PHOSPHOLIPASE A IN H U M A N BRAIN: A,-TYPE AT ALKALINE pH J. A. ROOKE’ and G. R. WEBSTER’ Department of Chemical Pathology, Guy’s Hospital Medical School, London, SEI 9RT. U.K (Rewired 23 J ~ m r1975. Accepted 5 April 1976)
Abstract-A novel phospholipase A , . present in human cerebral cortex and active at pH 9.25 upon ultrasonicated phosphatidylethanolamine is described. It has been purified 39-fold from acetone-dried powders of cortex by ammonium sulphate fractionation at 250.; saturation. followed by Sephadex GI50 gel filtration. A molecular weight of approx 500,OOO has been found by Sepharose 6B gel filtration. The enzyme was slightly stimulated by 2 mM-Ca*’ and was inhibited by 1 mM-HgZ+ and by all detergents tested. The enzyme hydrolysed phosphatidylcholine at I8”, of the rate for phosphatidylethanolamine, lysophospholipids to a lesser degree and neutral lipids not at all. There appeared to be a preference for fluid phosphatidylethanolamine substrates. The similarities of the enzyme to other phorpholipases A , are discussed.
THE PRESENCE of positionally-specific phosphatide I-acyl and 2-acyl hydrolases (EC 3.1.1.32 and 3.1.1.4) is now well established in most mammalian tissues including brain (VAN DEE” & DE HAAS. 1966). GATT(1968) first reported the isolation of purified preparations of phospholipase A, from rat and calf brain which were active at p H 4.0. Purified preparations of a similar phospholipase A, and of a phospholipase A2. also optimally active at low pH, were obtained from human nervous tissue in this laboratory (COOPER & WEBSTER,1970, 1972). Both of these enzymes with acid pH optima hydrolysed phosphatidylcholine (PC) more actively than phosphatidylethanolamine (PE). During this earlier work, some evidence was also found for phospholipase A , activity in the alkaline pH range, which appeared t o be due to a separate enzyme. Other experiments (MOORE& WEBSTERunpublished) confirmed this finding with rat brain and showed that for the alkaline phospholipase A , activity in this tissue. PE was the preferred substrate. The present study reports the isolation in soluble form from human cerebral cortex of an alkaline phospholipase A , activity, its partial purification and characterization. The enzyme differs appreciably in its properties from the phospholipase A, active at acid pH in human brain (COOPER & WERSTER.1970) and resembles the phospholipase A , in rat liver described & WAITE(1971. 1973). which is optiby NEWKIRK mally active at p H 9.0 with P E as preferred substrate. I
Deceased 4 November. 1974.
’ Present address: Department of
Biochemistry. Aberdeen University. Marischal College, Aberdeen. AB9 1AS. U.K .Ahhrei~ioriomi t r t d PC. phosphatidylcholine: IysoPC. Iq’sophosphatidylcholine: PE. phosphatidylethanolamine: IysoPE. lysophosphatidylethanolamine: GGIG: glycylglycine:glycine buffer (see Methods).
MATERIALS AND METHODS Chemicals. All chemicals were of Analar or best available grade. Methanol and CHCI, were redistilled before use. CHCI, being immediately restabilized with 20” (viv) methanol. Diethyl ether was dried over sodium metal and acetone over anhydrous Na,SO,. Radioactive fatty acids [l-tJC]arachidonic, linoleic. palmitic and [9.10(nt3H]stearic acids). glyceryl tri-[ 1 -‘*C]oleate. glyceryl tri-[l-”C]stearate and cholesteryl [l-”C]oleate were purchased from the Radiochemical Centre. Amersham. Bucks.. U.K. Pure unlabelled phospholipids for use as carriers were obtained from Lipid Products. Epsom. Surrey. U.K. and triolein. tristearin and cholesteryl oleate from Sigma Biochemical Co. (London), Ltd., Kingston-upon-Thames, Surrey. U.K. Bufer. Throughout this work. a buffer consisting of equimolar amounts of glycine and glycylglycine was used. Addition of NaOH to this mixture gave smooth buffering over the range pH 7.&10.5. The abbreviation G G G refers to the above mixture, the concentration stated being the total concentration of glycine plus glycylglycine. E.xrractioit of tvi;wie from bruiit. Human brain material was obtained at post mortem. usually within 24 h of death from cases of either sex showing no evidence of neurological disease. Samples of cortex were treated with acetone. butan-1-01 and diethyl ether as described by GALLAI-HATCHARD rt a/. (1962). The resulting acetone-dried powders were stored over silica gel at -70 C and yielded 108.3 mg powder!@ fresh weight 1s.D. = 5 . 2 : 10 preparations). The enzymic actibity was extracted by stirring the acetonedried powder with 0.5 w-NaCI buffered with 0.02 M-GGG . pH 8.0. at a concentratioii of 50 mg powder ml buffer. for I h at W C with magnetic stirring. The mixture was then centrifuged at 100.ooO g for I h and the resulting clear, amber-coloured supernatant fraction either used directly or further purified by (NH,),SO, fractionation and gel filtration. 4nttwntiint s t t l p h i r e /rwtiomirio)i. Solid (NH4)>S0, (0.141 g;ml). was added to the saline extract and after dissolution of the (NH&SO,. allowed to stand for 45 min at 0 4 C and then centrifuged at 3000 g for 20 mtn. The
