLA, 559 50 25 125
B (PLB) ~crmrrns PLB 0 0 62 19 18
Activity determined at pH 8 by automatic titration of fatty acid release from either egg yolk (PLAN or lysophatidylcholine (PLH). Results expressed as pM/min/mg .
The effect of heating upon the PLA, and PLB activities of two Hymenoptera venoms using egg yolk and purified lysophosphatidylcholine respectively as substrates is shown in Fig. 1 . The two activities decrease with increasing temperature at a similar rate for both venoms . These results could be explained by either a single molecule having both enzymatic activities or two separate proteins with PLA, and PLB activities respectively having the same heat stability characteristics.
59 6
J. E. FLETCHER, W. B. ELLIOTT, J. ISHAY and P. ROSENBERG so
a c É i
t 8
u a c n ä r!S 30
10
Temperature,
~C
FIG. 1. EFFECT OF HEATING UPON THE PHOSPHOLIPASE A, (PLAN AND PH03PHOLIPASE B (PLB) ACTIVTIIE4 OF V. völgarls AND V maCGlatO HORNET VENOM3.
Following 30 min heating at pH 6~2 (V. vulgaris) or 5~8 (V. maculata), enzymatic activity was measured at 40 °C and pH 8 by automatic titration of free fatty acids. Egg yolk (PLAN or lysophosphatidylcholine (PLB) were used as substrates : f- " PLA V. vuJgarts ; ~-~ PLH, V. vulgaris ; !:: O PLA,, V. maculata ; p-p PLB, V. maculata.
The PLB activities of yellow hornet (Y. arenaria) and baldface hornet (V. maculata) were determined at pH 7~4 and 9~2. The activities were 5-10 ~ greater at pH 7~4 than 9~2, in excellent agreement with results shown in Table 1. In Fig . 2 the PLA a and PLB activities of V. orientalis venom sacs are shown to be associated with different fractions collected on a tandem column . Therefore, it appears that separate proteins are responsible for the PLA s and PLB activities of the oriental hornet venom, even though these two proteins have similar heat stability characteristics (Table 2). DISCUSSION
We have found PLB activity in a variety of snake, Gila monster and Hymenoptera venoms. The richest source of PLB in snake venom (N. scutatus) was much less active than any of the Hymenoptera venoms with the exception of bee and mixed Bombes sp. venoms, which had little or no activity depending upon method used. The use of relatively large amounts of venom for many of our studies was necessary in order to detect the relatively low levels of PLB activity. Greater PLA Q activity was detected using one-sixth the amount of venom used in the PLB assay ; therefore, at least in the reptiles tested, the discovery of PLB activity is more of phylogenetic interest than for use as a potential source of the
Phospholipases A and B
597
c ô â
Fraction FiO . 2. SEPARATION OP FHOSPHOLIPASE A~ (PLAN AND PH03PHOLn'ASE B (PLB) ACrIVITIE4 OF V Or~ClJtaUs VENOM SAC EXTRACT. Y. orientalts venom sacs (1 ~25 g) were suspended and applied to a tandem column . Protein eluted was followed by absorption at 230 nm and 280 nm . Enzymatic activity was determined at pH 8 by automatic titration of fatty acid release from either egg yollc (PLAN or lysophosphatidalcholine (PLB) .
