ACTION

OF

SNAKE

A 2 ON A N A L O G S

OF

VENOM

PHOSPHOLIPASE

PHOSPHATIDYLCHOLINE

V. V . B e z u g l o v , Yul. a n d L . D. B e r g e l ' s o n

G.

Molotkovskii,

UDC 577.153

Phospholipase A z is an enzyme widely distributed in nature which selectively hydrolyzeS phosphatidylcholine (PC) (I) with theliberation of the acid occupying position 2. It has been shown previously that of the diol analogs of PC (III-V) only the ethylene glycol analog (III) is hydrolyzed by phospholipase A z at an appreciable rate, while other analogs undergo e n z y m o l y s i s only to an insignificant degree [1-3]. CIsH31COOCH.-

RCOOCH2

I

C1rH:~3COOCH I

(CH,,)n I

( ICH2). O il

0

I

rt

+

CH~-O --POCH~CH.~NMe, +

I O-

CH.,--O--F'OCH.:CH~NMe3

I

O1, n = 0 II, n--2

I11, n=0,

R=CI:H3r,

lV, n=2,

R--

Ci;,H3,,

V, n~:3, R~-C~zH3a C i6H2:~OOC t CH20 I ',1 ~CH:OPOCHeCH:NM e3 I OVI

The low rate of enzymatic h y d r o l y s i s of diol phospholipids can be explained by s e v e r a l factors. In the first place, in the diol analogs of PC the fatty acid is bound to a p r i m a r y hydroxyl, while in PC it is split off from a secondary hydroxyl. In the second place, P C contains, in addition to the e s t e r group attacked, a second e s t e r group unaffected by the enzyme but, possibly, activating the first one. In the third place, it is known that the p r e s e n c e of only one fatty-acid residue in the molecule of a diol phospholipid changes its physical p r o p e r t i e s , including the capacity for being included in micelles and the specific a r e a on the surface of a micelle, which is important for the c o u r s e of enzymatic h y d r o l y s i s [3, 4]. In o r d e r to investigate how the capacity of a phospholipid for being hydrolyzed by phospholipase A z depends on the distance between the phosphate and e s t e r groups when two e s t e r groups are present in its molecule, we have subjected to h y d r o l y s i s by snake venom phospholipase A z a pentanetriol analog of PC r a c - 4 - o l e o y l o x y - 5 - p a l m i t o y l o x y p e n t y l p h o s p h o r y l c h o l i n e (II) - i n which the phosphate group is separated from the c l o s e s t e s t e r group by two additional methylene links as c o m p a r e d with PC. In addition, synthetic PC - 2 - o l e o y l - l - p a l m i t o y l - 3 - s n - g l y c e r o p h o s p h o r y l c h o l i n e (I) - and also the butanediol (IV) and pentanediol (V) analogs of PC and another analog (VI) in which the diglyceride part has been r e p l a c e d by the residue of fl-hydroxypropionic acid esterfied with cetyl alcohol has been subjected to enzymatic hydrolysis. As can be seen from the r e s u l t s given (Table 1), the hydroxypropionyl analog of PC (VI) proved to be completely stable to the action of phospholipase A2, and the diol analogs (IV) and (V) were cleaved only slightly. At the same time, the pentanetriol analog of PC (II) was cleaved by the enzyme at an appreciable M. M. Shemyakin Institute of Bioorganic C h e m i s t r y , Academy of Sciences of the USSR. T r a n s l a t e d from Khimiya Priorodnykh Soedinenii, No. 3, pp. 413-415, May-June, 1975. Original article submitted April 11, 1974. ©19 76 Plenuoz Publishing Corporation. 22 7 West 1 7th Street, New York, N. Y. 10011. No part o f this publication may be reproduced, stored in a retrieval system, or trausmitted, in any form or by any means, electronic, mechanical, photocopying, rnicrofilming, recording or otherwise, without written permission o f the publisher. A copy o f this article is available front the publisher for $15.00.

421

TABLE 1. Cleavage of Phosphotipids by Phospholipase A 2

Phospholipid

Phosphatidylcholine (1) Diol analogs of PC butanediol analog (IV) pentanediol analog (V) Pentanetriol analog of PC (ll) Hydroxypropionyl analog of PC (VI)

Reaction Time

of fatty acid split off (means of three experiments)

7 rain

100 (oleic) 3 1 11 (palmitic)