613
J. A. RWKF and G. R. WEBSTER
614
supernatant fluid was discarded and the precipitate was redissolbed in 0.5 hi-NaC1. 002 hf-GG.G.. pH 8.0. usually in I 10 original volume of ths extract. The amount of (NH4)2S0, used corresponded to 25",, saturation on the nomogram of D I X O& ~ WEBB11958). Gel .filrrution. The redissolbed (NH,),SOd precipitate was applied to a 2.5 x 30 cm column containing Sephadex GI50 (Pharmacia. Sweden) equilibrated with 0.5 M-NaCI. 0.02 M-GG G. pH 8.0. and eluted with the same buffer. Molrcular . s i x . Crude NaCl extracts o r redissolved 25",, (NH,),SO, precipitates were filtered through a 2.6 x 82 cm column containing Sepharose 6B (Pharmacia. Sweden). equilibrated with 0.5 M-NaCI. 0.02 M-GGIG. pH 8.0. The column was calibrated with the following proteins: porcine thyroglobulin. bovine catalase, equine heart cytochrome c (Sigma Biochemical Co.(London). Ltd.) and bovine plasma albumin (Armour Pharmaceutical Co., Ltd. Eastbourne. Sussex. U.K.). Stokes radii (r.) for the markers were calculated from diffusion coefficients given by EDSALL(1953) and SMITH(1970). Blue Dextran 2ooO (Pharmacia. Sweden) was used as a void-volume marker. K , values for the marker proteins and for phospholipase A, were calculated from their elution volumes ( K , = ( V , - K)/(Y - V,) where V, = elution volume of the protein. V, = void volume. and = total bed volume). and K ; , , was plotted against Stokes radius according to the correlation of Porath as described by SIEGEL& MONTY(1966). The molecular weight of phospholipase A, was calculated from the determined Stokes radius using the equation (2) cited by SIEGEL & MONTY(1966), and assuming a partial specific volume of 0.73 cm3/g and a frictional ratio of 1.25. characteristic of gobular proteins (SMITH.1963). Siihsrrures. I-[9.10(~i)-'H]Stcaro~l. 2-acyl PC and PE. I-[I-'4C]palmitoyl. 2 acyl PE. I-acyl. 2-[l-"C]linoleoyl and I-acyl. 2-[1-'*C]arachidonyl PEs were prepared as described by SMITHrt n / . (1973). except that for the 2-acyl labelled PEs, I-acyl IysoPE (egg) replaced 1-acyl IysoPC as the acyl-acceptor group. l-[9.l0(nt['H]Stearoyl IysoPE and IysoPC were prepared by the action of the phospholipase A, of Agkistrodon p f s u r o r u s pisciooriis venom (Ross Allen's Reptile Institute. Silver Springs. Florida, U S A . ) on the appropriate labelled diacyl substrates and were freed from contaminating traces of free fatty acid on small silicic acid columns. The intramolecular distribution of the radioactive fatty acids in. the biosynthetic phospholipids was determined (SMITHer a/.. 1973) and at least 98",, of the label was in the C1 position of the 1-acyl-labelled lipids and in the C2 position of the 2-acyl labelled lipids. Specific
TABLE 1. SPECIFICRADIOACTIVITIESOF
Specific radioactivity (mCi/mmol)
Substrate I-[3H]Stearoyl, 2-acyl PE
LIPID SUBSTRATES
(I)
(ii) l-[14C]Palmitoyl, 2-acyl PE 1,2-[i4CIDipalmitoyl PE I-Acyl. 2-['4Cllinoleoyl PE 1-Acyl, 2-[i4C]arachidonyl PE I-['HIStearoyl, 2-acyl PC 1-['HIStearoyl lyu, PE I-['HIStearoyl lyso P C Glyceryl tri-[ 14CJoleate Glyceryl tri-CL4C]stearate Cholesteryl [I4CJoleate
312 449 1.6 0.03 6.6 1.0 405 449 72 13 37 10
radioactivities of the lipid substrates used are giien in Table 1. The [l-'T]dipalmitoyl PE was a gift from Dr. J. D. BILLIMORIA. Dept. of Chemical Pathology. Westminster Hospital. London, U.K. The lipids were stored at -15:C in CHCI,. For each experiment an appropriate volume of solution was pipetted into a test tube and the solvent removed at 50 C under N:. The lipid was dispersed in 0.01 M - G G G .pH 9.25 at 0 4 C by exposure for 2 niin in a 100 Watt ultrasonic disintegrator (Measuring and Scientific Equipment Ltd.. Horshum. Sussex. U . K . ) tuned to maximum amplitude. The dispersion contained I p i o l lipid,'ml and approx 10' c.p.m. ml. . 4 , w i ~ Incubations . were carried out in glass-stoppered tubes. Normally 1 ml contained 30 pmol GG,G. pH Y.25. 