Interactions of Phospholipase D with 1,2 Diacyl-sn-glycerol-3phosphorylcholine, dodecylsulfate, and Ca 2+ MICHAEL HELLER, NAVA MOZES, and IRENA PERI (ABRAMOVITZ), Department of Biochemistry, Hebrew UniversiW-Hadassah Medical School, Jerusalem, Israel
peanut seeds. The mol wt can vary up to 200,000 + 10,000 in multiples of 22,000 + Some properties of the pure, soluble 3,000 (1). CatalyticaUy active molecules, howp h o s p h o l i p a s e D (phosphatidycholine ever, were observed only with enzyme having phosphatido hydrolase, EC 3.1.4.4) interthe high mol wt (1,2). actions with phosphatidyl choline (1,2 We have studied primarily the hydrolysis of di a cyl-sn-glycerol-3-phosphoryl choline) egg-lecithin (phosphatidylcholine, or 1,27diin a system also containing dodecylsulfate acyl-sn-glycerol-3-phosphorylcholine), which and Ca2+ ions were studied. Concentraconsists of a mixture of molecules having b o t h tions of Ca 2+ greater than 50 mM were saturated and unsaturated fatty acid esters. necessary both for activity and adsorpOther phospholipids may be hydrolyzed, but tion of the enzyme to the "supersubno extensive studies have been reported (2-4). strate." Ethylenediamine tetraacetic acid Although the reaction may be stimulated by caused inhibition of activity, greater than various agents, detergents and especially doone would expect from its chelating decyl sulfate have been shown to be the best capacity. A nonlinear increase in activity activators (2). The reaction also requires Ca 2+. with the increase of enzyme protein was Optimal hydrolysis rates were obtained with a observed, suggesting a subunit aggregation phosphatidylcholine:dodecylsulfate molar ratio into a higher mol wt protein, catalytically of 2:1 (2,5,6). Under these circumstances, a more active. Upon centrifugation of the heterogeneous system is formed containing at supersubstrate-enzyme complex at 4.5 x least five components, i.e., enzyme, substrates l 0 s g ' m i n at 30 C, most of the substrate (phospholipid and water), and activators (determolecules sedimented regardless of the gent and metal cation). pH. The reverse was true when centrifugaWith the cabbage phospholipase D, Dawson tion was done at 1 C. Phospholipase D and Hernington (7) have carried out studies h y d r o l y z e d phosphatidylcholine moleinvolving similar components in an attempt to cules present in the supersubstrate at elaborate the concept of surface charge density temperatures around 0 C at a rate I/5 requirements for the action of phospholipases. that of a maximal value measured at Phospholipids, either alone or in association 30 C. The Arrhenius plot was linear in the with other amphipaths, may acquire different range from 0 to 30 C, and at that tempermacromolecular forms and may be treated for ature the curve broke with a smaller e n z y m o l o g i c a l considerations as "supersubslope. Activation energy of 9.1 Kcal/mol, strates" (4,8). This colorful term, which was below 30 C, was calculated. Adsorption coined by Brockerhoff (8), describes a "matrix of the enzyme to the sedimentable superin which a substrate molecule is embedded and substrate occurred at pH 8.0, regardless refers to a triglyceride droplet, a phospholipid of temperature. At pH 5.6, a considerable micelle or liposome, or an aggregate of many p o r t i o n o f p h o s p h a t i d y l c h o l l n e was substrate molecules with or without the includegraded at 30 C, thus minimizing the sion of nonsubstrate amphipaths" (4). capacity of the supersubstrate to adsorb We have chosen to study the reaction catat h e e n z y m e . Although Mg2+ could lyzed by the highly purified phospholipase D replace Ca2+ in the formation of sediacting on such a supersubstrate. Its composition mentable supersubstrate, it neither assists may be determined and controlled, but a in adsorption of the enzyme nor in activaknowledge of its structure is unfortunately tion of the phosphatidylcholine hydrolrather limited. ysis. H o w e v e r , this supersubstrate, a macrom o l e c u l a r complex made of phosphatidylI NTRODUCTI ON choline (PC), dodecylsulfate, and Ca 2+ ions, We have recently been able to purify a apparently binds the enzyme at the right soluble phospholipase D (phosphatidylcholine orientation to bring its active sites to the phosphatido hydrolase, EC 3.1.4.4) from dry vicinity of the reactive bonds in the molecules ABSTRACT
604
PHOSPHOLIPASE D--SUPERSUBSTRATE INTERACTIONS of the supersubstrate. Our data seem to indicate an adsorption of the enzyme molecules onto the surface of the supersubstrate, followed by some sort of size transformation into a catalytically more active enzyme. We describe some of the conditions for the formation of such a supersubstrate-enzyme complex. A preliminary account of these studies has been previously published (9). MATERIALS AND METHODS
The preparation of (3H) choline-labeled PC, the purification of phospholipase D, and the assay of its activity have been previously published (1,2). Interactions of phospholipase D with the PC-dodecylsulfate-Ca2+ complex were carried out at pH 5.6 or 8.0 as follows: 1. Reaction mixtures were prepared containing 50 mM acetate, pH 5.6, or TrisHC1, pH 8.0; 50 mM CaC12; 5 mM (3H) PC (the concentration of the lipid is b a s e d on phosphorous analysis); and 2.5 mM sodium dodecylsulfate in a final volume of 2 ml. 2. The tubes were incubated with continuous shaking in a thermostatic water bath at 1 C or 30 C for 10 min after the addition of the enzyme. 3. After the incubation, total radioactivity was determined on a small aliquot of the reaction mixture. 4. Partitioning of both PC and phosphol i p a s e D b e t w e e n sedimentable and soluble fractions was done as follows: (a) A n a l i q u o t was c e n t r i f u g e d at 3 0 , 0 0 0 x g f o r 15 m i n ( 4 . 5 x l 0 s g-min) at either 1 C or 30 C. The supernatant was carefully and completely removed, and the precipitate was resuspended in fresh buffer solution containing CaC12. (b) Determination of radioactivity was done on aliquots of the supernatant and precipitate. (c) Determination of PC in the supernatant and precipitate was performed. Aliquots of each were extracted with chloroform and methanol according to Folch et al. (10), and after phase separation, the radioactivity was determined in the upper (aqueous-methanolic) phase and in the lower (chloroform rich) phase. The former contains only (3H) choline, which is water soluble, whereas the latter contains the unhydrolyzed (3H) PC and the nonlabeled phosphatic acid.