20 h 2 (oleic) 0

rate, although considerably m o r e slowly than PC itself. In this p r o c e s s , the acid bound to the p r i m a r y h y droxyl (palmitic) was split off to a g r e a t e r degree than the acid e s t e r i f y i n g the s e e o n d a r y h y d r o x y group (oleic), while in the case of PC it is known that only the acid substituting the secondary hydroxyl is split off. These differences cannot be a s c r i b e d to the d i s s i m i l a r natures of the fatty-acid residues, since it is known that in the case of PC a change within small limits of the length and degree of unsaturation of the fatty acids s c a r c e l y affects the rate of their splitting off by phospholipase A2 [1]. Apparently, an important condition for the effective action of phospholipase A z is the vicinal position of the phosphate and e s t e r groups: Under conditions close to those described in the present paper the ethylene glycol analog of PC (III) is m o r e than 40 % hydrolyzed [2]. When, however, the e s t e r group is remote from the phosphate group, the splitting off of the fatty acid residue from a p r i m a r y hydroxyl takes place more readily than from a secondary, probably because of the s m a l l e r steric hindrance. The higher rate of enzymatic h y d r o l y s i s of the pentanetriol analog of PC (II) as c o m p a r e d with the diol analogs (V) and (IV) can be explained by a difference in the physical p r o p e r t i e s of the micelles which they form and, in the first place, by the different specific a r e a s occupied by the c o r r e s p o n d i n g molecules at the s u r f a c e o f separation of the phases. It has been shown by van Deenen et al. [4] that h y d r o l y s i s by phospholipase A 2 (pancreatic) o2 takes place fastest where the PC molecule occupies an area of about 90 A , which c o r r e s p o n d s to a PC with two caprylic acid residues. It is obvious that the pentanetriol analog (II) occupies an a r e a on the surface of the micelles which is c l o s e r to the optimum than the diol analogs (IV) and (V). EXPERIMENTAL

METHOD

The solvents were purified by standard methods. F o r t h i n - l a y e r c h r o m a t o g r a p h y (TLC) we used type KSK silica gel with 5% of gypsum. The pentanetriol (II) and hyroxypropionyl (VI) analogs of PC, and also the diol analogs (IV) and (V) were synthesized as described previously [5]. As the preparation of phospholipase A 2 (EC 3.1.1.4) we used lyophilized venom of the Central Asian cobra (Naja naja oxiana). A mixture of 1 mg of phospholipid (the accurate amount was calculated from the phosphorus content [6]), 0.7 ml of borate buffer (pH 7.5), 1 ml of ether, and 0.2 ml of a 0.8% aqueous solution of sodium deoxycholate was emulsified for 5 min with ultrasound (22 kHz, intensity 50 W) with ice cooling. To the resulting emulsion was added 0.2 mt of a 0.005 M solution of CaCI 2 and 1 mg of lyophilized c o b r a venom, and the mixture was incubated with shaking in a t h e r m o s t a t e d space (28° C). After the end of the reaction, a solution of the standard for GLC (margaric acid) was added to the mixture, it was evaporated to d r y n e s s in vacuum at 20 ° C, i ml of a mixture of c h l o r o f o r m and methanol (1 : 1) was added, and it was treated with an ethereal solution of diazomethane and evaporated. The residue was separated by p r e p a r a tive TLC in benzene (zones visible in UV light after treatment with morin). The zones containing the methyl e s t e r s of the fatty acids were identified by c o m p a r i s o n with the standard (methyl m a r g a r a t e ) and were separated off and eluted with c h l o r o f o r m . In the e p x e r i m e n t s with the hydroxypropionyl analog of PC (VI), a solution of m y r i s t y l alcohol was used as standard, the mixture was evaporated to dryness, and the residue was separated by preparative TLC in the b e n z e n e - e t h y l acetate (3 : 1) system, (the spots being revealed with morin). The zone containing the fatty alcohols was eluted with c h l o r o f o r m containing 5 ~7cof methanol, the eluate was evaporated to d r y n e s s , and the fatty acids were converted into their trimethylsilyl d e r i v a tives by treatment with chlorotrimethytsilane and hexamethyldisilazane in pyridine [7]. The amount of fatty acids (alcohols) was determined by GLC.

422

SUMMARY

The h y d r o l y s i s by phospholipase A 2 of the following synthetic phospholipids has been investigated: 2-oleoyl-l-palmitoyl-3-sn-glycerophosphorylcholine, 4-stearoyloxybutylphosphorylcholine, 4-oleoyloxy-5palmitoyloxypentyIphosphorylcholine, and 2-(hexadecyloxycarbonyl)ethylphosphorylcholine. LITERATURE 1o 2. 3. 4.

5. 6.

7.

CITED

L. L. M. van Deenen and G. H. d e H a a s , B i o c h i m . Biophys. Acta, 70_._,538 (1963}. V. I. Kozhukhov, Yul. G. Molotkovskii, and L. D. B e r g e l ' son, Biokhimiya, 34, 1236 (1969). P. P. M. Bonsen, W. A. P i e t e r s o n , and L. L. M. van Deenen, Biochim. Biophys. Acta, 23~9, 252 (1971}. G. H. de Haas, P. P. M. Bonsen, W. A. P i e t e r s o n , and L. L. M. van Deenen, Biochim. Biophys. Acta, 239, 252 (t97I}. L. F. Nikulina, V. I. Kozhukhov, Yul. G. Molotkovskii, and L. D. B e r g e l ' s o n , Izv. Akad. Nauk SSSR, Ser. Khim., 410 (1973). E. G e r l a c h and B. Deuticke, Biochem. Z., 337, 477 (1963}. C. C. Sweely, R. Bentley, M. Makita, and W. W. Wells, J. A m e r . Chem. Soc., 8_.55,2497 (1963}.

423