2 jimol CaCI,, an appropriate amount of lipid aubstrate (usually 100 nmol) and enzyme. The reaction was started by the addition of ice-cold enzyme to the tubes at 37 C and incubated for appropriate times. normally 10 min. The enzymic reaction was halted by adding 2.5 ml of methanol and the lipids were extracted according to BLIGH & DYFR (1959) by adding 1.5 ml of CHCI, (containing 2 p o l of carrier fatty acid). I.5 ml of water and 0. I ml of 1 wsodium acetate/acetic acid buFler. pH 4.0. Assays were carried out in duplicate and control tubes without enzyme were included in each experiment. Chromatographic separation of the radioactive products and their measurement were as described by COOPFR & WEBSTER (1970). Protein was estimated by the modification of the method of LOWRYer a / . (1951) described by HARTREF (1972). using bovine plasma albumin as standard. Absorption at ZSO nm was used to monitor the elution of protein from gelfiltration columns.
RESULTS Initial experiments were carried o u t with crude brain extracts using l-[3H]stearoyl, 2-acyl a n d I-acyl. 2-[14C]linoleoyl PEs as substrates to detine the types of phospholipase A activity detectable a t alkaline p H . In Table 2 a r e shown the results of three experiments each carried o u t with a different extract. In every case the principal radioactive products were free C3H]stearic acid a n d 2-[iJC]linoleoyl lyso P E ; relatively small amounts of free [iJC]linoleic acid a n d l-[3H]stearoyl IysoPE were formed. T h e major phospholipase A activity is quite clearly of the A, type; A, as assessed by formation of either [iJC]linoleic acid or l-[3H]stearoyl IysoPE averaged lo",, of the A , activity as shown in t h e last two columns of Table L.
In two further experiments (conditions as in Table 2), the hydrolysis of I-[3H]stearoyl IysoPE was compared with that of l-[3H]stearoyl, 2-acylPE. T h e amounts of C3H]stearic acid released from the IysoPE were 22";, a n d 470: of the fatty acid released from PE. These findings indicate that appreciable lysophospholipase activity (EC 3.1.1.5) at p H 9.25 also exists in the crude extracts. Whether this is d u e to an enzyme different from the A,-type activity cannot be determined from these results. In each of the three experiments in Table 2, the extent of hydrolysis of the fatty acid from the I-acyl position, a s expressed
Phospholipase A , activity at alkaline pH in human brain TABLE 2. HYOK~LYSIS OF ACYL-LABELLED PE Substrate
( i ) I-[3Hlstearoyl.2-acyl PE
['Hlfatty acid Product
BY CRUDE EXTRACTS OF HUMAN CEREBRAL CORTEX
lii) 1-acyl. 2-[14C]linoleoylPE
[3H]lyso PE (b)
(a)
615
['Qfatty-acid
['4C]lyso PE
(C)
Id)
Phospholipase A, activity as a percentage of total activity 100b/a lmjd
~
Experiment 7
0 58 1 49
3
0 90
1
0 08 004 0 03
0 09 0 07
0 47 0 92 0 61
0 06
194 78 10 1
144 28 28
Products are given in mU/mg protein. The amounts of protein assayed in experiments I. 2 and 3 were 725, 650 and X70 p p respectively. by 2-['4C]linoleoyl IysoPE formation. is consistently less than that estimated from [3H]stearic acid release. Although it is possible that some lysophospholipase action on [lJC]linoleoyl IysoPE may have occurred. the free [ ' 4 ~ l i n o l e i cacid formed is too small to account for this deficit. The protein content of the 0.5 wNaCI extracts of acetone-dried human cerebral cortex was 1Cl5 mg/ml. However the specific activity of the phospholipase A , enzyme was more variable: most values were in the range 0.42-1.00 mU/mg protein, which corresponds to 1&27 mU/g of fresh tissue. Substantial purification of the crude saline extracts of human cerebral cortex was achieved by precipitation with (NH,),SO, at 25':, saturation followed by gel tiltration of the redissolved (NH&SO, precipitate through Sephadex (3150 (Table 3). The increase in 0.7 (7 expts)] of the total phospholiactivity [ I S pase A , activity that occurs after the (NH&SO, fractionation appears to be due to removal of bulk protein which interferes with the assay system. Readdition of equivalent amounts of dialysed supernatant from the 25'; (NH,),SOJ fractionation reversed this increase in activity. However, subfractionation of the 25",, (NH,),SO, supernatant, by increasing (NH,),SO, saturation, did not produce any subfraction w i h a significantly increased inhibitory ability. The W , ,(NH,)2S0, precipitate was redissolved in one-tenth of the original volume of extract and applied to a Sephadex (3150 column. from which the activity was quantitatively recovered at the void volume of the gel bed. TABLE 3.
