Toxtem, 1977, Vol. 15, pp.347-354. Perpmon Pree. Printed in Great Britain

CATALYTIC PROPERTIES OF TWO PHOSPHOLIPASES A FROM COBRA (NAJA NIGRICOLLIS) VENOM J. DuPoNT Laboratoire de Biologie Végétale IV, 12 rue Cuvier, 75005, Paris, France (Accepted for publication 12 December 1976) J. DuPoNT. Catalytic properties of two phospholipases A from cobra (Naja nigrieollis) venom. Toxicon 15, 347-354, 1977 .-The catalytic properties of two proteins with phospholipase A activity, isolated from Noja n(gricollis venom, have been studied. They are of type A, (E .C. 3.1 .1 .4) toward phosphatidyl choline and phosphatidyl ethanolamine. These enzymes are active in the presence of sodium deoxycholate (DOC) or Triton X-100. Ca'* does not appear to be necessary for enzyme activity . Their optimum pH is 8-50 . INTRODUCTION

A (E.C. 3.1 .1 .4) catalyzes the hydrolysis of diacyl phosphoglycerides into their monoacyl analogues with the release of free fatty acids fromthe ßposition . This activity has been detected in the venoms of snakes as well as in several mammalian tissues (VAN DEENEN and DE HAAS, 1966) and plant organelles (DoucE and LANCE, 1972). SArro and HANAIIAN (1962) reported the isolation of two highly purified proteins with phospholipase A activity from the venom of Crotalus adamanteus . These enzymes differ significantly in their electrophoretic mobilities and isoelectric points, but exhibit the same catalytic behaviour. More recently, WAHLsTROM (1971) reported the purification of two phospholipases A from the venom of Naja nigricollis . One of these proteins appears to be acidic in nature, whereas the other appears to be a basic protein. The physical and toxicological properties of the basic protein have been recently published by DumAREY et al. (1975). The amino acid sequence of this basic protein has also been determined (]BAKER, 1976). In this paper we report on the comparison of the catalytic properties of both proteins . PHOSPHOLIPASE

MATERIALS AND METHODS The crude venom taken from the black-necked spitting cobra (Naja nigrïcollis) was collected in Ethiopia . The acidic and basic phospholipases were purified by Dr. Boquet, of the Pasteur Institute at Garches, France (DumARElt et al ., 1975). The purity of the two phospholipases A proteins was ascertained by several methods. In the ultracentrifuge a single symmetrical sedimentation peak was obtained for the two proteins . Moreover, each fraction gave a single band on disc electrophoresis (DumAREY et al., 1975). Phosphaddyl choline from egg yolk, phosphatidyl ethanolamine from yeast, phosphatidic acid from corn seedlings and cardiolipin (diphosphatidyl glycerol) from beef heart mitochondria, used as substrates in activity assays were gifts of Dr. Faure (Pasteur Institute, Paris) . The various substrates were checked for purity using deacylation with methanolic KOH (DAWSON, 1960). For the determination of the enzyme specificity, phosphatidyl choline, previously purified by TLC, was converted to 1-aryl-lyso phosphatidyl choline by the action of phospholipase A, from Vipera russelli (Sigma, St . Louis, Mo., U.S .A .) as described by SArro and HANmiAN (1962). Lipid analysis Fractionation of lipids was achieved by one-dimensionalpaperchromatography (Silica gel 81, Whatman) . A chloroform-methanol-water mixture (65 : 25 : 4, v/v/v) was used as the developing solvent. Identification 347