enzyme. In contrast, the hornets V. orientales, V. arenaria, Y. rnaculata and V. vulgaris are rich sources of PLB. The purity of the substrates was verified by thin layer chromatography to ensure that tha activity observed was due to PLA$ and PLB activities . It might be argued that some of the activity recorded as being due to PLAs is actually due to PLB activity since the product of PLA$ action (lysophospholipid) is the substrate for the PLB enzyme . This is not likely to be a significant source of error in the case of snakes, Gila monster or bee venoms, since with the low amounts of venoms used for the PLAs assay, little or no PLB activity would be observed. In order to assay PLB activity in these venoms, it was necessary to use 3~0 mg ofvenom rather than 0"5 mg used in the PLA, assay. However, for the hornet venoms, which have much greater PLB activity, a significant portion (but not all) of the activity listed as being due to PLAs may actually represent PLB activity. Since PLB activity in snakes is maximal in the range ofpH 8 "5-10"0 (DoStY and PBAxsox, 1964) and PLAß activity is optimal around pH 6"5-8~0 (CormxBA and DeVx~s, 1965), the crude venoms were incubated with substrate at both pH 7~4 and pH 9~4. Our experiments confirmed the earlier reports showing somewhat higher snake venom PLAs activity at pH 7"4 and slightly higher PLB activity at pH 9"4. In contrast, the hornet venoms show higher PLB activity at pH 7"4. There was no direct proportional relationship between PLA s and PLB activities in the various venoms.
59 8
J . E. FLETCHER, W. B . ELLIOTT, J . ISHAY and P . ROSENBERG
Several of the snake venom PLB values are not in agreement with the results of Do>r1tY and Plinxsoty (1964) . Most notably, higher relative activity was observed for N. scutatus venom and lower activity was observed for A. piscivorus venom. However, variations of enzyme activity within species as well as within individual snakes have been reported ($ARKBR and Dl:vl, 1968). Differences in experimental conditions could also explain differences between this and other reports. The heating experiments do not clearly indicate whether the PLAQ and PLB activities are associated with separate proteins or the same protein. With N. scutatus venom a clear difference in the heat sensitivity of the two enzymatic activities was observed . This suggests separate proteins as being responsible for the two enzymatic activities. A less likely possibility would be two active sites on a single protein. In contrast the PLA$ and PLB activities of several other venoms were affected similarly by heat . PLA$ activity is thought to be stable to boiling in an acidic medium and destroyed by boiling at an alkaline pH (COND1tEA and D>äVx»rs, 1965). In our experiments, however, the PLA$ activity was not always stable to boiling at pH 5~5 or to heating above 75°C for 30 min, at pH 5'8 or 6~2. D. polylepis and V. orientalis venoms lost greater than 80 ~ of their PLAp activities when boiled for 15 and 3 min, respectively, at pH 5~5. In contrast, B. caeruleus venom retained 95 ~ of its PLA$ activity after boiling at pH 5~5 for 15 min. The heat stability of PLAs at acidic pH cannot therefore be assumed, but must be determined for each venom. While the PLAP activity of several venoms was destroyed by boiling at pH 9~4, it is interesting that under these conditions N. scutatus venom retains about 30 ~ of its activity. In addition to varying stabilities of PLA$ to heating or boiling. PLB activity also demonstrated varying degrees of stability. PLB from Pseudechis porphyriacus venom was previously shown to be relatively stable to boiling for 2 hr at pH 7, retaining 21 ~ of its activity (D01?RY' and PSAxsoN, 1964). In our experiments the PLB activities were abolished in all venoms tested after 15 min of boiling at pH 9~4. After boiling or heating (80°C) at an acidic pH the venoms exhibited varying retention of activity, from greater than 80 ~ (B. caeruleus venom) to 0 ~ (D . polylepis and hornet venoms). We have found that in Y. orientalis venom the PLAs and PLB activities reside upon different molecules as determined by column separation and subsequent analysis of enzyme activity . The next logical step is to purify the enzymes of other Hymenoptera and snake venoms and conduct further analysis of the PLA, and PLB activities to determine conclusively whether these activities reside on the same protein. The results of this paper indicate that specific phospholipases from different venoms vary considerably in their properties, despite a similar catalytic site of action. Therefore, as mentioned by SHILOAH et al. (1973b), the classification of an enzyme as a PLA% may be far to restrictive in defining its properties . Acknowledgements-The authors acknowledge the skilled technical assistance of Mr . RoHexT Sam . This research was supported in part by grants from the University of Connecticut Research Foundation (P .R .) and the National Institutes of Health, NINCDS 1IR01 NS 1478801 (W.B .E .)] . REFERENCES i, G . B ., Hwwrxoxxe, J. N . and D~wsox, R . M . C. (Eds .) (1973) Form and Function ofPhospholipids AxsFi BBA Library, Vol . 3, second edn ., New York : Elsevier. Cormxen, E . and DEVt~s, A. (1965) Venom phospholipase A : a review. Toxlcon 2, 261 . DE Havas, G . H ., POSTEMA, N . M ., NIEUWENHUIZEN, W . and vnx DeElvex, L . L. M . (1968) Purification and properties of phospholipase A from porcine pancreas . Biochim. biophys. Acta 159, 103.
