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Hydrolysis of cardiolipin by snake venom phospholipase A
BIOCHIMICAET BIOPHYSICAACTA
55
BBA 55000
HYDROLYSIS OF CARDIOLIPIN BY SNAKE VENOM PHOSPHOLIPASE A* G. V. MARINETTI Biochemistry Department, University of Rochester School of Medicine, Rochester, N.Y. ( U.S.A .) (Received August 2nd, i963)
SUMMARY Phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) from snake venom has been shown to hydrolyze cardiolipin in wet ether and yield lyso-cardiolipin. The enzyme reaction is slow but is greatly stimulated by the addition of beef heart lecithin. The lysocardiolipin was characterized by chemical and chromatographic analysis and by its hydrolysis products. Lyso-cardiolipin has an ester :P ratio of o.96 and yields monoglycerides upon acetic acid hydrolysis. Cardiolipin, on the other hand, has an ester :P ratio of 1.9 and yields diglycerides upon acetic acid hydrolysis. The fatty acids on the cardiolipin and lyso-cardiolipin are the same and thus it appears that these fatty acids have a fairly random distribution. INTRODUCTION The action of snake venom phospholipase A (phosphatide acyl-hydrolase, EC 3. I.I.4) on lecithin and phosphatidylethanolamine is well documented 1. It seemed of interest to see if this enzyme also were capable of hydrolyzing cardiolipin. The results reported in this paper demonstrate that phospholipase A can hydrolyze cardiolipi n to its deacylated lysoderivative which shall be given the trivial name of lyso-cardiolipin. The reaction occurs in wet ether and is greatly stimulated by the addition of lecithin. METHODS AND REAGENTS Beef heart cardiolipin and beef heart lecithin were purchased from the Sylvana Corp. Snake venom (Naia naia) was purchased from the Ross Allen Reptile Institute, Silver Springs, Fla. Paper and column chromatography and ester and P analyses were carried out as described previously ~. 38 mg of cardiolipin and 41 mg of lecithin were dissolved in 50 ml of ethyl ether (Merck, peroxide free). To this solution were added 0.2 ml of N a i a naia venom in isotonic saline (I mg venom). The solution was well mixed and let stand at room temperature. At hour intervals 2o-/zl aliquots were removed for paper chromatographic analysis. Although the reaction was complete after 4 h, the solution was let stand for 24 h. After this time period the solution was evaporated to dryness under vacuum and " Part of this work was presented at the Gordon Research Conference on Lipid Metabolism, Meriden, Conn., June 1962. Biochim. Biophys. Acta, 84 (1964) 55-59
50
G. V. MARINETTI
the residue dissolved in 5 ml of chloroform. The lipids were chromatographed on a I.O cm diameter column containing 15 g of Unisil silicic acid. The silicic acid was first washed with 40 ml each of heptane, ethyl ether and chloroform before the 5-ml aliquot of lipid solution in chloroform was applied. The lipids were eluted by the following solvents in the order given: a, 5 ° m l of chloroform; b, 80 ml of 20% methanol in chloroform; c, 80 ml of 50 % methanol in chloroform ; d, 80 ml of absolute methanol; e, 80 ml of methanol-chloroform-water (9 ° :IO :5, v/v). Fractions of 5 ml were taken by means of an automatic fraction collector. A control sample of cardiolipin and lecithin was run through the same procedure. In the control sample the venom solution was replaced b y 0.2 ml of saline. Each column fraction was analyzed for P content and each fraction was also analyzed by paper chromatography on silicic acid impregnated paper. In addition, the major peaks from the control and from the venom-treated samples were analyzed for ester and P content, and were subjected to acetic acid hydrolysis. The data are given in Table I. TABLE I PROPERTIES
OF C A R D I O L I P I N
Rp* Column fractionation'" E s t e r :P P r o d u c t of a c e t i c acid hydrolysis'"
AND LYSO-CARDIOLIPIN
Cardiolipin
Lyso-cardiolipin
0. 7 t u b e No. 37 1.9
0.2 t u b e No. 43 o.96
diglyceride
monoglyceride
" R~, on silicic acid i m p r e g n a t e d p a p e r w i t h a s o l v e n t c o n s i s t i n g of d i i s o b u t y l k e t o n e a c e t i c a c i d - w a t e r (4 ° : 2o : 3, v/v). *" F r a c t i o n a f i o n on silicic acid. See t e x t for details. The t u b e n u m b e r r e p r e s e n t s t h e p e a k t u b e n u m b e r of t h e c o l u m n fraction. *'* H y d r o l y s i s w i t h 9 0 % a c e t i c acid in w a t e r a t 95 ° for I h.