605
(d) Assay of phospholipase D activity in the supematant and precipitate was done with aliquots of each fraction added as enzyme to a complete reaction mixture (2). RESULTS AND DISCUSSION
We considered the possibility that the enzyme is acting primarily on a "supersubs t r a t e " composed of ovolecithin, dodecylsulfate, and Ca2+ ions. Effect of Divalent Cations
The soluble phospholipase D requires Ca 2+ for activity, and maximal rates were obtained with 50 mM or higher CaCI 2 (2,5,11). Although with 50 or 60 mM Ca2+ the activity of the enzyme was 4.95 and 5.4 units/mg protein, respectively, the addition of only 1 0 m M Na2EDTA at pH 5.6 reduced the activity to 1.8 and 2 units, respectivelY. This is far below an anticipated decrease in activity based on t h e calculated ability of EDTA (ethylenediamine tetraacetic acid) to chelate only equimolar amounts of Ca 2+, e.g., 10 mM (12)o Dodecylsulfate is expected to bind also equivalent amounts of Ca2+. Therefore, under the experimental conditions, ca. 38.75 mM Ca 2+ should be free and available. During incubation at pH 5.6, very turbid suspensions were always formed. Such turbidities could be observed even if Ca2+ was replaced by Mg 2+. Qualitative analysis shows that Ca2+ or La3+ ions form insoluble salts with dodecylsulfate over a wide range of pH values. On the other hand, Mg2+ or Mn 2+ ions form soluble salts (M. Heller, unpublished observations). The occurrence of such turbid suspensions suggested a formation of aggregates of supersubstrate molecules which might serve as an adequate surface for enzyme binding. Such aggregates might be large enough and sediment at relatively low centrifugal forces. Table I shows that only when Ca 2+ ions are present does phospholipase D bind to the supersubstrate molecules and sediment at 4 . 5 - l 0 s g . m i n (Exp. 1). It is also obvious that although omission of Ca 2+ or replacing it with Mg2+ does not support enzymatic hydrolysis (Column B), it also prevents binding of appreciable amounts of enzyme to the sedimentable supersubstrate (compare columns C & D). The diminished recovered activities are due to the sensitivity of the enzyme to dodecylsulfate (1). Ca2+ ions seem to protect the enzyme from the detergent (2). We have included Exps. 5 and 6 to indicate absence of nonspecific adsorption of enzyme to LIPIDS, VOL. 11, NO. 8
M. HELLER, N. MOZES, AND I. PERI
606
TABLE I Effects of Ca 2+ or Mg2+ on the Sedimentation of Phospholipase Da Phospholipase D activity (mUnits) Before After centrifugation centrifugation PCT SUP (C) (D) (B)
Exp. no.
Composition (A)
1 2 3 4 5 6
Complete Omit Ca2+ Omit Ca2+ and enzyme Omit Ca2+ add Mg2+ Ditto, (Exp. 4) add 2 x enzyme Ditto, (Exp. 4) add 4 x enzyme
112 1.7 1.5 1.5 3.2 1.5
80 20 0 0 19 24
1.3 14 6.3 46 62 215
aphospholipase D (18.5 ~tg protein) was incubated at 1 C in a final volume of 2 ml containing S0 mM acetate pH 5.6, 50 mM CaCI2 or MgC12, 5 mM 3H-phosphatidylcholine, 2.5 mM sodium dodecylsulfate (except Exps. 5 and 6, which contained 37 #g and 74 ~tg enzymatic protein, respectively). After 10 rain of incubation, aliquots were removed for determination of phospholipase D activity, and the rest was centrifuged at 30,000 x g, 15 min at 4 C. The precipitates (PCT) were resuspended in acetate pH 5.6, and aliquots of the PCT and supernatants (SUP) were immediately withdrawn and assayed for activity (see Materials and Methods). TABLE II Effect of Ca 2+ or Mg2+ on the Sedimentation of Phosphatidylcholinea (3H) Phosphatidylcholine (/~mol) Exp. no.
Composition (A)
PCT (B)
SUP (C)
1 2 3 4 5 6
Complete Omit Ca2+ Omit Ca2+ omit enzyme Omit Ca2+, add Mg2+ Ditto, (Exp. 4) add 2 x enzyme Ditto, (Exp. 4) add 4 x enzyme
7.51 3.30 3.95 7.69 7.51 7.46
0.52 6.54 6.29 1.91 2.04 2.15
aThe conditions were identical to those described in Table I. At the end of the incubation, the tubes were centrifuged at 30,000 x g, 15 min at 4 C. The amounts of 3H-phosphatidylcholine in the precipitate (PCT) and supernatant (SUP) were determined in the chloroform rich, lower phase after extraction according to Folch et al. (10). an i n a d e q u a t e surface o f t h e s u p e r s u b s t r a t e . Since t h e e n z y m e d o e s n o t a d s o r b t o t h e s e d i m e n t in t h e a b s e n c e o f Ca 2+, we t h e n e x a m i n e d the c o m p o s i t i o n o f t h e s e d i m e n t i n g s u p e r s u b s t r a t e u n d e r t h e same c o n d i t i o n s d e s c r i b e d in Table I. The only c o n d i t i o n for a s e d i m e n t a b l e s u p e r s u b s t r a t e is t h e p r e s e n c e o f divalent cation (Table II, C o l u m n B). We may p o i n t t o t h e possible roles o f divalent cations in the catalysis o f p h o s p h o l i p a s e D: 1. Ca2+ m a y be n e e d e d f o r t h e actual catalytic h y d r o l y s i s similar t o t h e p r o c e s s a c c o m p l i s h e d by p h o s p h o l i p a s e A1 (13). 2. The excess i n h i b i t o r y a c t i o n b y E D T A m i g h t result f r o m t h e r e m o v a l o f an essential metalic cation f r o m the e n z y m e . This has b e e n s h o w n w i t h p h o s p h o l i p a s e C (14). LIPIDS, VOL. 11, NO. 8
