Phospholipases A and B activities of the oriental hornet (Vespa Orientalis) venom and venom apparatus

Toxltoe, 1977 . Vol. 15, pp. 141-136 . Peraamoa Pros. Printed in Drat Hrltain.

PHOSPHOLIPASES A AND B ACTIVITIES OF THE ORIENTAL HORNET (VESPA ORIENTALIS) VENOM AND VENOM APPARATUS PHILIP ROSBNBSRG,* JACOB ISHAY and SIMON GITTER Departments of Physiology and Pharmacology, Sacklex School of Medicine, Tel-Aviv University, Raurat-Aviv, Israel

(Accepted joy publication 22 September 1976) P. Rost:xealtG, J . ISI~IAY and S. Grrren . Phospholipases A and Bactivities of the oriental hornet (Vespa orientalis) venom and venom apparatus. Toxicon 15, 141-156, 1977 .-Oriental hornet venom is a rich source of both phospholipase A (PhA) and phospholipaso B (PhB) activities . This was shown by incubating venom with egg yolk or with pure lecithin and lysolecithin . Activity was measured by titrating liberated fatty acids and by phosphorus analyses of separated phospholipids from thin-layer chromatographicplates . Both lecithin and lysolecithin were rapidly hydrolyzed by venom at pH 4 and 8. With egg yolk as substrate, the optimum Ph activity was observed at pH S although considerable activity was observed from pH 3~5 to 9~5. In contrast purified substrates showed greateractivity at an alkaline pH, whether assay was in the presence of collidine-acetate or Tris buffer or in the presence or absence of ether. Ether dramatically changed the optimal pH for Ph activity, with egg yolk as substrate, from acidic to alkaline . It is not known whether these PhA and PhB activities are dual activities of a single enzyme or activities of two separate enzymes. The venom has neither PhC norlipase activities . PhA and PhB activities were observed not only in pure venom M, but also in venom sacs (VS) where the venom is stored, in the acid (venom) glands (H+) where the venom is produced, and in the alkaline (Dufotus) gland (OH-) whose function is unknown. The release of free fatty acids from egg yolk at pH S was in the ratios of 1(V) : 0~ 13 (VS) : 004 (H+) : 0'30 (OH-). Measurements of Ph activities in combinations of the above preparations showed that strong activators or inhibitors of the enzyme are notpresent. The mid-gut, fat body and hemolyrrtph of the hornet showed Ph activity only equal to about one-half of one per cent of that of the OH gland. Antisera produced against V, VS, H+ and OH - were cross-reactive and inhibited to varying degrees the Ph activities of each of the above preparations . The highest titre antisera were produced in rabbits injected with V or VS, in contrast to the low titres produced with H+ and OH - glands . Low Ph activity is detectable in the venom sacs 2 days prior to emergence, with 20-fold higher activities being observed at 5 days of age. The oriental hornet may be extremely useful as a rich source of not only PhA but also PhB activities. The effects of this latter enzyme on biological systems have not been thoroughly evaluated. The drastic disruption of phospholipid structure and hydrophobic binding forces between phospholipid and protein, by the combined PhA plus PhB activities, may be responsible for some of the pharmacological actions of hornet venom. INTRODUCTION

V13PiOMS and toxins are the richest sources of several phospholipase (Ph) enzymes ; catalytic proteins which hydrolyze phospholipids at specific sites (~IVSELL et al., 1973). For example, PhC (phosphatidylcholine cholinephosphohydrolase, EC 3 .1 .4.3), an enzyme which splits oifthe phosphorylated base from phospholipids, leaving a diglyceride, although present in low activity in animal tissues, has not been well studied in this source, and most of our knowledge concerning this enzyme is derived from studies on bacterial toxins *Permanent address : Section of Pharmacology and Toxicology, University of Connecticut, School of Pharmacy, Stoma, Connecticut 06268, U .S .A .

142

PHII.IP ROSENBERG, JACOB ISHAY and SIMON GITTER

(ANSELL et al., 1973). PhA, (phosphatide acyl hydrolase, EC 3.1 .1 .4) the enzyme which deacylates at the 2-position of the glycerol moiety of phospholipids is widely distributed throughout animal tissues; however, its richest sources are snake and hymenopteran (bee, wasp, hornet) venoms (ANSELL et al., 1973 ; CONDREA and DE VRIFS, 1965 ; HABERMANN, 1968, 1971 ; HANAHAN, 1971). PhB (lysophospholipase ; lysolecithin aryl hydrolase, EC 3.1 .1 .5) removes the remaining fatty acid from lysophospholipids, which, for example, are produced as a result of PhAs action (ANSELL et al., 1973). PhB seems to be less widely distributed than PhA, and where found it is usually present only in low activity. PhB activity has been detected in mammalian tissues (MARPLES and THOMPSON, 1960 ; Vnr DEN BOSCH et al., 1968 ; HORTNAGL et al., 1969 ; LEIBOVITZ-BEN CrERSHON et al., 1972 ; De JoNG et al., 1974), E. coli (Dot and NOJIMA, 1975) and Penicilliun: notatum (BEARS and KATES, 1967 ; KAWASAKI et al., 1975). PhB activity has also been reported to be present in several Australian and Egyptian snake venoms (DOERY and PEARSON, 1964 ; MoHAnrr:n c" r aL, 1969) as well as in purified enzymes isolated from V palaestinae and N. rraja venoms (SHILOAH et al., 1973a, 1973b). In the latter studies by $HiLOAH et al., it was clearly shown that the PhA$ and the much weaker PhB activities were due to a single enzyme and not activities of two separate enzymes. PhB activity, as noted in recent reviews (HABERMANN, 1968, 1971, 1972) has also been reported in wasp and hornet venoms (CONTARDt and LATZER, 1928 ; CONTARDI and ERCOLI, 1933 ; FRANCIOLA, 1937 ; ERCOLt, 1940), although this has not been studied recently using newer techniques . The effects of venom Ph on biological preparations have been extensively studied (MELDRUM, 1965 ; CONDREA arid DE VRIES, 1965 ; R~ENBERG, 1976, 1977) either to see what the Ph of the venom contributes to the overall venom action or as tools to help understand the contribution of phospholipid to the structure or function of a particular membrane, tissue or organ. In these studies it is essential to have rich sources of the Ph so that its biochemical and pharmacological properties can be well characterized .

Although, as noted above, PhA and PhB activities have been reported to be present in hornet venom, a thorough study has not yet been undertaken . We therefore studied the Ph activities of the venom and venom apparatus of the large social hornet Vespa orientalis (oriental hornet). This hornet is widely distributed throughout the Mediterranean region and the Near East (BODENHEIMf:R, 1930, 1933) and its life habits and biology have been extensively studied (ISIiAY, 1964, 1973 ; ISHAY et al., 1968 ; ISIiAY and RLJTrNER, 1971 ; ISHAY ând SCHWARTZ, 1973 ; IsHAY ând BROWN, 1975 ; IsHAY and $ADEH, 1975). We Wished to evaluate in this preliminary study whether this hornet may be a useful rich source of Ph enzymes. If it were found to be a rich source, it may then be worthwhile to carry out the isolation and purification of Ph enzymes from this hornet. It would then be possible to critically evaluate the contribution, if any, which the Ph makes to the varied pharmacological effects which this hornet venom has on blood (JOSHUA and ISHAY, 1973 ; ISHAY, 1975), central nervous system (ISHAY et al., 1974), cardiovascular system (KAPLINSKY et al., 1974), muscle (ISHAY et al., 1975), etc. (EDERY et al., 1972 ; 5ANDHANx et al., 1973). It was also of great interest to compare the Ph activity of the pure venom with that of the several components of the venom apparatus. The structure of the venom apparatus of Hymenoptera in general (PAWLOWSKY, 1927 ; MASCHWITZ and KLOFr, 1971) and of the oriental hornet, in particular (KANWAR and JsrHt, 1971 ; BARB-NEA of al., 1976) have been described. The venom apparatus is composed of the venom gland, also called the acid gland, which is thought to be the main-site of venom production, the venom sac, where the venom is stored, and the Dufours or alkaline gland, whose secretion was thought to be of a lubricating or alarming nature and non-toxic (MASCHWITZ and KLOFr, 1971).