To assess the purity of the phospholipase A, preparations after gel filtration, samples of the purified preparations were incubated with 1-['Hlstearoyl. 2-acyI PE. I-acyl. 2-['*C]Enoleoyl PE and I-['Hlstearoyl IysoPE as in Table 2. Both the phosphobpase A z and phospholipase activities were substantially reduced (Table 4). The phospholipase Az activity was reduced to an average of 1"; of the phospholipase A , activity: a 9-fold reduction as compared with the crude extract. The lysophospholipase activity was reduced to an average of So/,; a 4 to 5-fold reduction from the crude extract. Indeed when the lysophospholipase activity was measured in the presence of unlabelled PE (10nmol of 1-['Hlstearoyl IysoPE + 90 nmol of egg PE), the lysophospholipase activities in experiments 1 and 2 (Table 4) were 0.13 and 0.23 mU/mg protein, 0.2 and 1.1", respectively of the activity towards l-[3H]stearoyl, 2-acyl PE. The [L"C]linoleic acid production in Table 4. then probably represents the combined phospholipase A2 and lysophospholipase activities in purified preparations. As in Table 2, the ['"C]Iinoleoyl IysoPE formed was consistently less than the ['Hlstearic acid released. Smbility. Acetone-dried powders of human cortex were routinely stored at -20-C over silica gel. The phospholipase A, activity of freshly prepared extracts made from any powder remained constant during 4 months storage. Thus the enzyme appears to be stable in the powder form. In solution. however. the phospholipase A , was unstable. Crude saline extracts lost 1-20", activity per week at 0 or -2OC. Purified preparations following gel chromatography were
F'URIFICATION OF PHOSPHOLIPASE
A,
ACTIVITY
Protein Amount
Cnncen-
asayed
tration
Total
(mg!ml)
h.) Crude extract
Enzymic activity Specific activity
540-760
(rnUimg
Img)
Recovery V'J
Recovery
protein
r")
12.6 k 1.4
16@261
100
0.5-1.2
100
+ 2.9
14.5-_'6.9
Purification (X)
1
Redissolved (NH,),SO,
precipitate Sephadex GI50 peak fraction
75-170 35-70
11 2
0.7k0.1
Results are expressed. where applicable. as the mean in Methods.
1.3-2.7 S.D.
8.8
1.6
0.9+0.1
6.4-23.3 13.9-38.9
k 21
18 ? 6.2
35k7.7
39+11.1
151
from 7 experiments The method of purification is described
J . A. R ~ K and E G. R. WEBSTER
616 TABLr 4
H \ D R O L \ S I S O C ACYL-LABELLED
Subatrate
(I)
Products
PE
AND
LYSOPE BY
I-['Hlstearoyl. 2-acyl PE
(11)
PURIFIED EXTRACTS OF HUMAN CEREBRAL CORTEX
I-acyl, 2-[14CllinoleoylPE
(111)
l-['H]stearoyl lyso PE
['Hlfatty acid
['Hllyso PE
['4C]fatty acid
['4C]lyso PE
['Hllatty acid
59.6 21.2
0 0
0.9 0.1
19.3 7.6
2.8 2.5
Experiment I 7
Products are given in mU!mg protein. The amounts of protein assayed in experiments I and 2 were 34 and 44 lip respectively.