3. It is i n s u f f i c i e n t t o f o r m a s e d i m e n t a b l e s u p e r s u b s t r a t e (e.g., b y Mg 2+) as long as e i t h e r the right o r i e n t a t i o n o f t h e molecules in t h e s u p e r s u b s t r a t e is n o t a t t a i n e d or the d e n a t u r i n g negative charges o f t h e d o d e c y l sulfate a n i o n are n o t b l o c k e d (e.g., in the case o f soluble Mg 2+ salts). Effect of Protein Concentrations
We have s h o w n previously t h a t t h e e n z y m e is p r o b a b l y c o m p o s e d o f s u b u n i t s having a m i n i m a l mol wt o f 2 0 , 0 0 0 - 2 5 , 0 0 0 (1). However, only t h o s e molecules having a mol wt at 2 0 0 , 0 0 0 have b e e n f o u n d t o be catalytically active. F u r t h e r m o r e , a t i m e - d e p e n d e n t dissociat i o n o f the active e n z y m e having a mol w t o f 2 0 0 , 0 0 0 i n t o s u b u n i t s o f smaller m o l w t was o b s e r v e d (1). We r e a s o n e d t h a t t h e surface o f the sedi-
PHOSPHOLIPASE D-SUPERSUBSTRATE INTERACTIONS mentable supersubstrate composed of ovolecithin, dodecylsulfate, and Ca2§ ions might assist in the transformation of the noncatalytically active subunits into active, high mol wt species. Using the most purified enzyme (Step 5, Ref. 1), we have found a nonlinear increase in activity exhibiting an upward bend with the increase of protein concentrations (Fig. 1). This behavior was observed on different occasions with several batches of the enzyme. This was not always observed with the cruder enzyme (Step 4, Ref. 1) and the protein concentrations employed. Occasionally, the enzyme at an earlier stage of purification exhibited this kind of behavior. Dawson and Hemington (7), using a highly purified, soluble phospholipase D from cabbage leaves, showed a time-dependent nonlinear increase in activity with large lecithin particles in the presence of Ca 2+ ions (cf. Ref. 7, Fig. 2). They have ascribed this "autocatalysis" to the "stimulating formation of the product-phosphatidic acid," but the data could be alternatively interpreted as a time-dependent conversion of the enzyme into a more active form. In addition, in the same report, Dawson and Hemington have shown a nonlinear increase in the rate of hydrolysis with the increase in the concentrations of the purified enzyme (cf. Ref. 7, Fig. 11 [The straight lines drawn in this figure are apparently misleading, because the experimental points do not necessarily comprise a straight line] ). It therefore seems reasonable to assume an initial adsorption of enzyme molecules onto the surface of the supersubstrate, followed by an organizational step of conversion into higher mol wt species (ca. 200,000) which are catalytically more active. At this stage, it will be difficult to describe the exact mechanism for the role of the divalent cations in either the supersubstrate organization, enzyme adsorbance, orientation, or catalysis.
120 - ~oo
o
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--
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9
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-
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o ~
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FIG. 1. Effect of protein concentrations on phospholipase D activity The activity was assayed with enzyme preparations at step 4 (o-----o) and step 5 (A-----~) of purification (1,2) as a function of protein concentrations. Aliquots were taken from either enzyme stock solutions containing both 10 #g protein/ml into a final volume of 1 ml. Incubationslasted for 10 rain at 37 C. (The experiment is representative of four others.) Am
I00
80
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>
_4
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~o G.
2O
Effects of Temperature and pH
The organization of amphipathic phospholipids in an aqueous environment depends on t h e i r c o m p o s i t i o n , concentration, and on temperature (15). The rate of enzyme catalyzed r e a c t i o n s is also affected by temperature (16,17). Hydrolysis of PC "supersubstrate" in a complex with phospholipase D occurs even at temperatures around 0 C (Fig. 2). The rates increased linearly up to 30 C and then leveled off. Temperatures higher than 5 0 C cause i r r e v e r s i b l e d e n a t u r a t i o n of the enzyme ( 2,5, t t ). By plot ring the logarithm of the initial velocities vs. l/T, two straight lines are ob-
607
0
I 10
( I I 20 30 40 T i m e {rain)
I. 50
t 60
FIG. 2. Effect of temperature on phospholipase D activity. The activity was assayed in the presence of dodecylsulfate and Ca2+ ions with constant shaking, as outlined in Materials and Methods, at the temperatures indicated in the figure. 100% activity was taken as the value determined at 30 C after 60 rain of incubation. tained intersecting at 30 C, Arrhenius energies of 9.1 Kcal/mol below 30C. enzymes exhibit activation 10 Kcal/mol (16,17).
with calculated a c t i v a t i o n of Most hydrolytic energies of ca.