i?hospholipases A and B in the Hornet

143

However, recent studies suggest that the alkaline gland, containing its secretion, is lethal to bees and toxic to mice (BARR-NaA et al., 197 . It was therefore of interest to determine whether the alkaline gland secretion has any Ph activity. MATF?RiA~g AND METHpDS Obtaürirrg venom acrd venom apparatus The methods, previously described (Bnxx-Nr.~, et al., 197, may ba anm~++Ari~p d as follows : pure venom was collected directly from the stinger by exerting pressure on the abdomen of live hornets. About 2 mg (wet weight) of pure venom (0"3 mg of protein) were obtained from each hornet . Values of 3"68 and 104 mg for wet and dry weight of venom per hornet were previously reported (AHRAHAMa, 1955), which are much greater than the dried weight of 0"06 mg (Russer t, 1967) or 0" 11 mg (LAV~H and Vnr.~, 1939) or the wet weight of 0"3 mg (LAVTBR and Vxt~, 1939) venom reported as being obtained per bee (Apis mell(jera) . Venom glands were removed by pulling with forceps on the stinger of hornets which had been previously killed by decapitation .The stinging apparatus was removed together with the attached alkaline gland, venom sac and acid gland. Using a Zeiss stereomicroswpe it was possible to remove the glands uncontaminated by extraneous tissue. The average protein content (lig) per gland was reported (B.~xrt-NE.~, et al., 1976) to be as follows : acid gland,l0-20 ; alkaline gland, 5-15, and venom sacs 1000-2000. The glands were dissected in a large volume of isotonic 0"85 ~ sodium chloride solution and care was taken to prevent rupture of the venom sac or discharge of venom into the dissecting solution. We are therefore confident that activities observed are not due to contamination during dissection . The glands and the pure venom were immediately frozen, and separate batches for each day's dissection prepared. About 15 hornets could be dissected per hour . Batches of 50-100 acid or alkaline glands or 20 venom sacs were thawed and homogenized in a total volume of 1 ml (made up to volume with 0"85 sodium chloride) with a Potter-Elvehjem hand homogenizer. Aliquots of appropriate dilutions of these homogenates were taken as needed with the remainder kept frozen, for no longer than 3 weeks. Control experiments showed that the Ph activity of the frozen homogenates did not significantly decrease over a period of 1 month. Aliquots of the frozen pure venom were taken as heeded . Assays Protei~r was determined by the method of LOWRY et al. (1951) using purified bovine serum albumin as the standard . PhA arrd PIrB assays . Different substrates and procedures were used for evaluating Ph activity : titration of frce fatty acids (DOLE, 1956) liberated from a 1 :5 dilution of egg yolk in 0"85 ~ sodium chloride solution containing 0" 1 M Tris and adjusted to the appropriate pH, was carried out. Blanks without substrate and without enzyme source were always run simultaneously and used for correcting enzymatic data . Assays were routinely performed at 0, 5,10 and 20 minfollowing the addition of venom or tissue homgenates. 1?almitic acid (0 "01 N) served as standard . Even though Tris is not as effective buffer in the acid range, the pH of incubates between 3"5 and 5"5 did not change significantly even with high Ph activities, no doubt because the released fatty acids do not form more acidic solutions. Between pH 6 and 9 there was at moat a Oß pH unit decrease observed in 20 min with high Ph activity . In some experiments synthetic nrra phosphatidylcholine (lecithin), dipalmitoyl (crystalline, synthetic, 99 ~ purity) and rra lysophosphatidyl choline (lysolecithin, 98 ~ pure), mainly containing pahnitic and stearic acids, and prepared by reacting PhA, with egg yolk, were used as substrates . Both were obtained from Sigma Chemical Co ., St . Louis, Missouri . When these pure substrates were used (1-3 mg per ml) the incubation media (unless otherwise stated) consisted of 0" 05 M wllidine (2, 4, 6 trimethylpyridine) buffer, 0"05 M acetic acid-sodium acetate buffer and 0"5 mM calcium chloride, the media being adjusted to desired pH . The tubes containing lecithin wen; sonicated and a final concentration of about 9% ether maintained . As noted in the Results section some experiments were performed without ether, while in other experiments egg yolk was used as substrate with the collidine-acetate buffered system . All of the titration experiments are reported as pequiv free fatty acids liberated per time per a certain number of Rg of protein. In other experiments, rather than measuring 1?h activity by the titration of liberated free fatty acids, the individual phospholipids were separated and quantitated, after exposure of egg yolk or purified phospholipid substrate (see above) to venom or tissues. The lipids were extracted by 1 :3 chloroform- methanol (Manuac~rrt et a1.,1959), evaporated and re-extracted with 2 :1 chlorofor~methanol, according to the method described by For cx et al. (1957) . Portions of the lipid extracts were analyzed for total lipid phosphorus (BARTrETr', 1959) with the remainder being applied to and separated on thin-layer chromatographic plates using a twodimensional developing system of chloroform-methanol-water (65 :25:4 v/v) followed by 3-heptanoneacetic acid-water (80:60:10 v/v) (Coxnnr.,~ et al., 1967 ; LYSZ and RosErrasao, 1974). Spots were visualized by exposure to iodine vapors, scraped off of the plate and phosphorus determined (BAR~n.sr-r, 1959). The percentages of each individual phospholipid hydrolyzed were calculated . PhC and lipase assay Venom or venom gland tissues were incubated for 20 min at desired pH values, with a 1 :5 dilution of egg

144

PHILIP ROSENBERG, JACOB ISHAY and SIMON GITTER

yolk in saline as previously described . A lipid extract was prepared (see above) and the neutral lipids separated on thin-layer chromatographic plates by the method of KtsEts . and HnsatM (1963) . The following lipid classes are separated (from front to origin) cholesterol esters, triglycerides, fatty acids, cholesterol, diglycerides, monoglycerides and phospholipids. After visualization by exposure to iodine vapor, the spots were scxaped off and analyzed for glycerides (VAx I3At~Et. and Z1LV puciurr, 1957) and cholesterol (Zra-n.rs et al., 1953). A decrease in triglycerides would be expected to be observed if lipase activity were present, while PhC activity should give rise to an increase in the diglyoeride level. Cholesterol measurements were used as internal standards for the thin-layer separation . Preparation ofantisera Antisera were obtained by immuniting two rabbits with saline extracts of pure venom, venom sacs, acid gland or alkaline gland. Subcutaneous injections into rabbits of the above materials in saline and mixed with complete Freund's adjuvant were made at the rate of three injections per week over a S week period in a dose corresponding to 1150 kg of homogenized tissue. The last injection, a booster dose, contained 2050 times more material . After testing the immunological effect of the antisera, by immunodiffusion of a sample from each rabbit, the rabbits were bled to death by cannulation of the femoral artery and the serum separated by aseptic techniques and stored at -20°C until used . The production and effectiveness of rabbit antiserum against Vespa orfentalis venom has been previously described (IsenY et al ., 1971, 1972). RESULTS