J. DUPONT

34 8

of phospholipids was carried out by exposure to vapours of osmium tetroxide, using pure phospholipid samples as reference standards. Quantitative estimations of phospholipids were made by determination of phosphorus (DUCET and MENCL, 1957). Following the actions of phospholipases A from Naja nigricollis or phospholipase As from Vipera russelli, free fatty acids resulting from the hydrolysis of the phospholipid molecule, or the fatty acids of the 1-acyl-lyso phospholipid (each present in the organic phase, chloroformic or diethyl ether phases) were treated by methanolysis (methanol-sulfuric acid ; 100 : 2-5, v/v) at 83° for 90 min (Duporrr, 1975). Methyl esters were extracted by light petroleum and gas chromatographed at 180° on a polar phase of diethyleneglycol succinate (DupoNT, 1975). Phospholipase A assay The assayprocedure used was similar to that described by KRAMER et al . (1974) . The standard incubation mixture contained 0-1 M Tris-HCl (pH: 8-50), 6 mg per ml of Triton X-100 or 6 mg per ml of sodium deoxycholate (DOC) and 15 mM phosphatidyl chohne (12 mg per ml). The final volume was 0-5 ml . The reaction was started by adding a small volume of the enzyme preparation . After incubation at 22°, the reaction was stopped by the addition of 2 ml of chloroform-methanol (2 : 1 v/v)-HCI 0-05 N in order to inactivate the proteins . The homogenate was shaken vigorously for 30 sec and centrifuged (5 min, 3000 rev per min) for complete separation into two phases. The aqueous phase and the protein layer were removed by aspiration . For further analysis, the chloroform phase was collected, evaporated under nitrogen and the residue dissolved in 1 ml of methanol . When the incubation mixture was a diethyl ether solution, the phospholipids were evaporated under nitrogen and dissolved in 3 ml of this solvent. Addition of 0-3 ml of 0-1 M Tris-HCI (pH 8-50) permits the formation of an aqueous phase. The reaction was started by addition of the enzyme preparation . Atfer incubation, the aqueous and diethyl ether phases were removed for analysis by one-dimension chromatography on paper (Silica gel 81, Whatman) . Determination ofprotein Protein concentrations of the enzyme preparations were determined by the procedure of LowRY et al. (1951) with bovine serum albumin as standard . RESULTS

Specificity ofposition and substrate Phosphatidyl choline was the main phospholipid used for studying the specificity of position of the two phospholipases purified from Naja nigricollis. Phospholipase Ae from Vipera russelli was used as control . Table 1 illustrates the hydrolysis of phosphatidyl choline under these conditions. The action of phospholipase A$ from Vipera russelli shows that unsaturated fatty acids, oleic (018 : 1) and linoleic (C18 :$), are preferentially fixed on the ß position of the glycerol moiety of the phospholipid molecule (VAN DEENEN and Ds TABLE 1. DISTRIBUTION OF THE FATTY ACIDS OF PHOSPHATIDYL CHOLINE ISOLATED FROM EGG YOLK, AFTER HYDROLYSIS BY PHOSPHOLIPASE A. FROM Vipera russelli (A), ACIDIC PHOSPHOLIPASE (B) AND BASIC PHOSPHOLIPAsE (C) FROM Naja nigricollis

The hydrolysis of phosphatidyl choline by phospholipase A, from Vipera russelli is used as a reference for the determination of the a and ß positions of the glycerol molecule (TATTRIE,1959) . Fatty acids were estimated as indicated in Materials and Methods. The system used was a diethyl ether medium . Phospholipid Phosphatidyl choline Total

Per cent of total fatty acids

Czs:0

Cte:0

Cie:i

CIe:9

37.3

15-3

32-3

14-8

(A)

Position a Position ß

62-0 15-3

32-5 -

5-6 60-5

24-1

(B)

Position a Position 8

55-0 12ß

36-0 4-3

8-8 61-7

21-7

(C)