LIPIDS, VOL. 11, NO. 8
608
M. HELLER, N. MOZES, AND I. PERI TABLE III Phospholipase D Activity of the Sediment and Supernatant after Centrifugation a
pH
Shaking at: Temperature (C) (A)
5.6
1 30
8.0
30
1
Phospholipase D activity (mUnits) after centrifugation at the following temperatures IC 30C PCT SUP PCT SUP (B) (C) (D) (E) 305 288 32 60
0 48 10 13
0 0 191 97
398 530 0
0
aTen #tool of phosphatidylcholine, 5 #mol sodium dodecylsulfate, 100 #mol CaCI2, and 100/~mol acetate pH 5.6 or tris-HC! pH 8.0 in a final vllume of 2 ml were shaken for 10 rain in the presence of 18.5 #g phospholipase D at 1 C or 30 C. After incubation, aliquots were withdrawn and centrifuged at 30,000 x g, 15 min at 1 C or 30 C. The supernatant (SUP) was completely removed and the precipitate (PCT) was resuspended with acetate and CaC!2. Aliquots of each fraction were assayed for phospholipase D activity (see Materials and Methods) at a final pH of 5.6. From phase diagrams and Tm values of PC containing saturated and unsaturated acids, it seems unlikely that the breaking point at 30 C r e f l e c t s a phase transition of egg-lecithin (15,18-22). Actually, the high degree of unsaturation of egg-lecithin brings the Tm value below 0 C (23). Inclusion of dodecylsulfate, however, might affect the melting characteristics of this PC, yet lack of data on the effects of both dodecylsulfate and Ca 2+ on the melting profile of egg-lecithin prevents the drawing of a final conclusion. We have shown the ability of the calciumc o n t a i n i n g s u p e r s u b s t r a t e to adsorb the enzyme, and also the effects of temperature on the enzyme's activity. We consequently examined the effects of temperature and pH on the sedimentation characteristics of the enzyme with the supersubstrate and of the supersubstrate itself. The components were mixed at two temperatures (1 C or 30 C) and at two pH values (5.6 and 8.0). Table III shows the measured activities of phospholipase D which either adsorbed to the sedimenting "supersubstrate" or remained in solution. Since the centrifugation period equals that of the incubation period, the centrifugation was also carried out at these two temperatures. The incomplete recoveries of the enzymatic activities are, in part, due to instability displayed by the enzyme under these conditions. The enzyme adsorbed to the sedimenting supersubstrate almost regardless of temperature and pH. An exception was observed when the incubation was conducted at 1 C or 30 C at pH 5.6, then centrifuged at 30 C; the enzyme was found in solution and did not sediment (Table III, Column E). Since these conditions are optimal for efficient hydrolysis of PC in the LIPIDS, VOL. If, NO. 8
supersubstrate, a large portion of it was degraded, causing a change in the molar proportion of PC to dodecylsulfate D despite the reduction of PC in the supersubstrate, its sedimentation pattern was not affected (data not shown). Regardless of the pH, PC sedimented as a supersubstrate complex primarily at 30 C. Since at low temperature (ca. 0 C), or at pH 8.0, the degree of PC hydrolysis was considerably smaller compared t o p H 5.6 and 30 C, it was not surprising to obtain these results. We may thus summarize our conclusions as follows: An appropriate surface furnished by the supersubstrate composed of PC, dodecylsulfate, and a divalent cation (Ca 2+) adsorbs the enzyme molecules. Subsequently, a conversion into a macromolecular form occurs on the surface either by increasing protein concentrations or with time. With the right spatial organization of supersubstrate and the accumul a t i o n o f the appropriate macromolecular enzyme species, pH 5.6 and 30 C, maxiaml rates of hydrolysis are obtained. Our data do not exclude the possibility that any form of the enzyme might also hydrolyze another spatial organization of PC in a different supersubstrate arrangement (D. Lichtenberg and P. Greenzaid, unpublished observations). The model system presented in these studies may serve for subsequent studies of a more physiological supersubstrate complex in which the dodecylsulfate anion is replaced by other negatively charged amphipatic phospho- or g l y c o l i p i d s in large aggregates furnishing adequate surfaces. We have previously clearly shown that phospholipase D degraded phospholipids in biological membranes, e.g., rat liver microsomes or human erythrocyte membrane vesicles, without the need of any nonsubstrate
609
PHOSPHOLIPASE D - S U P E R S U B S T R A T E INTERACTIONS
amphipathic activator (2,24). Furthermore, in previous studies we described a "natural supersubstrate" in which dodecylsulfate was replaced b y r a t liver microsomes. Radioactive PC, microsomes, and Ca 2+ created a "natural" supersubstate with the right orientation for both binding of the enzyme and catalysis (24). On the other hand, forming a supersubstrate with another type of lipoprotein, e.g., plasma ~ - l i p o p r o t e i n , caused an inhibitory effect, probably due to a wrong orientation, improper binding, or lack of size transformations (24). REFERENCES 1. Heller, M., N. Mozes, I. Peri, and E. Maes, Bioehim. Biophys. Acta 396:397 (1974). 2. Heller, M., N. Mozes, and E. Maes, in " M e t h o d s in E n z y m o l o g y , " Vol. 35B, Edited by J.M. Lowenstein, Academic Press, New York, NY, 1975, pp. 226-232. 3. Strauss, H., Z. Leibovitz-Ben Gershon, and M. Heller, Lipids 11:442 (1976). 4. Brockerhoff, H., and R.G. Jensen, "Lipolytic E n z y m e s , " Academic Press, New York, NY, 1974, Pp. 282-288. 5. Aladjem, E., "Partial Purification and Properties of Phospholipase D f r o m Peanut Seeds," M.Sc. Thesis, Hebrew University, Jersusalem, Israel, 1_969,_ pp. 24-26. 6. Tzur, R., and B. Shapiro, Biochim. Biophys. Acta 2 8 0 : 2 9 0 (1972). 7. Dawson, 1LM.C., and N. Hemington~ Biochem. J. 102:76 (1967). 8. Brockerhoff, H., Bioorg. Chem. 3:176 (1974).
9. Heller, M., N. Mozes, and I. Peri, Proc. 18th ICBL, Graz. p. 42 (1975). 10. Folch, J., M. Lees, and G.H. Sloane-Stanley, J. Biol. Chem. 2 6 6 : 4 9 7 (1957). 11. Heller, M., E. Aladjem, and B. Shapiro, Bull. Soc. Chim. Biol. 50:1395 (1968). 12. West, T.S., and A.S. Sykes, Analytical Applications of EDTA, British Drug House Publication, Dorset, England, 1960. 13. Yabusaki, K.K., and M.A. Wells, Biochemistry 14:162 (1975). 14. Moskowitz, M., M.W. Devereil, and R. McKinney, Science 123:1077 (1956). 15. Chapman, D., and D.F.H. Wallach, in "Biological Membranes," Edited by D. C h a p m a n , Academic Press, London and New York, 1968, pp. 125-202. 16. Dixon, M., and E.C. Webb, " E n z y m e s , " 2nd Edition, Academic Press, New York, NY, 1964, pp. 150-166. 17. Netter, H., "Theoretical Biochemistry," Oliver and Boyd, Edinborough, Scotland, 1969, p. 608. 18. Ladbrooke, B.D., R.M. Williams, and D. Chapman, Biochim. Biophys. Acta 50:333 (1968). 19. Ladbrooke, B.D., and D. C h a p m a n , Chem. Phys. Lipids 3:304 (1969). 20. Philips, M.C., B.D. Ladbrooke, and D. Chapman, Biochim. Biophys. Acta 196:35 (1970). 21. Chen, J-S., a n d P.G. Barton, Can. J. Biochem 4 9 : 1 3 6 2 (1971). 22. Chen, J-S., "Studies of Dialkyl Ether Phospholipids," Ph.D. Thesis, University of Alberta, E d m u n d t o n , Alberta, Canada, 1972, pp. 54-58. 23. Bangham, A.D., Prog. Biophys. Mol. Biol. 18:29 (1968). 24. Heller, M., a n d R. Arad, Bioehim. Biophys. Acta 2 1 0 : 2 7 6 (1970).