Figure 1 shows the liberation of free fatty acids from an egg yolk substrate, at various pH values, by pure venom, and venom apparatus tissues. At the optimal pH of 5 the activities of venom, venom sacs, acid and alkaline glands were in the proportions of approximately 1 "0:0.13 :0.0"04 :0"03. Although optimal activity was observed in the acid range, considerable activity was observed throughout the pH range of 3"5-9~5. This method of Ph assay is not very sensitive and would not detect the low amounts of endogenous activity expected to be found in body tissues of the hornet . For example, at a concentration of 1 mg protein per ml, no liberation of fatty acids was produced by hornet fat body, midgut or hemolymph. It required concentrations of 10 mg per ml to give the same extent of fatty acid liberation (~Etequiv free fatty acid per 10 min) as observed with 50 ttg of acid gland. It is likely therefore that the activities recorded in Fig. 1 for venom sac, acid and alkaline gland are due to the venom or venom components contained in the lumen of these structures rather than the cells of the structures themselves. The results shown in Fig. 1 could be due to either PhA, PhB or lipase activity . We determined, however, that with egg yolk as substrate the pure venom has neither lipase nor PhC activity. For example, the control diglyceride values recorded at pH 4 and 8 were (pmole glycerol per 0"2 ml egg yolk) 0"73 ~ 0"08 and 055 ~ 018 (mean ~ S.E.). Corresponding values after 20 min exposure to 1 mg venom protein per ml were 0"54 ~ 005 and 0"39 f 011 . These results clearly show that the venom has no PhC activity ; if this enzyme were present it would increase the diglyceride level. While the small decrease in diglyceride levels might suggest some lipase activity, our triglyceride results clearly show that the enzyme has no lipase activity. The control triglyceride values at pH 4 and 8 were (ltmole glycerol per 0"2 ml egg yolk) 0"85 f 0-06 and 1"20 ~ 0"24 (mean ~ S.E.). Corresponding values after 20 min exposure to 1 mg venom protein per ml were 1 "01 ~ 0~17 and 0"98 ~ 0"12. Each of the above means is based upon four experiments. Even at this relatively very large concentration of venom, no definite lipase activity could be detected . In order to quantitate the extent of phospholipid splitting, lipid extracts of control and venom treated egg yolk incubation media were prepared and the phospholipids separated on thin-layer chromatograms (see Methods) . The control total lipid phosphorus value of I ml of a 1 :5 dilution of egg yolk is about 500 ltg, indicating that there was a total of about 33 ~.equiv offatty acids which would have been liberated if both fatty acids were hydrolyzed . Since venom shows a maximum activity much higher than 16"5 ltequiv liberated (~ 28 ; Fig. 1), this clearly indicates that PhB activity must be present in addition to PhA activity.

Phospholipases A and B in the Hornet 30 T

14 5

VENOM

x VENOM SAC u ACID GLAND

o

ALKALINE GLAND

25

u

5t

~~,,. _.x-_ __ e

pH FRO . 1 . LIBERATION OF FREE FATTY ACII>,Y FROM E00 YOLK BY VENOM, VENOM 9AC, ACID Aa~~ AT .KAT .ITiFi QLAND .

Egg yolk dilutions (1 :~ in saline containing 0' 1 M Tris were incubated for 0, 5, 10 and 20 miu with hornet venom and venom apparatus tissues at the pH values indicated . Results are recorded as llequiv free fatty acids liberated from 1 ml of egg yolk dilution in 10 min by 501Ig protein. The points are means of duplicate determinations all of which were within 10 % of each other.

The ~ distribution of phospholipids in control egg yolk was lecithin, 80 ; lysolecithin, 2"2 ; sphingomyelin, 0"7 ; phosphatidylinositol, 1 "5 and phosphatidylethanolamine, 15"3 . The total lipid phosphorus values of the tissues and pure venom in lIg per mg protein were venom, 0"5 ; venom sacs, 0"8 ; acid gland, 8~0 and alkaline gland 4"6. The extent ofhydrolysis of egg yolk phospholipids by venom and venom sacs is shown in Table 1. Knowing the amount of lecithin and phosphatidylethanolamine in control tissue and after treatment, the ~ hydrolysis is readily calculated . The difference in amounts of phospholipids before and after treatment is the amount of lysophosphatide expected to be produced from this extent ofphospholipid hydrolysis . The actual lysophosphatide values found after treatment

146

PHILIP ROSENBERG, JACOB ISHAY and SIMON GITTER TAHLE 1 . HYDROLYSIS OF EOG YOLK PHOSPHOLIPIDS HY HORNET VENOM AND VENOM SA('S

Treatment Venom

ug protein/ml S00 50

Venom sac

50

pH 4 8 4 8 4 8

TLP % loss 72 67 36 28 6 6

L 92 73 85 23 58 11

LL PE ~ hydrolysis _ . .__. -__ S9 76 97 SG H7 56 95 39 10 4S GS 2

LPE 63 100 100 81 18 100

One ml of a 1 :5 dilution of egg yolk in saline containing 0~1 M Tris was incubated for 20 min with or without addition of venom or venom sacs and then lipid extracts were prepared (see Methods) . Abbreviations : TLP = total lipid phosphorus ; L = lecithin, LL = lysolecithin, PE = phosphatidylethanolamine ; LPE _ lysophosphatidylethanolamine .

are subtracted from the expected values to give the ~ hydrolysis values shown for lysolecithin and lysophosphatidylethanolamine . These results clearly show the presence of both PhA and PhB activity . The hydrolysis of phospholipids shows the presence of PhA activity . The hydrolysis of phospholipids shows the presence of PhA activity ; however, if only PhA activity were present the total lipid phosphorus value would not have decreased, since lysophosphatides are also extracted in a lipid extract and would be included in the total lipid phosphorus value. The action of PhB would, however, give rise to glycerolphosphorylcholine and glycerol-phosphorylethanolamine which are aqueous, not lipid soluble. It is most interesting that PhA activity, that is phospholipid hydrolysis seems greater at pH 4 than at pH 8, whereas with the high concentration of venom and with venom sacs, lysophospholipids seem to be hydrolyzed better at the alkaline pH . To obtain direct evidenceforPhBactivitywecompared the release of free fatty acids from synthetic lecithin and lysolecithin (Table 2). The hydrolysis of lysolecithin appears about the same order of magnitude as that of lecithin, which could be interpreted to mean that PhA and PhB activities are about equal . However, some of the free fatty acids liberated from lecithin may be due to hydrolysis of the second fatty acid ester due to PhB activity. In all ofthe experiments at pH 4~2, lysolecithin was hydrolyzed slightly better than lecithin, whereas at pH 8, venom and venom sacs hydrolyzed lecithin to a somewhat greater extent than lysolecithin . Acid and alkaline glands hydrolyzed lecithin and lysolecithin about equally at pH 8. It should also be noted in Table 2 that in every case lecithin and lysolecithin were hydrolyzed better at pH 8 than at pH 4~2 ; the difference in activities being greater with lecithin. This is a marked contrast to the results obtained when egg yolk was used as substrate and hydrolysis was greater at pH 4 than at pH 8. To check the results obtained with venom in Table 2, and to determine whether lysolecithin accumulates when lecithin is used as substrate, the following experiment was performed . Lecithin and lysolecithin were incubated in collidine-acetate buffer with venom for 20 min, after which lipid extracts were prepared and phospholipids quantitated from thin-layer plates (Table 3). An amount of lecithin (1 mg per ml) and lysolecithin were used which theoretically should give about 40 !ag total lipid phosphorus. The actual values were very close to the theoretical, and the recovery from the plates in the control experiments was about 100 ~. The lecithin appeared to have about 2~5 % lysolecithin contamination with similar extent of lecithin contamination being observed in the lysolecithin sample. Both fatty acids were almost completely hydrolyzed at pH 8, whereas at pH 4 there was some accumulation of lysolecithin . Lysolecithin was almost completely hydrolyzed to glycerol-phosphorylcholine at both pH values . These results confirm our earlier data suggesting that hornet venom is a rich source of PhB activity .