more unstable, losing 8"" per day at -20°C. At -8O~C. however, this loss could be reduced to 2-37,; per day. Purified preparations were therefore stored in sealed plastic ampoules at -8OC. As might be expected. the enzyme was heat-labile. Crude saline extracts were inactivated by heating at 60°C for 5 min. The phospholipase A, was also sensitive to acid pH. Dialysis of a crude extract at W ' C , overnight against 0.5 M-NaCI, 0.02 M-Tris-malate buffer, pH 5.0, reduced the activity by 929,. This is in marked contrast to the acid phospholipase A, previously described (COOPER & WEBSTER,1970), which was stable to dialysis at pH 4.0. Drprndencr on p H . The hydrolysis of 1-C3H] stearoyl. 2-acyl PE by purified preparations of phospholipase A, was greatest in the range, pH 9.2-9.6. Very little activity could be detected at pH 8.0 or below (Fig. I). Subsequent studies were carried out at pH 9.25. cfecr of biralenr carioris. The effect of Ca2+ upon phospholipase A activities has been one of the chief tests by which they are differentiated. The phospholip a x A, activity of human cortex does not seem to have an absolute requirement for Caz+, since treatment with EDTA does not affect the enzymic activity.
-
Molecular size. The Sepharose 6B column was calibrated with a mixture of proteins as described in the Methods section. The elution volume of the phospholipase A , peak corresponds to a mean K d value of
100 -
60
However, the enzyme is always slightly and variably stimulated by 2 m - C a 2 + (2.5-fold; S.D. = 0.8: 7 expts) in purified preparations. Above this concentration the effect of CaZ+is variable. Sometimes there is further activation. sometimes no increase or even inhibition. Thus. 2 mM-Ca2+ was routinely used. No other bivalent cation had a greater stimulatory effect than Ca2+, although Mg2+ and Co2+ gave a slight activation. However, Cu2', Zn" and Hgz+ all exhibited some inhibitory activity, 1 mM-HgZ+ giving almost total inhibition of the phospholipase A, activity. This suggested the presence of a sulphydryl group which might be essential for enzymic activity. This idea is supported by the observation that 10 mMp-hydroxymercuribenzoate completely inhibits the phospholipase A, activity. EfjPct o j detergerits. Much work on phospholipase A activities has been carried out using phospholipiddetergent emulsions as substrates. It was important, therefore to determine the effect of various detergents on the alkaline phospholipase A,. The anionic delergents, sodium deoxycholate, sodium taurocholate and sodium dodecylsulphate; nonionic detergents. Tween 20 and Triton X-100and the cationic detergent. cetyl trimethylammonium bromide were tested at a con-
-
20 -
i /:
80
10-
I
I
90
00
FIG. 1. The effect of pH on phospholipase A, activity. Results are the means from 3 experiments. The activity at each pH was calculated as a percentage of the activity at pH 9.25. Activities corresponding to lOOo< were from 14.4 to 30 rnU/mg protein and 2 8 4 3 pg protein were assayed in individual experiments.
r,
, 4
FIG. 2. Calibration of Sepharose 68 column. Calculation of I(d and rs are described in Methods. The proteins arc: 1. Horse heart cytochrome c ; 2. Bovine serum albumin: 3. Bovine catalase; 4. Phospholipase A, or human cerebral cortex; 5 . Porcine thyroglobulin.
Phospholipase A , activity at alkaline pH in human brain TABLE
617
5. HYDROLYSIS OF ACYL-LABELLED
Activity P")
Phospholipid l-[3H]StearoyL 1-['HIStearoyl, l-[3H]Stearoyl, l-['H]Stearoyl,
PHOSPHOLIPIDS BY
A,
PHOSPHOLIPASE
100
2-acyl PE 2-acyl PC lyso PE lyso PC
17.6 & 2.1 9.1 f 1.8 1.4 I 0.5 ~
l/s,
pM-'
FIG. 3. Lineweaver-Burk plot for hydrolysis of PE by phospholipase A,. Assays. volume 1 ml, contained 30 pnol of GG/G, pH 9.25, 2 pmol of Ca" and appropriate amounts of 1-['Hlstearoyl, 2-acyl PE and enzyme. Incubation was for I min at 37 C. I/v Values are the mean values f S.D. from 6 experiments. with the hydrolysis at 50 ~ M - P set E at unity to normalize the data from indivi-
OF DIFFERENT TABLE6. HYDROLYSIS
4
0.31 1
5
I . Human cerebral cortex 7. Human cerebral cortex 3.
Human brain
4.
Rat brain Rat brain microsomes
5.
?"