[ Received March 26, 1976]
LIPIDS, VOL. 11, NO. 8
peanut seeds. The mol wt can vary up to 200,000 + 10,000 in multiples of 22,000 + Some properties of the pure, soluble 3,000 (1). CatalyticaUy active molecules, howp h o s p h o l i p a s e D (phosphatidycholine ever, were observed only with enzyme having phosphatido hydrolase, EC 3.1.4.4) interthe high mol wt (1,2). actions with phosphatidyl choline (1,2 We have studied primarily the hydrolysis of di a cyl-sn-glycerol-3-phosphoryl choline) egg-lecithin (phosphatidylcholine, or 1,27diin a system also containing dodecylsulfate acyl-sn-glycerol-3-phosphorylcholine), which and Ca2+ ions were studied. Concentraconsists of a mixture of molecules having b o t h tions of Ca 2+ greater than 50 mM were saturated and unsaturated fatty acid esters. necessary both for activity and adsorpOther phospholipids may be hydrolyzed, but tion of the enzyme to the "supersubno extensive studies have been reported (2-4). strate." Ethylenediamine tetraacetic acid Although the reaction may be stimulated by caused inhibition of activity, greater than various agents, detergents and especially doone would expect from its chelating decyl sulfate have been shown to be the best capacity. A nonlinear increase in activity activators (2). The reaction also requires Ca 2+. with the increase of enzyme protein was Optimal hydrolysis rates were obtained with a observed, suggesting a subunit aggregation phosphatidylcholine:dodecylsulfate molar ratio into a higher mol wt protein, catalytically of 2:1 (2,5,6). Under these circumstances, a more active. Upon centrifugation of the heterogeneous system is formed containing at supersubstrate-enzyme complex at 4.5 x least five components, i.e., enzyme, substrates l 0 s g ' m i n at 30 C, most of the substrate (phospholipid and water), and activators (determolecules sedimented regardless of the gent and metal cation). pH. The reverse was true when centrifugaWith the cabbage phospholipase D, Dawson tion was done at 1 C. Phospholipase D and Hernington (7) have carried out studies h y d r o l y z e d phosphatidylcholine moleinvolving similar components in an attempt to cules present in the supersubstrate at elaborate the concept of surface charge density temperatures around 0 C at a rate I/5 requirements for the action of phospholipases. that of a maximal value measured at Phospholipids, either alone or in association 30 C. The Arrhenius plot was linear in the with other amphipaths, may acquire different range from 0 to 30 C, and at that tempermacromolecular forms and may be treated for ature the curve broke with a smaller e n z y m o l o g i c a l considerations as "supersubslope. Activation energy of 9.1 Kcal/mol, strates" (4,8). This colorful term, which was below 30 C, was calculated. Adsorption coined by Brockerhoff (8), describes a "matrix of the enzyme to the sedimentable superin which a substrate molecule is embedded and substrate occurred at pH 8.0, regardless refers to a triglyceride droplet, a phospholipid of temperature. At pH 5.6, a considerable micelle or liposome, or an aggregate of many p o r t i o n o f p h o s p h a t i d y l c h o l l n e was substrate molecules with or without the includegraded at 30 C, thus minimizing the sion of nonsubstrate amphipaths" (4). capacity of the supersubstrate to adsorb We have chosen to study the reaction catat h e e n z y m e . Although Mg2+ could lyzed by the highly purified phospholipase D replace Ca2+ in the formation of sediacting on such a supersubstrate. Its composition mentable supersubstrate, it neither assists may be determined and controlled, but a in adsorption of the enzyme nor in activaknowledge of its structure is unfortunately tion of the phosphatidylcholine hydrolrather limited. ysis. H o w e v e r , this supersubstrate, a macrom o l e c u l a r complex made of phosphatidylI NTRODUCTI ON choline (PC), dodecylsulfate, and Ca 2+ ions, We have recently been able to purify a apparently binds the enzyme at the right soluble phospholipase D (phosphatidylcholine orientation to bring its active sites to the phosphatido hydrolase, EC 3.1.4.4) from dry vicinity of the reactive bonds in the molecules ABSTRACT
604
PHOSPHOLIPASE D--SUPERSUBSTRATE INTERACTIONS of the supersubstrate. Our data seem to indicate an adsorption of the enzyme molecules onto the surface of the supersubstrate, followed by some sort of size transformation into a catalytically more active enzyme. We describe some of the conditions for the formation of such a supersubstrate-enzyme complex. A preliminary account of these studies has been previously published (9). MATERIALS AND METHODS
The preparation of (3H) choline-labeled PC, the purification of phospholipase D, and the assay of its activity have been previously published (1,2). Interactions of phospholipase D with the PC-dodecylsulfate-Ca2+ complex were carried out at pH 5.6 or 8.0 as follows: 1. Reaction mixtures were prepared containing 50 mM acetate, pH 5.6, or TrisHC1, pH 8.0; 50 mM CaC12; 5 mM (3H) PC (the concentration of the lipid is b a s e d on phosphorous analysis); and 2.5 mM sodium dodecylsulfate in a final volume of 2 ml. 2. The tubes were incubated with continuous shaking in a thermostatic water bath at 1 C or 30 C for 10 min after the addition of the enzyme. 3. After the incubation, total radioactivity was determined on a small aliquot of the reaction mixture. 4. Partitioning of both PC and phosphol i p a s e D b e t w e e n sedimentable and soluble fractions was done as follows: (a) A n a l i q u o t was c e n t r i f u g e d at 3 0 , 0 0 0 x g f o r 15 m i n ( 4 . 5 x l 0 s g-min) at either 1 C or 30 C. The supernatant was carefully and completely removed, and the precipitate was resuspended in fresh buffer solution containing CaC12. (b) Determination of radioactivity was done on aliquots of the supernatant and precipitate. (c) Determination of PC in the supernatant and precipitate was performed. Aliquots of each were extracted with chloroform and methanol according to Folch et al. (10), and after phase separation, the radioactivity was determined in the upper (aqueous-methanolic) phase and in the lower (chloroform rich) phase. The former contains only (3H) choline, which is water soluble, whereas the latter contains the unhydrolyzed (3H) PC and the nonlabeled phosphatic acid.