Phospholipases A and B in the Hornet

147

TABLE 2 . RBLEASB OF FREE FATTY ACIDS (FFA) FROM LECITfnN (L) Arm LYSOIBCrrIUx (LL) BY soRNET vENOM (V), vexoM sACS (VS), Acro aLANDS (H+) AND ALx "" .~ aLANDS (OH- ) Enzyme source V

VS

H+

OH'

Conc . (lI8 protein/ml)

Phospholipid

Conc . (mg/mn

llequiv pH 4 " 2

FFA pH 8 " 0

100 100 600 600 100 100 600 600 100 100 280 280 SO 80 80

L LL L LL L LL L LL L LL L LL L L LL

1 1 3 3 1 1 3 3 1 1 3 3 1 3 3

0~8 1 "1 2"2 2"8 0"6 0~6 2" 3 2"9 0" 1 0~4 1"4 2"S 0"6 0"8 2" 1

2" 4 1~2 6"6 3"2 1 "4 1"0 S"9 3"9 1 "2 1"2 2"S 2"8 1 "3 2"S 2"2

% hydrolysis pH 4~2 pH 8~0 28 SO 27 41 21 28 28 44 4 18 17 37 21 10 32

88 S3 81 48 Sl 46 72 S9 45 S4 30 42 46 30 33

Phospholipids dissolved in collidine plus acetate buffer (see Methods) were incubated with enzyme source for 20 min and the released free fatty acids were then titrated . TABLE 3 . HYDROLYSLS OF SYNITIEI'IC LECITHIN (L) AND LYSOLECITImv (LL) BY HORNET VENOM Incubate

pH 4

TLP (llpa pH 8

L

40 " 0

36 " 4

L -t- V

18~4 17" 6

2"1 1 "7

4~04 44"0

40"4 44 " 8

L LL

4"2 S"0

1~2

L LL

LL LL + V

PL measured L LL L LL

pH 4 41 "2 41 " 3 0 "9 1 "2 Od 15 " 8 17 " 9 0"S 44" 8 39 " 0 0"0 2"2 2"S

ltg P

pH 8 32" 6 37"0 1~0 1~0 2" S 0" 8 1 "2 41-6 42"9 0" 6 0"0 0" S

Lecithin (1 mg per ml) and lysolecithin were incubated for 20 min in 1 ml of a collidine-acetate buffer in the absence or presence of 2S0 lIg (protein) hornet venom . Lipid extracts were prepared, a small aliquot analyzed for total phosphorus and the remainder spotted on thin-layer plates and the lecithin and lysolecithin spots analyzed for phosphorus . Results are expressed as lIg in original 1 ml incubation solution. TLP = total lipid phosphorus ; PL = phospholipid ; P = phosphorus .

In an effort to explain the marked difference in pH optima for hornet venom Ph activity, when egg yolk was used as substrate as contrasted to when purified phospholipids were used, the following experiment was performed . Egg yolk substrate solutions were prepared to collidine-acetate or Tris butFer with or without added ether, then 1 ml portions were incubated for 10 min with 50 or 100118 hornet venom, after which free fatty acids were extracted and titrated (Table 4). There was no difference in the activity of 50118 venom when the egg yolk substrate was in collidine-acetate or Tris buffer, and the activity (in the absence of ether) was over twice as great at pH 4"2 as compared to pH 8, which is in agreement with our previous findings (Fig. 1). The addition of ether, however, dramatically increased the Ph activity of hornet venom at pH 8 up to or even slightly higher than that at pH 4"2, in

148

PHILIP ROSENBERG, JACOB TSHAY and SIMON GITTER

TABLE 4 . RELaA.4E OF FREE FATTY ACIDS (FFA) FROM E00 YOLK SUBSTRATE PREPARED iN COLLiDINE-ACETATE (C-A) OR TRI3 BUFFER WITH AND WITHOUT ETHER

Venom ITg protein/ml

Buffer

Ether

pH

ITequiv FFA

100

C-A

-r __

4ß 4~3 7~9 7 .q

19~4 23~9 19~6 y. g

SO

C-A

42 4~2 8~0 H~U

12~3 18~4 21~3 ~6~8

SO

Tris

42 4~2 8~0 8~0

137 178 203 7~8

-

i__

Egg yolk substrate (1 :S dilution) solution (1 ml) was incubated for 10 min with hornet venom, then free fatty acids were extracted and titrated . Tris buffer, 0~1 M ; collidine-acetate buffer, 0'OS M in each ; ether, final concentration 9 % . TABLE S. RELEASE OF FREE FATIY ACH>3 (FFA~, BY VENOM SACS (SOO dig prOteln per mll, FROM LECITHIN (L) AND LYSOIBCITHIN (LL) SUBSTRATE(2 mg/per ml) PREPARED IN COLLIDINE- ACETATE (C - A) OR TAIS HUFFFR WITH AND WITHOUT ETHER

Substrate L

Buffer

Ether

kequiv pH 4~2

pH 7~8

Tris

+ --,1---{-

1~3 1~7 0~8 0~2 2~4 3~1 2~ 1 2~3

2~7 2~1 3~1 3~8 2'S 3~1 3~ 1 3~3

C-A LL

Tris C-A

FFA

~ hydrolysis pH 4~2 pH 7~8 29 33 IS 4 46 60 40 44

61 48 71 86 57 7U 70 73

One ml of substrate solution was incubated for 20 min with hornet venom sacs, then free fatty acids were extracted and titrated . Tris buffer, 0~1 M ; collidine-acetate buffer, 003 M in each ; ether, final concentration 9 ~ . the absence of ether. In contrast, ether decreased the Ph activity observed at pH 4~2 . In the presence of ether, hornet venom Ph shows greater activity at pH 8 than at pH 4, both with egg yolk (Table 4) or purified phospholipid (Table 2) as substrate. The results suggest that differences in the physical state of the phospholipid substrate are responsible for the differences in pH optima with egg yolk and with purified phospholipid substrates . We next carried out an analogous experiment to that shown in Table 4 ; instead of egg yolk, using the purified phospholipid substrates dissolved in collidine-acetate or Tris buffer in the presence and absence of ether (Table S) . The tubes containing lecithin were sonicated prior to incubation . Although venom sacs were used, the results would be expected to be similar to those for pure venom . In collidine-acetate buffer with added ether, the results are similar to those shown in Table 2, that is, Ph activity is greater at the alkaline pH than at the acidic pH . The activities were not markedly different in Tris or collidine acetate buffer. The addition of ether slightly decreased activity in 6 out of the 8 experiments, and somewhat increased the activity in the remaining two experiments . The results clearly show that as long as the lecithin containing incubation solutions are sonicated, the addition of ether is not required . Ether, under the conditions described in Table S, does not drastically

Phospholipases A and B in the Hornet

149

alter Ph activity in contrast to the results obtained in Table 4. These experiments have made it clear that the apparent pH optima observed with Ph is critically dependent upon the choice and availability of substrate. The function of the alkaline (Dufour's) gland is obscure ; several suggestions are noted in the review by M~scxwrrz and IClolrr (1971) . One ofthe suggestions is that the secretions of the alkaline gland neutralize those of the acid gland, in the process either increasing or decreasing toxicity . We carried out an experiment to see if there are any inhibitors of Ph activity in the glands of the venom apparatus . We measured the liberation of free fatty acids by venom, venom sac, acid and alkaline glands and calculated theoretical values expected from combinations . These theoretical values were then compared with the actual values observed when venom and venom gland materials were combined (Table ~. Most of the actual activities found in the combinations were not markedly different from the theoretical values expected, indicating that no inhibitors or activators of Ph are present. The apparent exception is the combination of venom plus venom sac which showed activities lower than expected at both pH values . To check for immunologic cross-reactivity between the Ph enzymes from these various sources, antisera were produced in rabbits, and the ability of these antisera to inhibit Ph activity was measured (Table 7). In a volume of 0" 1 ml control rabbit sera had no effect on the Ph activity from any of the hornet tissues or on that of the pure venom. The titres of the venom and venom sac antisera were much higher than those of the acid and alkaline TABLE

t1 .