I-['H]Stearoyl. 2-acyl PE I-[3H]Palmitoyl. 2-acyl PE 1, 2-[1JClDipalmitoyl PE I-Acvl. 2-r'4Cllinoleovl PE I-Acyl. 2-['4Clarachidonvl . PE L
>
82.4 f 7.0
DISCUSSION
OF NERVOUS TISSUE
optimum
500,000 48,300
9.25 4.60
Ultrasonicated PE Taurocholate emulsified PC
75.000
4.30
-
4.00
TaurocholateiTriton X-100 emulsified PC Triton X-100 emulsified PC Taurocholate! Deoxycholate emulsified PC
6.80
51.9 f 11.0
The present work describes a phospholipase A, activity not previously found in nervous tissue. It is distinct from the phospholipase A, described in several earlier reports (Table 7). The probable reasons why such an activity has not been detected before are the inhibitory action of detergents on this enzyme. which occurs at concentrations that other workers have used t o emulsify their phospholipid substrates. and the alkaline pH optimum. Another feature which has proved useful in the purfication procedure is the large molecular size of the phospholipase A,, which
Mol wt
~
LOO I03 6 f 8.1 0.2 f 0.1
The I-acyl hydrolysis was estimated as formation of free fatty acid for nos I . 2. 3 and of IysoPE for nos 4 and 5. Results are expressed as a percentage of the hydrolysis of substrate no. 1 with mean and S.E.M. from 3 experiments. The activities corresponding to 100", activity were 2C-35 mU/mg protein and the amounts of protein assayed. 3C-55 p g protein in individual experiments.
7. PHOSPHOLIPASES A , TABLE
Source
PE's
Hydrolysis (46)
PE
No.
1 2 3
(s.D. = 0.02) in 5 determinations giving a mean Stokes radius of 65.3A (Fig. 2.). From these data a molecular weight of about 0.5 x lo6 was calculated. K , t d u e . An estimate of the K , for phospholipase A, with PE as substrate was made. A value of 51 phi (s.D. = 21 ~ I M in ) 5 experiments was obtained (Fig. 3). Enzyme protein. For the normal range of enzyme protein concentrations (2C-lo0 p g protein) used with purified phospholipase A , preparations the rate of hydrolysis was essentially linear. Substrate spec#ificity. Various lipids were tested in the standard assay conditions. Table 5 shows that the specificity is directed towards phospholipids with PE as the optimal substrate. Neutral lipids (glyceryl trioleate, tristearate and cholesteryl oleate) were also tested in the same conditions. N o activity was observed using these substrates. Differently acyllabelled PEs were also examined (Table 6). IrStearoyl and palmitoyl, 2-acyl PEs were hydrolysed at similar rates. However, the nature of the C2 fatty acyl ester appeared to affect the hydrolysis of the I-acyl group. The most unsaturated fatty acid, arachidonic acid, was the preferred C2 ester group.
ACYL-LABELLED
BY PHOSPHOLIPASE A,
dual experiments.
NO.
~~
Results are means from 6 experiments f S.D. The activity is given as a percentage of the hydrolysis with diacyl PE as substrate. The activities equivalent to 100°,, were 18.3-31.7 mU/mg protein and the amount of protein assayed 3C-50 pg in individual experiments.
Preferred substrate
Reference Present work COOPER(1971). COOPER& WEBSTER (1970) WOELKer a / . (1972) G A(1968) ~ WOELK& PORCELLATi (1973)
J. A. RWKF and G. R. WFRSTER
61x
suggests that i t exist^ in an aggregated form. By using Sephadei G 150. which completely excludes molecules of this size. the phospholipase A , could be eluted quickly at the void volume of the gel bed during purification. This prevented loss in activity due to the instabilit) of the enzyme at 0 C and allowed a quantitative recovery of the activity from Sephadex G150. The total exclusion of phospholipase A , from Sephadex G150 also enabled a complete separation from the other phospholipases A , in human brain of molecular weights 49,500 and 76.000 reported by COOPER (1971) and by WOELK('I t i / . (1972) respectively. Crude saline extracts of acetone-dried brain powders also contained a lysophospholipase activity. This was partially removed during the purification of phospholipase A, and i t i s possible that this activity may be due to two different enzymes. The actibity lost on purification could be attributed to the lqsophospholipase enzyme. molecular weight approv 30.000 (COOPER 6r WEBSTER.unpublished). found by COOPER& WEBSER (1970) in their 0.9,, NaCl extracts of acetone-dried brain powders. This lysophospholipase may be analogous to that described & GATT(1968) which also exhibited a by LELBOMTZ neutral pH optimum and low molecular weight (1S,Oo(t-20.000). The activity which remained associated with the phospholipase A , was probably due to the phospholipase A , itself. .since the ratio of lysophospholipase activities on IysoPC compared with IysoPE (0.16)is similar to the ratio of phospholipase A , activities on PC compared with PE (0.181. The 39-fold purification of the phospholipase A , obtained was related to the activity present in 0.5 wNaCI extracts of acetone-dried powders. ANSELL 11961) estimated that the protein content of human cerebral cortex was 75 mg pr0tein.g fresh weight. Since the protein content of the crude saline extracts ( I 1-14 m g h l ) corresponded to 25-30 mg protein extracted/g fresh weight (I ml extract i s equivalent to 0.46 g fresh weight). an additional purification factor of 2.S3-fold can be added to give an overall purification or 100-120-fold for the phospholipase A , activity when related 10 fresh weight. BAZAN(1971). in the only previous work which demonstrated the existence of brain phospholipase A activity using ultrasonicated phospholipids as substrates, showed that this phospholipase A, activity
,
TABLE 8 COMPARISON OF ALKALINE ~~
was localized i n the microsomal fraction of rat brain. This finding i s consistent with data that hake been obtained with rat liver. Phospholipase A , activities which are similar to the present enzyme were first described in microsomes b) WAITE6r VAN DEENEN (19671 and in Golgi bodies by VA": GOLDEvr trl. (1971). The existence of these activities was confirmed by NACHRAURul. (1972) and extended by NE\VKIRK & WAiTE (1973). N E W K I R 8K 2. WAITE (1971. 1973) found that the phospholipaw A , activities in microsomes and plasma membranes were almo5t identical and the alkaline phospholipase A , in brain described here resembles them in several respects (Table 8). Furthermore. both TORQUELIIAU-COLAKD PI t r l . (19701 and NACHBACIR Y I ( I / . (1973) showed that the alkaline phospholipase A , activity in rat liver could be extracted by NaCl and KCI from the membrane, a property that the cortical phospholipase A , showed on extraction from acetone-dried powders WAITE& Sissor; (1973~1)have also solubilized the phospholipase A, from rat liver plasma membrane with heparin. a property not shared by the phospholipase A , from rat-liver microsomes. They showed that the postulate of ZIEVE& Z l E V E (1972) that the plasma membrane phospholipase A , is identical with postheparin serum phospholipase A , (VOGEL& ZIEM. 1964) is correct. However. this heparin-solubilized phospholipase A , differs in several respects from the phospholipase A , described in this study; it appears to be smaller and heterogeneous in size (mol wt 1OO,OOO1 it is inhibited b l free fatty acid. and hydrolyses IysoPE at the same rate as PE (WAI: & S~SSCIX, 1973h). Whether the phospholipase A , of human brain has any transacylating activity similar to the heparin-solubilized plasma membrane activity (WAITE & SISSON. 1973h. 1974) has yet to be determined. Thus. on balance. the phospholipase A , of human cerebral cortex seems to resemble in particular the NaCI-solubilized phospholipase A of rat liver mrcrosomes and plasma membranes. The phospholipase A , described here would appear from Tables 5 and 6 to be specific for phospholipids and to possess some selectivity towards certain molecular species of PE. The discrepancy between phospholipase A , activities measured by free ratty acid formation from I-acyl labelled PE and IysoPE from 2-acyl labelled PE is real. It i s not caused by contaminating lysophospholipase or phospholipase A 2 (71
,
,
PHOSPHOLIPASE A OF CEREBRAL CORTEX wim THE PHOSPHOLIPASES A , 01 R A T L I V E R LLICROSOMES AND PL4SMA MEMBRANES ~
~
~
~~~
PH optimum
Preferred substrate
Mol wt
Human cerebral cortex
9.25
Ultrasonicated PE
500.000
51 11x1 Present work
Rat liver microsomes and plasma membranes
9.25
Uitraaonicated PE
5M).000
30
Enzyme
Reference
K,,
~
I'M
NEUklRL;
& WAITI; (1971.