Position a Position 0

61-9 11-3

31-4 3-5

6-7 61-3

23-8

Phospholipases from Naja

nigricollis

349

Venom

HAAS, 1966), whereas the saturated fatty acids, palmitic (Cle:o) and stearic C19 :o), are

preferentially fixed on the a position of the same molecule . The fatty acids released during hydrolysis are ascribed to position ß and those found in the 1-acyl-lyso phosphatidyl choline spot, to position a. The same conclusions can be drawn from the examination of the hydrolysis of the same phospholipid after action of the acidic and basic phospholipases from Naja nigricollis. We can then conclude that the two phospholipases A isolated from Naja nigricollis (WAmsTR6m, 1971) are specific for the fatty acids at the ß position of phosphatidyl choline . Such a result was also obtained with phosphatidyl ethanolamine as substrate. Similar experiments with phosphatidic acid or diphosphatidyl glycerol as substrates (in our experimental conditions, i.e. in a diethyl ether medium) show that no hydrolysis of fatty acids can be obtained. This point is currently under further investigation . The enzymatic action of these two phospholipases A$ on phosphatidyl choline was followed in the same diethyl ether medium and the composition of the liberated fatty acids at different time intervals was determined . At fixed intervals, the phospholipids (phosphatidyl choline and 1-acyl-lyso phosphatidyl choline) and the fatty acids liberated during hydrolysis were separated, after exposure of the plates to iodine vapour. The only products of this enzymatic reaction were 1-acyl-lyso phosphatidyl choline and free fatty acids (at pH 8-50) . The gas chromatographic analysis indicates that only unsaturated fatty acids, oleic (018,1) and linoleic (C18:ß), are hydrolyzed during the time of incubation. In addition, these results seem to indicate that no lysophospholipase is present in the enzymatic preparation, because no saturated fatty acids such as palmitic acid (Cle:u) appeared in high proportion during the incubation, even after a long time (KRAM et aL,1974) . Nevertheless, this conclusion should be carefully formulated, since other studies (SHII .OAH et al., 1973) showed lysophospholipase activity when the phospholipase A from Vipera palestinae venom was tested at a different pH. Effect of incubation time Figure 1 illustrates a time course ofhydrolysis ofphosphatidyl choline by Naja nigricollis basic phospholipase A, It is apparent that although the reaction proceeds very rapidly 0 N 5.0 w

J

4.0

óÉ 2.0 i-

Q a u) O x n. O

_}i

1.0

0

I

20

I

40

Time (minules)

I

60

FIG. 1. TIME-COURSE OF HYDROLYSIS OF PHOSPHATMYL CHOLINE BY BASK PHOSPHOLIPASE A, FRom Naja nigricollis vENom.

In a final volume of 0-5 ml, the incubation mixture contained 3 mg DOC, 15 mM phosphatidyl choline and 40 pg of protein.

350

J. DUPONT

(50% of the reaction being completed in the first 10 min) it does not go to completion even after 60 min of incubation (complete hydrolysis = 6 mg of 1-acyl-lyso phosphatidyl choline released). Paper chromatography analyses performed at each of the time points illustrated in Fig. 1 shows that similar results can be obtained with the acidic phospholipase A,: the reaction proceeds very rapidly but the substrate spot was still visible after 60 min incubation. Effect of enzyme concentration

At low concentrations, the reaction rate of these two phospholipases As from Naja nigricollis appears to increase linearly with the enzyme concentration . Even when the

enzyme concentration in the incubation mixture is high, the reaction does not go to com pletion (complete hydrolysis = 9 mg of 1-acyl-lyso phosphatidyl choline released) (Fig. 2). The inhibitory effect of very high concentrations of enzyme protein on the hydrolysis of phosphatidyl choline seems to be in accordance with results of AuGusTYN and ELLicrr (1970). 7.5

W Z O S U 0, E

O a O

0

200

400 600 Proteins (pg)

600

1000

FIG. 2. EFPHGT OF INCREASING CONCENTRATION OF BASIC PHOSPHOLIPAsE A, FRom VENOM ON THE HYDROLYSIS OF PHOSPHA11 DYL CHOLINE .

Naja nigricollis

Reaction conditions are as described in Fig. 1 . The medium contained 9-0 mg of substrate (22-5 mM). Time of incubation was 5 min.