RELEASE OF FREE FA1TY ACIDS (FFA) PROM EGG YOLK HY VENOM (V), VENOM SACS (VS), ACID GLAND (H + ) AND Ai r~SixE GLAND (OH - ) ALONE AND IN CO~iHINATiON

Enzyme source

V VS H+ OH V + HF V -F OHV + H+ ~- OHV + VS VS + H+ VS + OH' VS -I- H+ + OH' H+ + OH-

Theoretical 12 "3 10~8 13 "3 16"6 9~4 7"9 10 "5 3"6

Actual pH 4"2 9"7 6~9 2"6 1"1 11 "8 10~7 1l " 1 11 "3 8~8 8~5 9"4 3"9

uequiv . FFA ~ Diff Theoretical -4 -1 -17 -32 -7 +7 --l0 +8

4~7 4~0 5"8 3"8 3"0 4"4 I"9

Actual pH 8" 1 3"4 2"4 I~4 0"6 4"0 3"7 4"6 3"2 2"8 3"5 I"8

~ Diff -16 -6 -21 -16 -9 -20 -8

The concentrations used (~Ig protein per ml) were : V, 5 ; VS, 50 ; H+, 50 ; OH - ,25 . One ml of egg yolk substrate (1 :5 dilution) containing 0'1 M Tris was incubated for 20 min with indicated enzyme source after which fatty acids were extracted and tittered . Some of the theoretical values do not exactly equal thoseobtained from theaddition of the"actual" values because the data in the table has been rounded off to one decimal place.

gland antisera. Both the venom and venom sac antisera caused marked inhibition of the Ph activity from all four sources, with some indication that the acid gland Ph activity was most inhibited and the maximum per cent inhibition seemed least with alkaline gland Ph. Acid and alkaline gland antisera also inhibited Ph activity from all four sources although the extent of inhibition was much less than with the venom and venom sac antisera . Both the acid and alkaline gland antisera seemed to be most effective in inhibiting the alkaline gland Ph activity. These results indicate a great deal of immunologic similarity in the Ph enzyme from these four sources. We checked the Ph activity of venom sacs as a function of age. It can be seen in Table 8

150

PHiLII' ROSENBERG, JACOB ISHAY AND SIMON GITTER

that some slight amount of Ph activity is observed in venom sacs even at 2 days prior to emergence, with about 20 times higher activity being reached by 5 days after emergence. These results suggest that venom secretion begins prior to emergence. DISCUSSION

Our results clearly show that oriental hornet venom is a rich source of both PhA and PhB (lysophospholipase) activities . This conclusion is based on the following evidence : 1 . The number of Etequiv free fatty acids liberated from 1 ml of a 1 :5 diluton of egg yolk by 50 ug of venom is about 28 (Fig. 1), whereas there is a total fatty acid content of only about 33 )tequiv. Any liberation above 16"5 indicates therefore hydrolysis of the second fatty acid of the phospholipid molecule. 2. Preparation of a lipid extract of the egg yolk and separation of the phospholipids after exposure to venom and venom sacs shows a decrease in total lipid phosphorus, which would only be observed if Ph activity were present. Phospholipid analysis also showed that both the lecithin of the egg yolk, and the lysolecithin TAHLE 7. INHIBITION OF Ph ACIIVIIY OF HORNEI VENOM (V), VENOM SAC (VS), ACID GLAND (H' ) AND ALKALINE GLAND (OH') HY ANTISERA PRODUCED AOAINSr FACH

Antisera V

VS

H+

OH'

Enzyme kg protein/ml source V 10

0 14 "9

VS

30

15 "5

H+

100

5"7

OH -

25

1 " 13

V

10

14 "9

VS

30

15 "5

H*

100

5"7

OH'

25

1 " 13

V

10

14"9

VS

30

15 "5

H+

100

5~7

OH'

25

1 "13

V

10

14" 9

VS

30

15 "5

H*

100

5~7

OH'

25

1"13

0" 1 021 (1 0"42 (3 0 (0 0"21 (19 0"64 ( 4 0"85 (5 0"21 ( 4 0"21 (19 10~6 ( 71 13 "4 ( 86 4"S ( 79 0"42 ( 37 11 " 0 ( 74 11 "9 ( 77 4"9 ( 86 0"32 (28

uequiv. FFA ml of Antsera 001 0'03 1 "3 0~42 3 9 1 "9 2"3 12 15 0"42 1 "3 7 23 0~42 37 1~70 3"4 23 11 2" 8 5"3 18 34 0"21 0"42 4 7 0"42 37 11 ~7 12 "5 79 84 l42 15 " 1 92 97 5" 1 5~1 89 89 0" 64 -57 12~5 12 "5 84 84 13 "4 13 "4 86 86 4"9 5.7 86 100 0"64 57 -

0001 10~0 67)* 11 "9 77) 1~3 23) 0"64 Sn I1 " 7 79) 12~7 82) 2"1 37) 0~42 37) 12 "7 86) 15~1 97) 5~3 93) 0" 85 75) 13 "2 89) 15"5 100) 5"9 104) 0"85 75)

The enzyme source (in 0" 1 ml) was incubated with the antisera for 15 min and then 1 ml of a 1 :5 dilution of egg yolk in 0"85 % saline plus 0" 1 M Tris (pH 4) was added. Incubation with substrate was for 10 min with V and VS and for 20 min with H+ and OH-. Free fatty acids (FFA) were then extracted and titrated. The Antsera alone caused no measurable release of fatty acids. The control values (0 ml antisera) are means of 4 experiments, all of which were within ~ 10 ~ of each other. `Percentages of control activity (0 ml antisera) are shown in parenthesis.