A portion of each major peak was hydrolyzed with sodium methoxide a n d the liberated f a t t y acid methyl ester subjected to gas chromatographic analysis. The data are given in Table II. TABLE II F A T T Y ACID A N A L Y S I S OF C A R D I O L I P I N
AND LYSO-CARDIOLIPIN
F a t t y acid a n a l y s i s w a s c a r r i e d o u t on a P e r k i n - E l m e r I 5 4 D F r a c t o m e t e r . The c o l u m n c o n s i s t e d of b u t a n e d i o l s u c c i n a t e on c h r o m o s o r b W (z m e t e r c o l u m n , 0.25 i n c h d i a m e t e r ) . Te mp. -- I8o °, H e l i u m p r e s s u r e : 3o lb, H y d r o g e n p r e s s u r e : 23 lb, air p r e s s u r e : 25 lb. D e t e c t o r - f l a m e i o n i z a t i o n . The a n a l y s e s were c a l c u l a t e d b y m e a n s of a n a u t o m a t i c p r i n t i n g i n t e g r a t o r . Fatty. acid
Linoleic * Oleic Palmitic Stearic Myristic Palmitoleic
Cardiolipin (%)
L ysocardiolipin (%)
63 15 8 6 3 2
64 15 7 6 3 2
* The v a l u e for linoleic a c i d is a m i n i m a l one since t h e m o l a r re s pons e b y linoleic a c i d is lower t h a n t h a t of t h e o t h e r f a t t y acids. Biochim.
Biophys.
A c t a , 84 (1964) 55-59
ENZYMIC HYDROLYSIS OF CARDIOLIPIN
,57
RESULTS
Snake venom phospholipase A is capable of hydrolyzing beef heart cardiolipin to yield a lyso-derivative in which 2 fatty acids have been removed. The reaction occurs in wet ether. However, unless lecithin is added to the system the hydrolysis by the enzyme is very slow and occurs to only a few per cent. When lecithin is added the reaction is rapid and goes to completion. In this case both the lecithin and cardiolipin are hydrolyzed to their corresponding lyso-compounds. The lyso-cardiolipin is characterized by the following data: lyso-cardiolipin has a longer retention time on column chromatography and has a lower mobility on silicic acid impregnated paper than does cardiolipin. Lyso-cardiolipin yields monoglycerides on acetic acid hydrolysis whereas cardiolipin yields diglycerides. Lyso-cardiolipin has an ester:P ratio of 1.0 whereas cardiolipin has an ester:P ratio of 2.o. These results are apparent from the reactions given below: O
O
IL
&
C H s - - O - - C - - R 1 CH s. O. .ip . O
CH 2
L
O
O-
O HAc
[i
CHOC--R 2
i
CHOH
CHO--C--R s
o
O
JL
CH~--O--~P--O--CH2
CH2--O--C--R 4
i
O-Cardio|ipin
0
CH2--O--C--R l
CH2OPOsH ~
O
Phospholipase A
CHIOH
!