Effect of pH and ionic environment

The effect of pH on the phospholipase Aa activity was assessed using phosphatidyl choline as substrate. As can be seen from Fig. 3, acidic and basic phospholipases A. from Naja nigricollis exhibit an optimal pH of 8-50 in our conditions of incubation. A very low activity can be found when the pH is lower than 7-0. The specific activities of the two phospholipases A are practically identical in the pH range 7-5-9-0.

Phospholipases from Nqja nigricollis Venom

35 1

6.0

0

Q W W W Z

5 .0

CA)

4.0

O x

J E3 .0 0

Q x o.

u> O x n. O

ï

2.0

1 .0

0

I

6.0

I

7.0

pH

8.0

9.0

FIG. 3. EFFECT OF pH ON HYDROLYSIS OF PH09PHATIDYL CHOI.IIQE BY ACIDIC PHOgPHOLIPAW A, (A) AND BASIC mospHoLmAm A, (B) FRom Nçja nigricollis vENom . Incubation medium is described under Fig. 1, except for the acidic phospholipase A, (A) where Triton X-100 (3 Ing) is used instead of DOC . Time of hydrolysis is 10 min for each

phospholipase . Medium (A) contained 50 pg of acidic phospholipase A, and medium (B) contained 60 pg of basic phospholipase A, . Phospholipase A from several venoms has been shown to be activated by the presence of Call+ in the reaction mixture (LONG and PENNY, 1957 ; ROHOLT and ScHLAmowrrz, 1961) . When Caß+ was added to reaction mixtures containing acidic and basic phospholi pases A ß from Naja nigricollis, only a slight stimulation of the activities of the enzymes occurred (20 and 3 %, respectively, for the acidic and basic phospholipases A) . Such a result can be obtained with the two enzymes when Caß+ is added to the reaction mixtures in concentration range from 5 to 50 mM . Although these results cannot be considered to be conclusive, they can be compared to those published by AUGusTYN and ELLIOT (1970) in which phospholipase A a from Agkistrodon piscivores appeared to be inhibited in presence of Cat+ . Further investigations seem to be needed on this point .

Determination of kinetic constants

The effect of the substrate concentration on the initial reaction velocity of the acidic and basic phospholipases A $ of Naja nigricollis is shown in Fig. 4 . The initial values are plotted according to the Lineweaver-Bark method . The reactions apparently follow Michaelis-Menten kinetics with K, of 17 and 25 gM, respectively, for the acidic and basic proteins . These values seem to reflect the high degree of purity of the enzymatic preparations (DumAREY et al., 1975) . Nevertheless, it is the first time that such low values of K, were obtained, in vitro, for phospholipases A, in presence of the natural phosphatidyl choline as substrate (DELORI, 1972 ; KRAMER et al., 1974 ; SArro and HANAHAN, 1962) .

352

J. DUPONT

K m=25 PM

0

0.1

0,2

1/5

4.

LINEWEAVER-BURK PLOT OF PHOSPHATIDYL CHOLINE HYDROLYSIS BY ACIDIC PHOSPHOLIPASE A; (A) AND BASIC PHOSPHOLIPASE A, (B) FROM Naja nigrlCOIlis VENOM . Varying amounts of phosphatidyl choline were incubated for 10 min in the presence of 50 pg of acidic phospholipase A_ (A) or 601Ig of basic phospholipase A= (B) Incubation media are FIG .

Effects of detergents

described in Fig .

3.

In order to determine the optimum incubation conditions, the action of two detergents commonly used in the biochemistry of phospholipids : Triton X-100 and sodium deoxycholate (DOC) were tested . Only low phospholipase activity was obtained in the standard reaction mixtures without detergents . Addition of DOC or Triton X-100 to the medium greatly enhanced the activity of the enzyme system. Table 2 shows the activating effect of DOC and Triton X-100 on hydrolysis of phosphatidyl choline . Optimal activity was obtained with 3 mg per 0-5 ml of standard mixture of both detergents. As shown in Table 2, Triton X-100 was more effective as an activator, compared to DOC for acidic phospholipase A2- On the other hand, DOC was more effective, compared to Triton X-100 for basic phospholipase A2. TABLE

2.