lhospholipases A and B in the Hornet TABLE 8. EFFECT OF AGE OF HORNET ON

Age (days) -2 -1 0 1 2 4 5 6

uequiv 0"4 0"4 0"8 8"3 6"7 6"6 10 "8 8" 3

Ph

151

ACl'IVrrY OF VENOM SACS

FFA 0~8 0"8 1~0 8"9 7"S 6" 7 11 "S 10 "0

Venom sacs (100 ug protein) were incubated for 10 min with a 1 :5 dilution of egg yolk in 0"85 ~ saline (pH 4"~ containing 0"1 M Tris. Free fatty acids (FFA) were extracted and titrated . Duplicate determinations at each age are shown in table. formed from it were hydrolyzed by the venom and venom sacs . 3. Fatty acids were hydrolyzed from both pure lecithin and lysolecithin . This was demonstrated both by titration of liberated fatty acids, and by phospholipid measurements on separated spots scraped from thin-layer plates . We have not studied whether there are two Ph enzymes, that is a PhA and a lysophos~ pholipase or whether both fatty acids are removed by a single enzyme. In V. palaestinae and N. raja snake venom it was shown that a single enzyme was responsible for both PhA, and PhB activities (SHiLOAH et al., 1973a, b) . In Penicillium notattan also, monoacyl and diacyl hydrolyse activity was associated with a single enzyme with the monoacyl (lysophospholipase) activity being 100-fold greater than the diacyl (KAwASnxl et al., 1975 ; BEARS and ICATES, 1967 ; $AII'O and KA~s, 1974). They suggested that a single enzyme (PhB) was responsible for the diacyl hydrolase activity and simultaneously hydrolyzed both aryl groups (at the l and 2 positions) with no intermediate lysophospholipid being observed . This is obviously not the case with hornet venom, since we did detect lysolecithin when lecithin was used as substrate (Table 3) . In mammalian systems only separate PhA and lysophoapholipase activities have been detected ; the simultaneous hydrolysis of both fatty acids is apparently not observed (ANSELL et al ., 1973). In the studies with snake venoms, PhB activity was much less than the PhA~ activities. In our studies, hornet venom appears about equally rich in both PhA and lysophospholipase activities, that is, both fatty acids are readily split by hornet venom, with no apparent great differences in the potencies of these two enzymatic activities . It will require separation and purification from hornet venom of proteins showing Ph activities in order to critically determine whether we are dealing with one or two enzymes. If there are two enzymes, it is likely that the PhA enzyme is of the Aa type since PhAt has never been found in venoms or toxins .

Our results show dramatically the marked effects which the physical state of the phospholipid substrate may have on the observed activity of Ph enzymes. Pure lecithin and lysolecithin were hydrolyzed by hornet venom better at pH 8 than at pH 4 regardless of whether collidine--acetate or Tris buffer were used or whether ether was present or absent (as long as the lecithin solutions were sonicated) . An alkaline pH optimum has also been noted for bee venom PhA using synthetic lecithin as substrate (IVIi7NJAL and ELLIOTT, 1972), whereas PhB from Penicillium notatum shows optimal activity at pH 4 (BEARE and KATES, 1967), while mammalian sources of this enzyme have a neutral or alkaline pH optimum (Els$xHERG et al., 1968 ; VArr DEx BoscH er al., 1973 ; L>aEOVrrz and GA'1-r, 1968). In marked contrast to our results with pure substrates we found that when egg yolk was the substrate, with either Tris or collidine buffer, hydrolysis proceeded much more rapidly at an acidic pH . The addition of ether to the egg yolk, however, increased hydrolysis at pH 8 and

152

PHILIP ROSENBERG, JACOB ISHAY AND SIMON GIT?ER

decreasedhydrolysis atpH 4 thereby reversing the pH potency profile so that hydrolysis now proceeded more rapidly at the alkaline pH. An inhibitory effect of ether on snake venom (DoexY and Penxsox, 1964 ; MAHOMED et al., 1969) and Penicillium notatum (KAWASAfiI et al., 1975) PhB activity has been reported . The results with the pure substrates show that ether is probably not having an effect on the enzyme . The exact physicochemical basis for the differences in the effects of ether at pH 4 and 8 have yet to be studied. Our results with pure lecithin show that Ph activity of hornet venom can readily be demonstrated in the absence of ether as long as the lecithin solution is sonicated. It was similarly found with a PhB from Pentcillium notatum, which hydrolyzes both aryl groups, that ultrasonically dispersed lecithin could be hydrolyzed in the absence of lipid activators such as ether (1CATE5 et al., 1965). The requirement for ether, in the absence of sonication, is well known and has been related to increased exposure of the phospholipid substrate and removal of the liberated fatty acids (DAWSON, 1963, 1973). The Ph activity was highest in the pure hornet venom, followed in order of potency by venom sacs, acid gland and alkaline gland . The Ph activity of the alkaline gland while only about 3 ~ of that of the pure venom (each at 50 ~g protein per ml, Fig. l), is still about 200 times greater than that of the fat body, midgut or hemolymph ofthe hornet all of which showed activity only at 10 mg protein per ml. The Ph activities of the venom sac, acid and alkaline gland appear due to the enzymatic activities of the material contained in their lumen. The much lower Ph activities (per pg protein) in the acid glands, alkaline glands and venom sacs than in the pure venom clearly indicates that the cells where the venom is produced or stored have little Ph activity. The Ph activities ofcombinations of venom, venom sac, acid and alkaline gland when compared to their separate activities (Table 6) show that there are no strong activators or inhibitors of Ph in these preparatons which could be responsible for their markedly different activities . We felt that it was important to check this point since, for example, extracts of venom sac contain many more proteins and other constituents than contained in pure venom (O'Cox~:oR et al., 1964; HSIANG and ELLi07"r, 1975 ; ROSENBROOK, JR . and O'CoxxoR, 1964). The presence of Ph activity in the alkaline (Dufours) gland is of special interest because this gland has not been classically considered as a venom producing gland, and indeed its function is obscure (MascHwrrz and KLOFr, 1971). Our results suggest that the possible contribution of the alkaline gland to production of venom constituents should be re-evaluated . The Ph enzymes from the various hornet sources show a considerable degree of immunologic cross-reactivity as evidenced by the inhibition of Ph activity from all sources by all antisera, although there were some differences in the relative sensitivities (Table 7). Both Ph and hyaluronidase of hymenopteran venoms are known to be highly antigenic, whereas the lower molecular weight constituents show little or no antigenicity (HAHERMANN, 1971). We were able to develop much higher titres with venom and venom sac than with acid and alkaline gland because of the difficulty in obtaining adequate amounts of these glands for injection into rabbits. The actual amount of venom contained in the lumens of these glands is also very small, so that, for example, I mg of these glands would contain much less Ph antigenic protein than would 1 mg of pure venom or even 1 mg of venom sac. It is of interest that venom secretion begins apparently even before emergence of the hornet (Table 8). It has also been noted in bees that venom secretion begins prior to emergence and reaches a maximum at about 10 days of age (AUTRUM and KNEITZ, 1959). In the bee the protein content of the venom gland falls after the first week of life and this precedes degeneration of the secretory cells (OwEx and BRIDGES, 1976). It is not known whether a similar phenomenon occurs in the hornet .