iJ
CHO--C--R 2
i
CHOH
+
+
l
0
Ji
CHOC--R 8
I
0
II
CH~OH O
O
II
I'
CHzOC--R I
II
CH2OC--R l
I O--
CHOH
CHOH
o
r
CHI--O--P--O--CH 2
I
O---
CH~OC--R
O
•
CH2--O--P---O--CH i
t
CHs0POsH ~
[ CHOH
HAc ------~
l i l
CH~OPOaH 2
CH2OH
i
CHOH
+
CHOH
+
CHOH
o CH~--O--C--R 4
O
l
CH~OH
CH2OPO3H 2
CH2OC--I~
~- other products Lyso-cardiolipin
+ R2COOH + R3COOH
Since phospholipase A is known to remove the fl-linked fatty acid of lecithin and phosphatidylethanolamine, it is assumed that it acts in the same manner on cardiolipin. Biochim. Biophys. Acta, 84 (1964) 55-59
58
G.V. MARINETTI
With natural lecithin it is the general finding that the Ê-linked f a t t y acids removed by the enzyme are predominantly unsaturated whereas the a-linked f a t t y acids are predominantly saturated. However, such cannot be the case with cardiolipin inasmuch as the f a t t y acid analysis of ox heart cardiolipin ~ shows this phosphatide contains 72 % linoleic acid, 8 % linolenic acid and 11% oleic acid. Ox liver cardiolipin contains 63~7o% linoleic acid, 12-15% linolenic acid and 6 - 7 % oleic acid 4. Hence the unsaturated f a t t y acids must be distributed fairly randomly on both the a- andfl-positions. This was borne out by analysis of the f a t t y acids of cardiolipin used here and its lysoderivative. The f a t t y acid analysis of each was essentially identical. Linoleic acid was the major f a t t y acid in this sample of cardiolipin and accounted for 65% of the total f a t t y acids. The f a t t y acid distribution of cardiolipin thus differs from that of lecithin not only qualitatively but also in positioning. DISCUSSION
This paper reports for the first time the hydrolysis of cardiolipin b y snake venom phospholipase A. I f it is assumed that phospholipase A hydrolyses the fl-linked fatty acids on cardiolipin as it does with lecithin and phosphatidylethanolamine, then unlike these latter two phosphatides, the f a t t y acid distribution in cardiolipin is essentially random. The cardiolipin used in these experiments (obtained from the Sylvana Corp.) showed 2 peaks b y column chromatography on silicic acid. These peaks represented the salt form and free acid form. The salt form was the major component. Treatment of an ether solution of cardiolipin with aqueous o. i N HC1 converted the salt form to the acid form. It is noteworthy that although these forms moved differently on silicic acid columns in which non-ionic solvents were used for elution, these forms move the same on silicic acid paper where ionic solvents are employed. This observation also has been reported by us previously for phosphatidylserine 5. The cardiolipin used here contained one m a j o r component as ascertained b y paper chromatography. The other two minor components have not been identified but are also acidic phosphatides which are ninhydrin negative and do not contain choline. One of these components is believed to be phosphatidylglycerol, since when it is subjected to the DAWSON hydrolysis 6 it yields a phosphate ester which behaves like glycerylphosphorylglycerol. Moreover, the component believed to be phosphatidylglycerol gives the expected periodateSchiff test whereas cardiolipin does not. The lyso-cardiolipin fraction contained one major component and 2 minor components. This is to be expected since ~[he cardiolipin fraction also had 3 components each of which can yield a lyso-derivative. The beef heart lecithin employed here was a mixture of the diester and monoester monovinyl ether analogues. This latter form will be referred to as the choline plasmalogen. Hence snake venom phospholipase A hydrolysis yielded two lyso-compounds, namely lyso-lecithin and lyso-choline plasmalogen. In the systems used here (either for paper or column chromatography), lecithin and choline plasmalogen are not resolved and lyso-lecithin and lyso-choline plasmalogen are not resolved. Hence these showed a single peak b y column fractionation and a single spot b y paper chromatography. The manner in which beef heart lecithin stimulates the venom hydrolysis of Biochim. Biophys. Acta, 84 (1964) 55-59