EFFECTS OF DETERGENTS ON THE ACTIVITY OF ACIDIC AND BASIC PHOSPHOLIPASES n4gricollis VENOM

A.

FROM

Naja

1-acyl-lyso phosphatidyl choline released mg per 10 min per 60 pg of protein Acidic phospholipase Standard +

DOC 3 mg per 0-5 ml Triton X-100 3 mg per 0-5 ml

1-60 3-50 420

Basic phospholipase

1-45 4-70 2-71

Ultrasonication

Basic phospholipase A2 was assayed following exposure of the substrate, i.e. phosphatidyl choline (suspended in an aqueous medium) to 30 kHz ultrasound for varying times at 2° (Bronwill, Biosonik III) . Sonication up to 60 sec gradually increases the enzyme activity (up to 25 % of the control) . Nevertheless, this increase is not comparable in intensity to that observed in the presence of detergents (Table 2). DISCUSSION

The present study demonstrates that the two phospholipases A isolated from Naja

Phospholipases from Naja nigricollis Venom

35 3

nigricollis venom catalyse the hydrolysis of exogenous phosphatidyl choline into free fatty acids and 1-acyl-lyso phosphatidyl choline. The findings of two distinct phospholipase A proteins in the venom of Naja nigricollis are similar to observations on other enzyme systems (BOMAN and KALETTA, 1957 ; PmRPDINT, 1957 ; TANFORD and HAuENsTEIN, 1956). In the field of snake venoms, SAITO and HANAHAN (1962) have isolated two enzymes from Crotalus adamanteus venom, which differed significantly in their electrophoretic mobilities and isoelectric points, but exhibit the same mode of action . Numerous other examples can be found in literature about the presence of two or more phospholipases A in the venoms of snakes (CuRRm et al., 1968 ; WELLS and HANAHAN, 1969). The acidic and basic phospholipases purified from Naja nigricollis venom show several properties classically related to phospholipase A activity and they show identical behaviour with phosphatidyl choline as substrate. It was observed that the fatty acids liberated by the two phospholipases A were predominantly unsaturated, mainly oleic and linoleic acids. Qualitatively, this distribution of fatty acids remains the same during the entire incubation period (60 min). This means that the proteins are not contaminated by a phospholipase of type A1 (i.e. which hydrolyses the a position of phosphatidyl choline), or by a lyso phospholipase A, with the restriction mentioned above. The Michaelis constants K, for these two proteins are not very different : 17 and 25 N.M, respectively, for the acidic and basic phospholipases A8. These values are somewhat different from those generally expected for hydrolytic enzymes. Our observations support the conclusion that acidic and basic phospholipases A have similar phospholipase As activity but differ primarily in their physical and chemical structures or both. These two phospholipases Aß isolated from Naja nigricollis venom possess similar molecular weights but have significantly different electrophoretic mobilities and isoelectric points (DumAREY et al., 1975 ; WELLS and HANAHAN, 1969). It is thus not likely that these two enzymatically active proteins are artifacts of the isolation technique. Acknowledgements-I am greatly indebted to Dr. P. BOQUET for valuable discussions, to Miss C. DumAny for the extraction and purification of phospholipases of Naja nigricollis venom and to Dr. M. FAURE for

the gift of phospholipid controls .