Phospholipases A and B in the Hornet

153

PhA, and PhC have been extensively used as tools to evaluate the biological effects of removing the fatty acid ester at the second position of phospholipids or removing the phosphorylated base of phospholipids (for references, see Rosi rtBt?RG, 1976) . These studies have shown that disruption of hydrophobic forces of interaction between phospholipid and protein with PhA $ have much more drastic effects on membrane structure and function than does disruption of hydrophilic forces of interaction by the use of PhC . The effects of PhB, plus PhA, which would cause even more drastic disruption of hydrophobic forces of interaction, by hydrolysis of both fatty acids, has not been studied because no rich source of PhB was readily available . If purified from hornet venom, PhB should be an extremely useful took for studying the function of phospholipids in membranes . Since hornet venom is such a rich source of Ph enzymatic activity, the possible contribution of this enzyme to the many and varied pharmacological effects of hornet venom must be carefully considered . For example, the oriental hornet venom has anticoagulant and hemolytic properties (JOSHUA and Ist-uY, 1973, 197, causes lesion development in the muscle transverse tubular system (ISHAY et al., 1975), causes hyperglycemia (ISHAY, 1975), has marked effects on the cardiovascular and central nervous systems (KAPLINSKY ft al ., 1974 ; IsrIAY et al., 1974) and, in addition, has other pharmacological actions (EDERY et al ., 1972) . It c:an no longer be automatically assumed that Ph enzymes are non-toxic constituents of venom. Some PhA Y enzymes from snakes and bees have potent pharmacological effects (see RossNS>;ttG, 1977, and HABERMANN, 1971, for references) . It should now be determined whether the Ph enzymes) in hornet venom are toxic or have major pharmacological effects, which could explain the effects of the whole venom . Bee venom PhA has been reported have an i .v . hDSU in mice and rabbits of 7 mg per kg and < 0~5 mg per kg, respectively eHABERMANN, 1971) . The effects of the other known constituents of hornet venom besides PhA and PhB, such as histamine, serotonin, acetylcholine, kinin and hyaluronidase eHABBRMANN, 1968, 1971, 1972) would not appear to be able to explain all of the effects of the crude venom . Acknowledgements-One of the authors (P.R.) expresses his deep thanks to Dr. $IMON GrrreR, Chairman of the Departments of Physiology and Pharmacology, Sacklet School of Medicine, Tel-Aviv University, Israel, for helping him obtain a United Nations, World Health OrganiTation Special Consultantship during his year's sabbatical stay in Israel . He is also grateful to Professor AxnRe De VRn?s and Er.eoxoRA CONDREA of the RogoffWellcome Research Institute, Beilinson Hospital, Petach Tikva, Israel, for making available their research facilities at which many of these studies were performed . REFERENCES AHRAI3AM5, G . (1955) Uber Gewinnung und pharmakologische Wirkungen von Hornfesen- und Wespengift . Inaug .- Diss. Wurzburg (cited in HASeRrrexrr,1968) . ANSer .L, G . B ., HAw-rEroRNe, J. N, and DAwsox, R. M . C . (Eds.) (1973) Form and Function of Phospholipids BBA Library, Vol . 3, second ed . Elsevier, New York . AvrRU~r, H . and ICrrerrz, H. (1959) Die Giftsekretion in der Giftdruse der Honigbiene in Abhangigkeit vom Lebensalter. Biol . 261 .78, 598 . BARB-Nee, L ., RoseNSeRa, P. and IsrrAV, J. (1976) The venom apparatus of Vespa orlentalis : morphology and cytology . Toxicon 14, 65 . BARTLEI'T, G . R . {1959) Phosphorus assay in column chromatography . J . blot Chem . 234, 466 . BEARS, l . L . and Iü~s, M . (1967) Properties of the phospholipase B from Penlclllium notatum . J. Biochem. 45, 101 . Bonet~n~reR, F . S . (1930) Schadlingsfauna . Palestine . In : Citrus Entomology, p . 400 . Dr . W . Junk, Gravenhage . BODENHSIMER, F . S . (1933) Über die Aktivitat von Vespa orientalis F. im Johresverlauf. 1n : Palestine . Zool. Anz.101, 135 . %ONDReA, E . and De VR.IFB, A . (1965) Venom phospholipase A : a review. Toxicon 2, 261 . CONDREA, E., RosermeRO, P . and DETTSARN W.-D . (1967) Demonstration of phospholipid splitting as the factor responsible for increased permeability and block of axonal conduction induced by snake venom . Blochlm. biophys . Acta . 135, 669 .

15 4

PHILIP ROSENBERG, JACOB ISHAY and SIMON GTITER

CONTARDI A. and ERCOI.I, A. (1933) Uber die enzymatische Spaltung der Lecithine and Lysolecithine. Biochim. Z, 261, 275. CONTARDI, A. and LATZER, P. (1928) Die tierischen Gifte in der Chemic . Blochem. Z. 197, 222. DAWSON, R. M. C. (1%3) On the mechanism of action of phospholipase A. Blochem. J. 8B, 414. DAWSON, R. M. C. (1973) Specificity of enzymes involved in the metabolism of phospholipids. In : Form and Fmrctlorl of Phosphollpids, BBA Library, Vol. 3 pp . 97-116, (ANSELL, G. B., HAWTHORNS, J. N. and DAWSON, R. M. C., Eds.) . New York : Elsevier . DE JONC, J . G . N ., VAN DEN Boscx, H., RUKEN, D. and VAN DSENEN, L. L. M. (1974) Studies on Lysophospholipases IiI . The complete purification of two proteins ~rith lysophospholipase activity from beef liver. Biochim. biophys. Acta 369, 50. DOERY, H. M, and PEARSON, J . E . (1964) Phospholipasc B in snake venoms and bee venom . Biocharr . J. 92, 599. Dot, O. and NOJIMA, S. (1975) Lysophospholipase of Escherichia coil . J. biol. Chenr. 250, 5208 . DOLE, V. P. (1956) A relation between non-esterified fatty acid in plasma and the metabolism of glucose. J. clin . Invest . 35, 150. EDERY, H. ISHAY, J.. LASS, I . and GITTER, S. (1972) Pharnracological activity of oriental hornet (Vespa orientalis) venom . Toxlcon 10, 13 . EISENHERG, S .,STEIx, Y. and STEIN, O. (1968) Phospholipases in arterial tissue . II . Phosphatide aryl-hydra lass and lysophosphatide aryl-hydrolase activity in human and rat arteries . Biochim. biophys. Acta 164, 205. ERCOLI, A. (1940) Lecithasen . In : Handbuch der Enzymologie, Vol. I, pp . 480-494 (NORD, F. F. and WEIOSNHAGEN, R., Eds.) . Leipzig: Akad . Verlagsges. FOt ctt, J., Lees, M . and SLOANE-STANLEY, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues . J. biol. Chenr. 226, 497. FRANCIOLA, M . (1937) Contributo alla conoscenza dell lecitasi A e B. Eirzynrologia 3, 204. HABERMANN, E. (1968) Biochemie, pharmakologic and toxikologie der Inhaltsstoffe von hymcnoptcrengiften . Ergeb. Physlol. 60, 220. HAHERMANN, E. (1971) Chemistry, pharmacology, and toxicology of bee, wasp and hornet venoms . ln : Venomous Animals and Their Verronrs, Vol . IL1, Venomous Im~ertebrates, pp . 61-93, (BUCHERL, E. and Bucxl.EV, E. E., Eds.) . New York : Academic Press. HABERMANN, E. (1972) Bee and wasp venoms. Science 177, 314. HANAHAN, D. J. (1971) Phospholipases . In : Tire Enzymes, Vol. V, Hydrolysis, pp . 71-85, (BOYER, P. D., Ed .) . New York : Academic Press. HORTNAGL, H ., WINKLER, H. and HORTNA(iL, H. (1969) The subcellular distribution of lysophospholipasc in bovine adrenal medulla. Eur. J. Biochim. 10, 243. HSIANG, H. K . and ELUOTr, W. B. (1975) Differences in honey bec (Apis mellijera) venom obtained by venom sac extraction and electrical milking. Toxicor:l3, 145. ISHAY, J. (1964) Observations sur la biologie de la guepe orientale Vespa orientalis F. I~rsectes soc. 11, 193. ISHAY, J. (1973) The influence of cooling and queen phenomone on cell building and nest architecture by Vespa orientalis (Vespinae, hymenoptera) . liuectes soc. 20, 243. Isxnv, J. (1975) Hyperglycemia produced by Vespa orientalis venom sac extract . Toxlcon 13, 221 . isxAY, J. and BROWN, M. B. (1975) Pattern in the hunger signal of hornet larvae (Vespa orientalis). Experientia 31, 1044 . L4HAY, J. and RUTTNSR, F. (1971) Thermoregulation in Hornissennest . Z. vergl. Physlol. 72, 423. isxnv, J. and SADSx, D. (1975) Direction-finding by hornets under gravitational and centrifugal forces . Science 190, 802. ISxÂY, J. and SCHWARrL A. (1973) Acoustical communication between the members of the oriental hornet (Vespa orientalis) colony . J. acoust . Soc . An:. 53, 640. Isxav, J., BYrIN31Cl-$ALZ and Sxtn.ov, A. (1968) Contributions to the bionomics of the Oriental hornet (Vespa orieraalls FAB) . Israel J. Errt. 2, 45. IstrAY, J., GITTER, S. and FrscFU., J. (1971) The production and effectivity of rabbit antiserum against Péspa orientalis venom. Acta allerg. 26, 286. ISHAY, J., FISCHL, J. and GITTER, S. (1972) Investigation of wasp venom : antigenic relationship . Actrr pharmac. tox. 31, 71 . ISHAY, J., LASS, Y., BEN-SCHACHAR, D., GITTER, S. and SANDHANK, LI . (1974) The effects of hornet venom sac extract on the electrical activity of the cat brain. Toxico» 12, 159. ISHAY, J., Lass, Y. and SANneANx, U. (1975) A lesion of muscle transverse tubular system by oriental hornet (Vespa orientalis) venom : electron microscopic and histological study. Toxlcon 13, 57. JOSHUA, H. and IsxnY, J. (1973) The hemolytic properties of the oriental hornet venom. Acta phar»rac . tux. 33, 42 . JosxuA, H . and Isl-tAY, J. (1975) The anti-coagulant properties of an extract from the venom sac of the oriental hornet . Toxlcon l3, 11 . KANWAR, K. C. and Ssrttt, R. C. (1971) Sudenophilic and PAS-positive granules in venom gland of Vesper orierrtali.s . Toxlcon 9, 179.