ENZYMIC HYDROLYSIS OF CARDIOLIPIN
59
c a r d i o l i p i n is n o t u n d e r s t o o d . B o t h cardiolipin a n d lecithin dissolve c o m p l e t e l y in wet ether. H e n c e a p h y s i c a l effect o f solubilizing cardiolipin in e t h e r is n o t t h e answer, H o w e v e r , i f t h e r e a c t i o n occurs a t a w a t e r - e t h e r interface (which is v e r y l i k e l y since t h e s y s t e m h a d these t w o l i q u i d phases), t h e lecithin m a y act to m a k e t h e c a r d i o l i p i n m o r e a v a i l a b l e a t this interface a n d hence p r o m o t e t h e f o r m a t i o n of a n e n z y m e s u b s t r a t e complex. This seems plausible since lecithin is a m u c h b e t t e r emulsifying a g e n t t h a n cardiolipin. I n d e e d t h e c a r d i o l i p i n used here was v e r y difficult to disperse in water. I t is i n t e r e s t i n g to n o t e in this r e g a r d t h a t DAWSON f o u n d a q u i t e different effect on t h e h y d r o l y s i s of lecithin b y p h o s p h o l i p a s e B (see refs. 7 a n d 8). ACKNOWLEDGEMENT This w o r k was s u p p o r t e d in p a r t b y a g r a n t H E o2063-09 from t h e N a t i o n a l I n s t i t u t e s of Health, National Heart Institute.
REFERENCES i M. KATES,in K. BLOCH, Lipid Metabolism, John Wiley and Sons, N.Y., 196o, p. I65.
2 G. V. MARINETTI, J. Lipid Res., 3 (1962) I. MACFARLANEAND C-. M. GRAY, Biochem. J., 67 (1957) 25 P. 4 M. G. MACFARLANI~,Biochem. J., 78 (1961) 44. 5 G. V. MARINETTI, J. ERBLAND AND E. STOTZ, Biochim. Biophys. Acta, 3° (1958) 41. 6 R. l~I. C. DAWSON,Biochem. J., 75 (196o) 45. R. M. C. DAWSON,Biochem. J., 68 (1958) 352. s A. n. BANGHAMAND R. M. C. DAWSON, Biochem. J., 72 (1959) 486. s M. G.
Biochirn. Biophys. Acta, 84 (1964) 55-59
55
BBA 55000
HYDROLYSIS OF CARDIOLIPIN BY SNAKE VENOM PHOSPHOLIPASE A* G. V. MARINETTI Biochemistry Department, University of Rochester School of Medicine, Rochester, N.Y. ( U.S.A .) (Received August 2nd, i963)
SUMMARY Phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) from snake venom has been shown to hydrolyze cardiolipin in wet ether and yield lyso-cardiolipin. The enzyme reaction is slow but is greatly stimulated by the addition of beef heart lecithin. The lysocardiolipin was characterized by chemical and chromatographic analysis and by its hydrolysis products. Lyso-cardiolipin has an ester :P ratio of o.96 and yields monoglycerides upon acetic acid hydrolysis. Cardiolipin, on the other hand, has an ester :P ratio of 1.9 and yields diglycerides upon acetic acid hydrolysis. The fatty acids on the cardiolipin and lyso-cardiolipin are the same and thus it appears that these fatty acids have a fairly random distribution. INTRODUCTION The action of snake venom phospholipase A (phosphatide acyl-hydrolase, EC 3. I.I.4) on lecithin and phosphatidylethanolamine is well documented 1. It seemed of interest to see if this enzyme also were capable of hydrolyzing cardiolipin. The results reported in this paper demonstrate that phospholipase A can hydrolyze cardiolipi n to its deacylated lysoderivative which shall be given the trivial name of lyso-cardiolipin. The reaction occurs in wet ether and is greatly stimulated by the addition of lecithin. METHODS AND REAGENTS Beef heart cardiolipin and beef heart lecithin were purchased from the Sylvana Corp. Snake venom (Naia naia) was purchased from the Ross Allen Reptile Institute, Silver Springs, Fla. Paper and column chromatography and ester and P analyses were carried out as described previously ~. 