REFERENCES

Auaum-YN, J. M. and EuLioT, W. B. (1970) Isolation of a phospholipase A from Agkistrodon piscivorus venom. Biochim . biophys . Acta 206, 98. BomAN, H. G. and KALETrA, U. (1957) Chromatography of rattlesnake venom. A. Separation of three phosphodiesterases . Biochim . biophys . Acta 24, 619. CuRRIE, B. T., OAKLEY, D. E. and BRoomxmw, C. A. (1968) Crystalline phospholipase A associated with a cobra venom toxin . Nature, Lond. 220, 371 . DAwsoN, R. M. C. (1960) A hydrolytic procedure for the identification and estimation of individual phospholipids in biological samples. Biochem. .7. 75, 45. DEENEN VAN, L. L. M . and DE HAAS, G. H. (1966) Phosphoglycerides and phospholipases . A . Rev . Biochem . 35,157 . DELoRi, P. (1972) Purification et propriétés d'une phospholipase A, toxique isolée d'un venin de Viperidae: Vipera berus . Thèse de Doctorat Ingénieur, Faculté des Sciences, Paris, 74 pp. no. A.O. 6367. DoucE, R. and LANCE, C. (1972) Altération des activités oxydatives et phosphorylantes des mitochondries de Chou fleur sous l'action de phospholipases et du vieillissement . Physiol. Veg.10,181 . DUCHT, G. and MENCL, 1. (1957) Séparation et dosage de faibles quantités de composés phosphorés acidosolubles . AnnIs. agron . 2Bis, 17 . DumAREV, C., SxET, D., JosEPH, G. and BOQUET, P. (1975) Etude d'une phospholipase basique du venin de Naja nigricollis. C.R. hebd. Séanc . Sci ., Paris 280, 1633. DupoNT, J. (1975) Incorporation et accumulation du phosphate dans les membranes des mitochondries végétales isolées . Thése de Doctorat de 3éme Cycle, Université Pierre et Marie Curie, Paris, 91 pp . EAxm, D. (1976) Snake venom toxins reacting post- and pre-synaptically at the neuromuscular junction . Bull. Inst. Pasteur 74, 9. KRANii R, R., JuNai, B. and ZAHLER, P. (1974) Some characteristics of a phospholipase A, from sheep red cell membranes. Biochim. biophys. Acta 373, 404.

35 4

J. DUPONT

LONG, C. and PENNY, 1. F. (1957) The structure of the naturally occurring phosphoglycerides-Ill. Action of moccasin-venom phospholipase A on ovolecithin and related substances . Biochem. J. 65, 382. LowRY, D. H., RosEHRouoH, N. J., FARR, A. L. and RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem . 193, 265. PiERroimr, W. S. (1957) The phosphatase and metaphosphatase activities of pea extracts . Biochem . J. 65, 67. ROHOLT, O. A. and SCHLAMOwrrz, M. (1961) Studies of the use of dihexanoyllecithin and other lecithins as substrates for phospholipase A with addendum on aspects of micelle properties of dihexanoyllecithin . Archs Blochem. Biophys. 94, 364. SArro, K. and HANAHAN, D. J. (1962) A study of the purification and properties of the phospholipase A of Crotalus adamanteus venom. Biochemistry 1, 521 . SHILoAn, J., KuBANsKY, C., DE VRIEB, A. and BERGER, A. (1973) Phospholipase B activity of a purified phospholipase A from Vipera palestinae venom. J. Lipid Res. 14, 267. TANFoRD, C. and HAuENmiN, J. D. (1956) Identification of the chemical difference between chromatographic components of ribonuclease. Biochim. biophys. Acta 19, 535. TATTRrE, N. H. (1959) Positional distribution of saturated and unsaturated fatty acids on egg lecithin . J. Lipid Res. 1, 60 . WAfusnsáxt, A. (1971) Purification and characterization of phospholipase A from the venom of Naja nigricollis. Toxicon 9, 45 . WEDS, M. A. and HANAHAN, D. J. (1969) Studies on phospholipase A-1. Isolation and characterization of two enzymes from Crotalus adamanteus venom. Biochemistry 8, 414.