605
(d) Assay of phospholipase D activity in the supematant and precipitate was done with aliquots of each fraction added as enzyme to a complete reaction mixture (2). RESULTS AND DISCUSSION
We considered the possibility that the enzyme is acting primarily on a "supersubs t r a t e " composed of ovolecithin, dodecylsulfate, and Ca2+ ions. Effect of Divalent Cations
The soluble phospholipase D requires Ca 2+ for activity, and maximal rates were obtained with 50 mM or higher CaCI 2 (2,5,11). Although with 50 or 60 mM Ca2+ the activity of the enzyme was 4.95 and 5.4 units/mg protein, respectively, the addition of only 1 0 m M Na2EDTA at pH 5.6 reduced the activity to 1.8 and 2 units, respectivelY. This is far below an anticipated decrease in activity based on t h e calculated ability of EDTA (ethylenediamine tetraacetic acid) to chelate only equimolar amounts of Ca 2+, e.g., 10 mM (12)o Dodecylsulfate is expected to bind also equivalent amounts of Ca2+. Therefore, under the experimental conditions, ca. 38.75 mM Ca 2+ should be free and available. During incubation at pH 5.6, very turbid suspensions were always formed. Such turbidities could be observed even if Ca2+ was replaced by Mg 2+. Qualitative analysis shows that Ca2+ or La3+ ions form insoluble salts with dodecylsulfate over a wide range of pH values. On the other hand, Mg2+ or Mn 2+ ions form soluble salts (M. Heller, unpublished observations). The occurrence of such turbid suspensions suggested a formation of aggregates of supersubstrate molecules which might serve as an adequate surface for enzyme binding. Such aggregates might be large enough and sediment at relatively low centrifugal forces. Table I shows that only when Ca 2+ ions are present does phospholipase D bind to the supersubstrate molecules and sediment at 4 . 5 - l 0 s g . m i n (Exp. 1). It is also obvious that although omission of Ca 2+ or replacing it with Mg2+ does not support enzymatic hydrolysis (Column B), it also prevents binding of appreciable amounts of enzyme to the sedimentable supersubstrate (compare columns C & D). The diminished recovered activities are due to the sensitivity of the enzyme to dodecylsulfate (1). Ca2+ ions seem to protect the enzyme from the detergent (2). We have included Exps. 5 and 6 to indicate absence of nonspecific adsorption of enzyme to LIPIDS, VOL. 11, NO. 8
M. HELLER, N. MOZES, AND I. PERI
606
TABLE I Effects of Ca 2+ or Mg2+ on the Sedimentation of Phospholipase Da Phospholipase D activity (mUnits) Before After centrifugation centrifugation PCT SUP (C) (D) (B)
Exp. no.
Composition (A)
1 2 3 4 5 6
Complete Omit Ca2+ Omit Ca2+ and enzyme Omit Ca2+ add Mg2+ Ditto, (Exp. 4) add 2 x enzyme Ditto, (Exp. 4) add 4 x enzyme
112 1.7 1.5 1.5 3.2 1.5
80 20 0 0 19 24
1.3 14 6.3 46 62 215
aphospholipase D (18.5 ~tg protein) was incubated at 1 C in a final volume of 2 ml containing S0 mM acetate pH 5.6, 50 mM CaCI2 or MgC12, 5 mM 3H-phosphatidylcholine, 2.5 mM sodium dodecylsulfate (except Exps. 5 and 6, which contained 37 #g and 74 ~tg enzymatic protein, respectively). After 10 rain of incubation, aliquots were removed for determination of phospholipase D activity, and the rest was centrifuged at 30,000 x g, 15 min at 4 C. The precipitates (PCT) were resuspended in acetate pH 5.6, and aliquots of the PCT and supernatants (SUP) were immediately withdrawn and assayed for activity (see Materials and Methods). TABLE II Effect of Ca 2+ or Mg2+ on the Sedimentation of Phosphatidylcholinea (3H) Phosphatidylcholine (/~mol) Exp. no.
Composition (A)
PCT (B)
SUP (C)
1 2 3 4 5 6
Complete Omit Ca2+ Omit Ca2+ omit enzyme Omit Ca2+, add Mg2+ Ditto, (Exp. 4) add 2 x enzyme Ditto, (Exp. 4) add 4 x enzyme
7.51 3.30 3.95 7.69 7.51 7.46
0.52 6.54 6.29 1.91 2.04 2.15
aThe conditions were identical to those described in Table I. At the end of the incubation, the tubes were centrifuged at 30,000 x g, 15 min at 4 C. The amounts of 3H-phosphatidylcholine in the precipitate (PCT) and supernatant (SUP) were determined in the chloroform rich, lower phase after extraction according to Folch et al. (10). an i n a d e q u a t e surface o f t h e s u p e r s u b s t r a t e . Since t h e e n z y m e d o e s n o t a d s o r b t o t h e s e d i m e n t in t h e a b s e n c e o f Ca 2+, we t h e n e x a m i n e d the c o m p o s i t i o n o f t h e s e d i m e n t i n g s u p e r s u b s t r a t e u n d e r t h e same c o n d i t i o n s d e s c r i b e d in Table I. The only c o n d i t i o n for a s e d i m e n t a b l e s u p e r s u b s t r a t e is t h e p r e s e n c e o f divalent cation (Table II, C o l u m n B). We may p o i n t t o t h e possible roles o f divalent cations in the catalysis o f p h o s p h o l i p a s e D: 1. Ca2+ m a y be n e e d e d f o r t h e actual catalytic h y d r o l y s i s similar t o t h e p r o c e s s a c c o m p l i s h e d by p h o s p h o l i p a s e A1 (13). 2. The excess i n h i b i t o r y a c t i o n b y E D T A m i g h t result f r o m t h e r e m o v a l o f an essential metalic cation f r o m the e n z y m e . This has b e e n s h o w n w i t h p h o s p h o l i p a s e C (14). LIPIDS, VOL. 11, NO. 8
3. It is i n s u f f i c i e n t t o f o r m a s e d i m e n t a b l e s u p e r s u b s t r a t e (e.g., b y Mg 2+) as long as e i t h e r the right o r i e n t a t i o n o f t h e molecules in t h e s u p e r s u b s t r a t e is n o t a t t a i n e d or the d e n a t u r i n g negative charges o f t h e d o d e c y l sulfate a n i o n are n o t b l o c k e d (e.g., in the case o f soluble Mg 2+ salts). Effect of Protein Concentrations