38 mg of cardiolipin and 41 mg of lecithin were dissolved in 50 ml of ethyl ether (Merck, peroxide free). To this solution were added 0.2 ml of N a i a naia venom in isotonic saline (I mg venom). The solution was well mixed and let stand at room temperature. At hour intervals 2o-/zl aliquots were removed for paper chromatographic analysis. Although the reaction was complete after 4 h, the solution was let stand for 24 h. After this time period the solution was evaporated to dryness under vacuum and " Part of this work was presented at the Gordon Research Conference on Lipid Metabolism, Meriden, Conn., June 1962. Biochim. Biophys. Acta, 84 (1964) 55-59
50
G. V. MARINETTI
the residue dissolved in 5 ml of chloroform. The lipids were chromatographed on a I.O cm diameter column containing 15 g of Unisil silicic acid. The silicic acid was first washed with 40 ml each of heptane, ethyl ether and chloroform before the 5-ml aliquot of lipid solution in chloroform was applied. The lipids were eluted by the following solvents in the order given: a, 5 ° m l of chloroform; b, 80 ml of 20% methanol in chloroform; c, 80 ml of 50 % methanol in chloroform ; d, 80 ml of absolute methanol; e, 80 ml of methanol-chloroform-water (9 ° :IO :5, v/v). Fractions of 5 ml were taken by means of an automatic fraction collector. A control sample of cardiolipin and lecithin was run through the same procedure. In the control sample the venom solution was replaced b y 0.2 ml of saline. Each column fraction was analyzed for P content and each fraction was also analyzed by paper chromatography on silicic acid impregnated paper. In addition, the major peaks from the control and from the venom-treated samples were analyzed for ester and P content, and were subjected to acetic acid hydrolysis. The data are given in Table I. TABLE I PROPERTIES
OF C A R D I O L I P I N
Rp* Column fractionation'" E s t e r :P P r o d u c t of a c e t i c acid hydrolysis'"
AND LYSO-CARDIOLIPIN
Cardiolipin
Lyso-cardiolipin
0. 7 t u b e No. 37 1.9
0.2 t u b e No. 43 o.96
diglyceride
monoglyceride
" R~, on silicic acid i m p r e g n a t e d p a p e r w i t h a s o l v e n t c o n s i s t i n g of d i i s o b u t y l k e t o n e a c e t i c a c i d - w a t e r (4 ° : 2o : 3, v/v). *" F r a c t i o n a f i o n on silicic acid. See t e x t for details. The t u b e n u m b e r r e p r e s e n t s t h e p e a k t u b e n u m b e r of t h e c o l u m n fraction. *'* H y d r o l y s i s w i t h 9 0 % a c e t i c acid in w a t e r a t 95 ° for I h.
A portion of each major peak was hydrolyzed with sodium methoxide a n d the liberated f a t t y acid methyl ester subjected to gas chromatographic analysis. The data are given in Table II. TABLE II F A T T Y ACID A N A L Y S I S OF C A R D I O L I P I N
AND LYSO-CARDIOLIPIN
F a t t y acid a n a l y s i s w a s c a r r i e d o u t on a P e r k i n - E l m e r I 5 4 D F r a c t o m e t e r . The c o l u m n c o n s i s t e d of b u t a n e d i o l s u c c i n a t e on c h r o m o s o r b W (z m e t e r c o l u m n , 0.25 i n c h d i a m e t e r ) . Te mp. -- I8o °, H e l i u m p r e s s u r e : 3o lb, H y d r o g e n p r e s s u r e : 23 lb, air p r e s s u r e : 25 lb. D e t e c t o r - f l a m e i o n i z a t i o n . The a n a l y s e s were c a l c u l a t e d b y m e a n s of a n a u t o m a t i c p r i n t i n g i n t e g r a t o r . Fatty. acid
Linoleic * Oleic Palmitic Stearic Myristic Palmitoleic
Cardiolipin (%)
L ysocardiolipin (%)
63 15 8 6 3 2
64 15 7 6 3 2
* The v a l u e for linoleic a c i d is a m i n i m a l one since t h e m o l a r re s pons e b y linoleic a c i d is lower t h a n t h a t of t h e o t h e r f a t t y acids. Biochim.