Phospholipases A and B
59 9
Do~eY, H. M. and Peaesox, J. E. (1964) Phospholipase B in snake venoms and bee venom. Biochem. J. 92, 599. Dow, V. P. (1956) A relation between non~sterified fatty acid in plasma and the metabolism of glucose. J. clln. Invest. 3S, 150. Eutoz-r, W. B. (1978) The chemistry and immunology of reptilian venom. In : Biology of the Reptilia, Vol. 8B, p. 163, (Gnxs, C. and Gnxs, R. A., Eds.). London : Academic Press. Kn~s, M. (1960) Lipolytic enzymes. In : LipidMetabolism, p. 165, (BLOCii, K., Ed .). New York : Wiley. Kocxou.~n, W. F., LEDFORD, E. B., Dnr,v, J. G. and BILLINGS, T. A. (1971) Toxicity and some enzymatic properties and activities in the venoms of Crotalidae, Elapidae and Viperidae. Toxicon 9, 131 . Lowxv, O. H., RoseHxoucx, N. J., Fnltx, O. L. and RnrrnnL.r.., R. J. (1951) Protein measurement with the Folin phenol reagent . J. biol. Chem . 193, 265. MOIiaMEn, A. H., Ka~L, A. and AYOBB, M. H. (1969) Studies of Phospholipase A and B activities of Egyptian snake venoms and a scorpion toxin. Toxin 6, 293. Nf+ue, B. C., NaIIe, C., DENNE, S., WYPYCH, J., AIeHesnfnx, C. E. and ELLIOrr, W. B. (1976) Immunologic comparison of phospholipases A present in Hymenoptera insect venoms. J. Allergy clip . Imn:un. 58, 101. RosExsexa, P. (1976) Bacterial and snake venom phospholipases : enzymatic proves in the study of structure and function in bicelectrically excitable tissues. ln : Animal, Plant acrd Microbial Tozins, Vol. 2, p. 229, (Oxsaxn, A., Ed.). New York : Plenum . RosexHSxG, P., IsIInY, J. and GrrrER, S. (1977) Phospholipases A and B activities of the oriental hornet (Vespa orientalis) venom and venom apparatus. Toxicon 15, 141 . Snlex.a,a, N. K. and D$vI, A. (1968) Enzymes in snake venoms. In : Venomous Animals and their Venoms, Vol. 1, p. 167, (BUCHERL, W., BucICLev, E. E. and DouLOFeu, V., Eds.). New York : Academic Press. SxILOnII, J., Kt>etxsxv, C., DsVxiFS, A. and B~GeR, A. (1973a) Phospholipase B activity of a purified Phospholipase from Vipera palaestinae venom. J. Lipid Res. 14, 267. Sxrtoax, J., KLIHANSKY, C. and DEVxIFS, A. (1973b) Phospholipase iscenzymes from Naja naja venom. II Phospholipase A and B activities . Toxicon 11, 491. Tu, A. (1977) Venoms : Chemistry and Molecular Biology. New York : Wiley. ZwaaL, R F. A., RoeLOrsex, B. and Cou.sY, C. M. (1973) Localization of red cell membrane constituents . Biochim, biophys. Acta 300, 159.