We have s h o w n previously t h a t t h e e n z y m e is p r o b a b l y c o m p o s e d o f s u b u n i t s having a m i n i m a l mol wt o f 2 0 , 0 0 0 - 2 5 , 0 0 0 (1). However, only t h o s e molecules having a mol wt at 2 0 0 , 0 0 0 have b e e n f o u n d t o be catalytically active. F u r t h e r m o r e , a t i m e - d e p e n d e n t dissociat i o n o f the active e n z y m e having a mol w t o f 2 0 0 , 0 0 0 i n t o s u b u n i t s o f smaller m o l w t was o b s e r v e d (1). We r e a s o n e d t h a t t h e surface o f the sedi-
PHOSPHOLIPASE D-SUPERSUBSTRATE INTERACTIONS mentable supersubstrate composed of ovolecithin, dodecylsulfate, and Ca2§ ions might assist in the transformation of the noncatalytically active subunits into active, high mol wt species. Using the most purified enzyme (Step 5, Ref. 1), we have found a nonlinear increase in activity exhibiting an upward bend with the increase of protein concentrations (Fig. 1). This behavior was observed on different occasions with several batches of the enzyme. This was not always observed with the cruder enzyme (Step 4, Ref. 1) and the protein concentrations employed. Occasionally, the enzyme at an earlier stage of purification exhibited this kind of behavior. Dawson and Hemington (7), using a highly purified, soluble phospholipase D from cabbage leaves, showed a time-dependent nonlinear increase in activity with large lecithin particles in the presence of Ca 2+ ions (cf. Ref. 7, Fig. 2). They have ascribed this "autocatalysis" to the "stimulating formation of the product-phosphatidic acid," but the data could be alternatively interpreted as a time-dependent conversion of the enzyme into a more active form. In addition, in the same report, Dawson and Hemington have shown a nonlinear increase in the rate of hydrolysis with the increase in the concentrations of the purified enzyme (cf. Ref. 7, Fig. 11 [The straight lines drawn in this figure are apparently misleading, because the experimental points do not necessarily comprise a straight line] ). It therefore seems reasonable to assume an initial adsorption of enzyme molecules onto the surface of the supersubstrate, followed by an organizational step of conversion into higher mol wt species (ca. 200,000) which are catalytically more active. At this stage, it will be difficult to describe the exact mechanism for the role of the divalent cations in either the supersubstrate organization, enzyme adsorbance, orientation, or catalysis.
120 - ~oo
o
t-
/9
i
--
80--
/
9
/ r O
60--
I" _
E =
/
9
~0--
/
-
/
20-
o ~
/A / r
0
t
i
I
i
I
1
2
3
4
5
.ug
protein
FIG. 1. Effect of protein concentrations on phospholipase D activity The activity was assayed with enzyme preparations at step 4 (o-----o) and step 5 (A-----~) of purification (1,2) as a function of protein concentrations. Aliquots were taken from either enzyme stock solutions containing both 10 #g protein/ml into a final volume of 1 ml. Incubationslasted for 10 rain at 37 C. (The experiment is representative of four others.) Am
I00
80
Z~3o,
>
_4
6o
~o G.
2O
Effects of Temperature and pH
The organization of amphipathic phospholipids in an aqueous environment depends on t h e i r c o m p o s i t i o n , concentration, and on temperature (15). The rate of enzyme catalyzed r e a c t i o n s is also affected by temperature (16,17). Hydrolysis of PC "supersubstrate" in a complex with phospholipase D occurs even at temperatures around 0 C (Fig. 2). The rates increased linearly up to 30 C and then leveled off. Temperatures higher than 5 0 C cause i r r e v e r s i b l e d e n a t u r a t i o n of the enzyme ( 2,5, t t ). By plot ring the logarithm of the initial velocities vs. l/T, two straight lines are ob-
607
0
I 10
( I I 20 30 40 T i m e {rain)
I. 50
t 60
FIG. 2. Effect of temperature on phospholipase D activity. The activity was assayed in the presence of dodecylsulfate and Ca2+ ions with constant shaking, as outlined in Materials and Methods, at the temperatures indicated in the figure. 100% activity was taken as the value determined at 30 C after 60 rain of incubation. tained intersecting at 30 C, Arrhenius energies of 9.1 Kcal/mol below 30C. enzymes exhibit activation 10 Kcal/mol (16,17).
with calculated a c t i v a t i o n of Most hydrolytic energies of ca.
LIPIDS, VOL. 11, NO. 8
608
M. HELLER, N. MOZES, AND I. PERI TABLE III Phospholipase D Activity of the Sediment and Supernatant after Centrifugation a
pH
Shaking at: Temperature (C) (A)
5.6
1 30
8.0
30
1
Phospholipase D activity (mUnits) after centrifugation at the following temperatures IC 30C PCT SUP PCT SUP (B) (C) (D) (E) 305 288 32 60
0 48 10 13
0 0 191 97
398 530 0
0
aTen #tool of phosphatidylcholine, 5 #mol sodium dodecylsulfate, 100 #mol CaCI2, and 100/~mol acetate pH 5.6 or tris-HC! pH 8.0 in a final vllume of 2 ml were shaken for 10 rain in the presence of 18.5 #g phospholipase D at 1 C or 30 C. After incubation, aliquots were withdrawn and centrifuged at 30,000 x g, 15 min at 1 C or 30 C. The supernatant (SUP) was completely removed and the precipitate (PCT) was resuspended with acetate and CaC!2. Aliquots of each fraction were assayed for phospholipase D activity (see Materials and Methods) at a final pH of 5.6. From phase diagrams and Tm values of PC containing saturated and unsaturated acids, it seems unlikely that the breaking point at 30 C r e f l e c t s a phase transition of egg-lecithin (15,18-22). Actually, the high degree of unsaturation of egg-lecithin brings the Tm value below 0 C (23). Inclusion of dodecylsulfate, however, might affect the melting characteristics of this PC, yet lack of data on the effects of both dodecylsulfate and Ca 2+ on the melting profile of egg-lecithin prevents the drawing of a final conclusion. We have shown the ability of the calciumc o n t a i n i n g s u p e r s u b s t r a t e to adsorb the enzyme, and also the effects of temperature on the enzyme's