Biophys.
A c t a , 84 (1964) 55-59
ENZYMIC HYDROLYSIS OF CARDIOLIPIN
,57
RESULTS
Snake venom phospholipase A is capable of hydrolyzing beef heart cardiolipin to yield a lyso-derivative in which 2 fatty acids have been removed. The reaction occurs in wet ether. However, unless lecithin is added to the system the hydrolysis by the enzyme is very slow and occurs to only a few per cent. When lecithin is added the reaction is rapid and goes to completion. In this case both the lecithin and cardiolipin are hydrolyzed to their corresponding lyso-compounds. The lyso-cardiolipin is characterized by the following data: lyso-cardiolipin has a longer retention time on column chromatography and has a lower mobility on silicic acid impregnated paper than does cardiolipin. Lyso-cardiolipin yields monoglycerides on acetic acid hydrolysis whereas cardiolipin yields diglycerides. Lyso-cardiolipin has an ester:P ratio of 1.0 whereas cardiolipin has an ester:P ratio of 2.o. These results are apparent from the reactions given below: O
O
IL
&
C H s - - O - - C - - R 1 CH s. O. .ip . O
CH 2
L
O
O-
O HAc
[i
CHOC--R 2
i
CHOH
CHO--C--R s
o
O
JL
CH~--O--~P--O--CH2
CH2--O--C--R 4
i
O-Cardio|ipin
0
CH2--O--C--R l
CH2OPOsH ~
O
Phospholipase A
CHIOH
!
iJ
CHO--C--R 2
i
CHOH
+
+
l
0
Ji
CHOC--R 8
I
0
II
CH~OH O
O
II
I'
CHzOC--R I
II
CH2OC--R l
I O--
CHOH
CHOH
o
r
CHI--O--P--O--CH 2
I
O---
CH~OC--R
O
•
CH2--O--P---O--CH i
t
CHs0POsH ~
[ CHOH
HAc ------~
l i l
CH~OPOaH 2
CH2OH
i
CHOH
+
CHOH
+
CHOH
o CH~--O--C--R 4
O
l
CH~OH
CH2OPO3H 2
CH2OC--I~
~- other products Lyso-cardiolipin
+ R2COOH + R3COOH
Since phospholipase A is known to remove the fl-linked fatty acid of lecithin and phosphatidylethanolamine, it is assumed that it acts in the same manner on cardiolipin. Biochim. Biophys. Acta, 84 (1964) 55-59
58
G.V. MARINETTI
With natural lecithin it is the general finding that the Ê-linked f a t t y acids removed by the enzyme are predominantly unsaturated whereas the a-linked f a t t y acids are predominantly saturated. However, such cannot be the case with cardiolipin inasmuch as the f a t t y acid analysis of ox heart cardiolipin ~ shows this phosphatide contains 72 % linoleic acid, 8 % linolenic acid and 11% oleic acid. Ox liver cardiolipin contains 63~7o% linoleic acid, 12-15% linolenic acid and 6 - 7 % oleic acid 4. Hence the unsaturated f a t t y acids must be distributed fairly randomly on both the a- andfl-positions. This was borne out by analysis of the f a t t y acids of cardiolipin used here and its lysoderivative. The f a t t y acid analysis of each was essentially identical. Linoleic acid was the major f a t t y acid in this sample of cardiolipin and accounted for 65% of the total f a t t y acids. The f a t t y acid distribution of cardiolipin thus differs from that of lecithin not only qualitatively but also in positioning. DISCUSSION