Phospholipases A and B in the Hornet

1 SS

KAPLIIdSKY, E., ISHAY, J. and GIrrER, S. (1974) Oriental hornet venom: effects on cardiovascular dynamics . Toxlcon 12, 69 . ICATes, M., MADELEY, J. R. and BeeRe, J. L. (1965) Action of phospholipase B on ultrasonically dispersed lecithin . Biochlm. blophys. Acta .106, 630. Kewesexr, N., Suaerertt, J. and SArro, K. (1975) Studies on a phospholipasc B from Penicilllum rrotatunr. Purification, properties and mode of action . J. Biochem. 77,1233. Kteat.t, K. and HestttM, S. A. (1963) Measurement of serum triglycerides by thin layer chromatography and infrared spectrophotometry. J. Lipid Res. 4, 407. LAUTER, W. M . and V1LLA, V. L. (1939) Factors influencing the formation of the venom of the honeybee . J. coon . F,nt. 32, 806. LEIBOVI7Z, Z. and Gerr, S. (1968) isolation of lysophospholipase, free of phospholipase activity from rat brain. Biochim. blophys. Acta 164, 439. Lemovrrz_BErt GexsxoN, Z., Kostrex, I. and Gerr, S. (1972) Lysophospholipases of rat brain. J. biol. Chem . 247, 6840 . Lownv, O. H., RoseNattoucx, N. J., FAtte, O. L. and RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Cheor.193, 265. LYSZ, T. W. and RosexeExa, P. (1974) Conwlsant activity of Naja naja venom and its phospholipase A component. Toxlcon 12, 253. Mexu~n-rn, G. V., At..eteECttr, M., Fonn, T. and Srorz, E. (1959) Analysis of human plasma phosphatides by paper chromatography. Blochlm. blophys. Acta 36, 4. MARPLES, E. A. and Tttonti'sox, R. H. S. (1960) The distribution of phospholipase B in mammalian tissues . Blochem. J. 74 .123. MASCxwtrz, U. W. J. and Kt oFr W. (1971) Morphology and function of the venom apparatus of insects, bees, wasps, ants and caterpillars . In : Venomous Animalsand 79reir Venoms, Vol. III, Venomous Invertebrates, Chapter 44, pp . 1-60, BucaEta., W. and BUCKIEY, E. E., Eds.) . New York : Academic Press. MEt.oxU~t, B. S. (196 The actions of snake venoms on nerve and muscle . The pharmacology of phospholipase A and of polypeptide toxins . Pharmac. Rev. 17, 393 . MOHAMED, A. H., KAMEL, A.andAYOHe, M. H. (1969) Studies of phospholipase A and B activities of Egyptian snake venoms and a swrpion toxin. Toricon 6, 293. Murer., D. and EL110IT, W. B. (1972) Further studies on the properties of phospholipasc A from honeybee (Apls melllfera) venom. Toxlcon 10, 367. O'CoNNOtt, R., RoseNaxoox, W. and EntcxsoN, R. W. (1964) Disc electrophoresis of Hymenoptera venom and body proteins. Science 145,1320 . OweN, M. D. and BRIDGES, A. R. (1976) Aging in the venom glands of queen and worker honey bees (Apes mell{Jera L.) : some morphological and chemical observations . Toxlcon 14,1 . PAWLOWSKY, E. N. (1927) Hymenoptera-HautjIrlgler (die giftgen Hautdrusen dieser Insekten stehem rnit der abgeanderten L,egerohredem Stachel-in Verbürdung), pp . 78-108 . Jena : Giftiere . Roser~eRG, P. (1976) Bacterial and snake venom phospholipases : enzymaticprobes in the study of structure and function in bicelectrically excitable tissues. In- Animal, Plant and Microbial Toxins, Vol. 2, pp . 229-261, (OIiSAKe, A,, Ed .) . New York : Plenum . ROSENBERG, P. (in press) Pharmacology of phospholipase A, from snake venoms . In : Snake Venoms . Handbook of Experimental Pharmacology (Lee, C. Y., Ed .). New York : Springer. ROSENBROOK, W., JR . and O'CoNrroR, R. (1964) The venom of the mud-dauber wasp . III Sceliphron caementarium : general character, Can. J. Biodiem. 42, 1567 . Rvsseu,, F. E. (1967) Comparative pharmacology of some animal toxins . Ferl. Proc. Fedn. Am . Socs exp. Blol. 26,1206. SANDSANx, U., lSt1AY, J. and GCI-rER, S. (1973) Kidney changes in mice due to oriental hornet (Vespa orientalls) venom: histological and electron microscopical study. Acta pharmac. tox. 32, 442. Snrro, K. and KATES, M. (1974) Substrate specificity of a highly purified phospholipase B from Perricilliur» notatunr . Blochim. blophys. Acta 369, 245. $ttII.OAH, J., KIIBANSKY, C., De VRiGS, A. and BERGeR, A. (1973a) Phospholipase B activity of a purified phospholipase from Vipera palaestürae venom . J. Lipid Res. 14, 267. St~uLOAx, J., Kttaexsxv, C. and Ds VRIAS, A. (1973b) Phospholipase isoenzymes from Naja raja venom 11 Phospholipase A and B activities . Toxlcon 11, 491 . Vex DEN Bosch, H., AeRSn~,N, A. J ., Storeoorn, A. J . and VAN DEENEN, L. L. M . (1968) On the specificity of rat-liver lysophospholipase. Biochlm. blophys. Acta 164, 215. Vex DEN Bosctl, H., AARSMAN, A. J., DE JoxG, J. G. N. and Vex DEENV.N, L. L. M. (1973) Studies on Lysophospholipases I. Purification and some properties of a lysophospholipase from beef pancreas . Biochlm. blophys. Acta 296, 94 . VAN HANDSL, E. and ZuveRSt~ttT, D. B. (1957) Micro-method for the direct determination of serum triglycerides. J. Lab. clln. Med. 50,152. Zl,erxls, A., Zex, B. and Bosrt.s, A. G. (1953) A new method for the direct determination of serum cholesterol. J. Lab. clip . Med. 41,486.