activity. We consequently examined the effects of temperature and pH on the sedimentation characteristics of the enzyme with the supersubstrate and of the supersubstrate itself. The components were mixed at two temperatures (1 C or 30 C) and at two pH values (5.6 and 8.0). Table III shows the measured activities of phospholipase D which either adsorbed to the sedimenting "supersubstrate" or remained in solution. Since the centrifugation period equals that of the incubation period, the centrifugation was also carried out at these two temperatures. The incomplete recoveries of the enzymatic activities are, in part, due to instability displayed by the enzyme under these conditions. The enzyme adsorbed to the sedimenting supersubstrate almost regardless of temperature and pH. An exception was observed when the incubation was conducted at 1 C or 30 C at pH 5.6, then centrifuged at 30 C; the enzyme was found in solution and did not sediment (Table III, Column E). Since these conditions are optimal for efficient hydrolysis of PC in the LIPIDS, VOL. If, NO. 8
supersubstrate, a large portion of it was degraded, causing a change in the molar proportion of PC to dodecylsulfate D despite the reduction of PC in the supersubstrate, its sedimentation pattern was not affected (data not shown). Regardless of the pH, PC sedimented as a supersubstrate complex primarily at 30 C. Since at low temperature (ca. 0 C), or at pH 8.0, the degree of PC hydrolysis was considerably smaller compared t o p H 5.6 and 30 C, it was not surprising to obtain these results. We may thus summarize our conclusions as follows: An appropriate surface furnished by the supersubstrate composed of PC, dodecylsulfate, and a divalent cation (Ca 2+) adsorbs the enzyme molecules. Subsequently, a conversion into a macromolecular form occurs on the surface either by increasing protein concentrations or with time. With the right spatial organization of supersubstrate and the accumul a t i o n o f the appropriate macromolecular enzyme species, pH 5.6 and 30 C, maxiaml rates of hydrolysis are obtained. Our data do not exclude the possibility that any form of the enzyme might also hydrolyze another spatial organization of PC in a different supersubstrate arrangement (D. Lichtenberg and P. Greenzaid, unpublished observations). The model system presented in these studies may serve for subsequent studies of a more physiological supersubstrate complex in which the dodecylsulfate anion is replaced by other negatively charged amphipatic phospho- or g l y c o l i p i d s in large aggregates furnishing adequate surfaces. We have previously clearly shown that phospholipase D degraded phospholipids in biological membranes, e.g., rat liver microsomes or human erythrocyte membrane vesicles, without the need of any nonsubstrate
609
PHOSPHOLIPASE D - S U P E R S U B S T R A T E INTERACTIONS
amphipathic activator (2,24). Furthermore, in previous studies we described a "natural supersubstrate" in which dodecylsulfate was replaced b y r a t liver microsomes. Radioactive PC, microsomes, and Ca 2+ created a "natural" supersubstate with the right orientation for both binding of the enzyme and catalysis (24). On the other hand, forming a supersubstrate with another type of lipoprotein, e.g., plasma ~ - l i p o p r o t e i n , caused an inhibitory effect, probably due to a wrong orientation, improper binding, or lack of size transformations (24). REFERENCES 1. Heller, M., N. Mozes, I. Peri, and E. Maes, Bioehim. Biophys. Acta 396:397 (1974). 2. Heller, M., N. Mozes, and E. Maes, in " M e t h o d s in E n z y m o l o g y , " Vol. 35B, Edited by J.M. Lowenstein, Academic Press, New York, NY, 1975, pp. 226-232. 3. Strauss, H., Z. Leibovitz-Ben Gershon, and M. Heller, Lipids 11:442 (1976). 4. Brockerhoff, H., and R.G. Jensen, "Lipolytic E n z y m e s , " Academic Press, New York, NY, 1974, Pp. 282-288. 5. Aladjem, E., "Partial Purification and Properties of Phospholipase D f r o m Peanut Seeds," M.Sc. Thesis, Hebrew University, Jersusalem, Israel, 1_969,_ pp. 24-26. 6. Tzur, R., and B. Shapiro, Biochim. Biophys. Acta 2 8 0 : 2 9 0 (1972). 7. Dawson, 1LM.C., and N. Hemington~ Biochem. J. 102:76 (1967). 8. Brockerhoff, H., Bioorg. Chem. 3:176 (1974).
9. Heller, M., N. Mozes, and I. Peri, Proc. 18th ICBL, Graz. p. 42 (1975). 10. Folch, J., M. Lees, and G.H. Sloane-Stanley, J. Biol. Chem. 2 6 6 : 4 9 7 (1957). 11. Heller, M., E. Aladjem, and B. Shapiro, Bull. Soc. Chim. Biol. 50:1395 (1968). 12. West, T.S., and A.S. Sykes, Analytical Applications of EDTA, British Drug House Publication, Dorset, England, 1960. 13. Yabusaki, K.K., and M.A. Wells, Biochemistry 14:162 (1975). 14. Moskowitz, M., M.W. Devereil, and R. McKinney, Science 123:1077 (1956). 15. Chapman, D., and D.F.H. Wallach, in "Biological Membranes," Edited by D. C h a p m a n , Academic Press, London and New York, 1968, pp. 125-202. 16. Dixon, M., and E.C. Webb, " E n z y m e s , " 2nd Edition, Academic Press, New York, NY, 1964, pp. 150-166. 17. Netter, H., "Theoretical Biochemistry," Oliver and Boyd, Edinborough, Scotland, 1969, p. 608. 18. Ladbrooke, B.D., R.M. Williams, and D. Chapman, Biochim. Biophys. Acta 50:333 (1968). 19. Ladbrooke, B.D., and D. C h a p m a n , Chem. Phys. Lipids 3:304 (1969). 20. Philips, M.C., B.D. Ladbrooke, and D. Chapman, Biochim. Biophys. Acta 196:35 (1970). 21. Chen, J-S., a n d P.G. Barton, Can. J. Biochem 4 9 : 1 3 6 2 (1971). 22. Chen, J-S., "Studies of Dialkyl Ether Phospholipids," Ph.D. Thesis, University of Alberta, E d m u n d t o n , Alberta, Canada, 1972, pp. 54-58. 23. Bangham, A.D., Prog. Biophys. Mol. Biol. 18:29 (1968). 24. Heller, M., a n d R. Arad, Bioehim. Biophys. Acta 2 1 0 : 2 7 6 (1970).
[ Received March 26, 1976]
LIPIDS, VOL. 11, NO. 8