This paper reports for the first time the hydrolysis of cardiolipin b y snake venom phospholipase A. I f it is assumed that phospholipase A hydrolyses the fl-linked fatty acids on cardiolipin as it does with lecithin and phosphatidylethanolamine, then unlike these latter two phosphatides, the f a t t y acid distribution in cardiolipin is essentially random. The cardiolipin used in these experiments (obtained from the Sylvana Corp.) showed 2 peaks b y column chromatography on silicic acid. These peaks represented the salt form and free acid form. The salt form was the major component. Treatment of an ether solution of cardiolipin with aqueous o. i N HC1 converted the salt form to the acid form. It is noteworthy that although these forms moved differently on silicic acid columns in which non-ionic solvents were used for elution, these forms move the same on silicic acid paper where ionic solvents are employed. This observation also has been reported by us previously for phosphatidylserine 5. The cardiolipin used here contained one m a j o r component as ascertained b y paper chromatography. The other two minor components have not been identified but are also acidic phosphatides which are ninhydrin negative and do not contain choline. One of these components is believed to be phosphatidylglycerol, since when it is subjected to the DAWSON hydrolysis 6 it yields a phosphate ester which behaves like glycerylphosphorylglycerol. Moreover, the component believed to be phosphatidylglycerol gives the expected periodateSchiff test whereas cardiolipin does not. The lyso-cardiolipin fraction contained one major component and 2 minor components. This is to be expected since ~[he cardiolipin fraction also had 3 components each of which can yield a lyso-derivative. The beef heart lecithin employed here was a mixture of the diester and monoester monovinyl ether analogues. This latter form will be referred to as the choline plasmalogen. Hence snake venom phospholipase A hydrolysis yielded two lyso-compounds, namely lyso-lecithin and lyso-choline plasmalogen. In the systems used here (either for paper or column chromatography), lecithin and choline plasmalogen are not resolved and lyso-lecithin and lyso-choline plasmalogen are not resolved. Hence these showed a single peak b y column fractionation and a single spot b y paper chromatography. The manner in which beef heart lecithin stimulates the venom hydrolysis of Biochim. Biophys. Acta, 84 (1964) 55-59
ENZYMIC HYDROLYSIS OF CARDIOLIPIN
59
c a r d i o l i p i n is n o t u n d e r s t o o d . B o t h cardiolipin a n d lecithin dissolve c o m p l e t e l y in wet ether. H e n c e a p h y s i c a l effect o f solubilizing cardiolipin in e t h e r is n o t t h e answer, H o w e v e r , i f t h e r e a c t i o n occurs a t a w a t e r - e t h e r interface (which is v e r y l i k e l y since t h e s y s t e m h a d these t w o l i q u i d phases), t h e lecithin m a y act to m a k e t h e c a r d i o l i p i n m o r e a v a i l a b l e a t this interface a n d hence p r o m o t e t h e f o r m a t i o n of a n e n z y m e s u b s t r a t e complex. This seems plausible since lecithin is a m u c h b e t t e r emulsifying a g e n t t h a n cardiolipin. I n d e e d t h e c a r d i o l i p i n used here was v e r y difficult to disperse in water. I t is i n t e r e s t i n g to n o t e in this r e g a r d t h a t DAWSON f o u n d a q u i t e different effect on t h e h y d r o l y s i s of lecithin b y p h o s p h o l i p a s e B (see refs. 7 a n d 8). ACKNOWLEDGEMENT This w o r k was s u p p o r t e d in p a r t b y a g r a n t H E o2063-09 from t h e N a t i o n a l I n s t i t u t e s of Health, National Heart Institute.
REFERENCES i M. KATES,in K. BLOCH, Lipid Metabolism, John Wiley and Sons, N.Y., 196o, p. I65.
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