IY73)
Rat liver microsomes and plasma membranes
8.5CL9.00
Ultrasonicated PE
~-
40 )IM NACHRAIIR 1'1 O f . (1977)
Phosphohpase A , activity at alkaline pH in human brain activities. For example, when 1 -acyl, 2-[1JC]linoleoyl PE is hydrolysed (Table 4). the contribution from lysophospholipase and phospholipase A2 activities as measured by ['4C]linoleic acid formation. would account lor less than 5"" of this discrepancy which must therefore be reflected in the substrate composition. Except for the pure dipdlmitoyl PE, the substrates in Table 6 consist of a mixture of molecular species of differing acyl composition. All the substrates have largely saturated fatty acids at the C1 position since substrates 4 and 5 (unlabelled i n the C1 position) are senthesized from hen egg I-acyl IysoPE whose composition is a mixture of palmitic and stearic acids. Therefore the differing rates of hydrolysis must be a reflection of the C2 acyl-composition. The data for substrates 3-5 (Table 6) suggest that the more unsaturated the C2 fatty acid, the greater the hydrolysis. The I-acyl labelled substrates (nos I and 2) were synthesized using rat liver homogenates and endogenous 2-acyl IysoPE as the acyl acceptor. So these PEs would presumably have the 2-acyl composition of rat liver PE. HOPKINSer [ I / . (1968) gave the 2-acyl composition of rat liver PE as arachidonic acid, 47"b docosahexaenoic acid, 33"; and linoleic acid, 10"; and these acids occur as tetraenoic, hexaenoic and dienoic species, respectively (KANOH,1969). Therefore the higher hydrolysis rates observed with the I-stearoyl and palmitoyl labelled PEs may have been due to the high content of docosahexaenoic acid in these substrates. However, the conclusion that the phospholipase A , possesses a greater specificity for PE species with polyunsaturated C2 acyl group needs substantiation by further experimental work using more clearly defined substrates. At this stage. a more likely explanation is that the polyunsaturated fatty acids affect the micellar substrates in such a way as to facilitate hydrolysis. Increasingly unsaturated phospholipids occupy greater cross-sectional areas at air-water interfaces (PAPAHADJOPOIJLOS, 1973): 'melt' at lower temperatures (CHAPMAN. 1973) and give more permeable synthetic bilayer membranes (DE GIFRef d..1966). This increase in fluidity of phospholipid structure accompanying increasing unsaturation in acyl composition could explain the apparent specificity of the phospholipase A , for polyunsaturated molecular species of PE. as micelles which contain these unsaturated species might be more easily bound by the enzyme o r else the ester linkage may be more accessible to the phospholiprrse A , . Physiologically. the function of this new phospholipase A, must remain obscure until more is known about its subcellular site in nervous tissues. By analogy with the phospholipase A, active in the microsomes and plasma membrane fractions of rat liter, i t is tempting to assign a similar site in ncrvous tissue and to suggest that the phospholipase A , may have a role in the modification of phospholipids. synthesized t l r 11o1u in the microsomes of nervous tissue. rici the deacylation reacylation cycle postulatcd by
619
LANDS& HART(1964) and thus play a role in defining and maintaining the characteristics of the nerve cell membrane. Adriou'leti~ri~ierits-Thiswork was made possible by facilities provided in the Department by the Wellcome Trust and The Medical Research Council. J.R. is grateful to Professor C . LONG.Institute of Basic Medical Sciences, Royal College of Surgeons, London. for invaluable advice is preparing the text and to Miss GLYNIS LAYTON for technical assistance during this work.
REFERENCES ANSELLG. B. (1961) in Biochrmisrs' Handbook (LONGC., ed.) p. 641. Spon, London. BAZAN N. G.. JR. (1971) Actn Pliysiol. lor. omrr. 21, 101 106. -
BLIGH E. G . & DYER W. J. (1959) Cur,. J. Bioclirm. Physiol. 37,911-917.
CHAPMAN D. (1973) in Fomi (ind F L I I I C I ~ofO IP/imp/io/ipidS I (AZISELLG. B.. DAWSOY R. M. C. & HAWTHORNE J. N., eds.). 2nd revised ed. pp. 117-123. Elsevier. Amsterdam. COOPER M. F. 11971) Ph.D. Tlieris, Drptrrrnirnr of Chemical Parholoyj. Guy's Hospital Medical School, London. COOPER M. F. & WEBSTER G. R. (1970) J. Nruroclterti. 17, 1543-1554.
COOPER M. F. & WERSTERG. R. (1972) J . Nrrfrochmi. 19, 333-340. Dt GIERJ., MANDERSLWT J. G. & VAN DEEKEN L. L. M. 11966) Biuc~lrt~~t. hiophj.\. rlrrci 1% 666675. Dixox M. & WEBBE. C . (19.58)EnrTnirs p. 49. Longmans. Green. London. EDSALLJ. T. (I9531 in 'The Proreiris (NEURATH H. & BAILEY K., eds.) Vol. I. p. 549. Academic Press. New York. GALLAI-HATCHARD J.. MACEEW. L.. THOMPWN R. H. S. & WEBSTERG. R. (1962) J . Neirrochmi. 9, 545-554. GATTS . (1968) Bioch~m.hioplij,. Artf7 159, 3M-316. HARTREE E. F. (1972) .4rio/j.r. Biocheni 48, 4 2 2 4 2 7 . Homius S. M.. SHEEHAK G . & LYMANR. L. (1968) Biochim. hrophys. .Acre 164. 272--277. KAWH H (1969) Bioc/iefri. hiiipli!.s. Acrlc 176, 75663. L.ANDSW C M. 6: HARTP. (1964) J . Lipid Res. 5. 81-87. Lclllo\lrL Z . & G ~ T s. T (1968) B f o d ~ i n fhtuphi.s. . .4m 164. 439 441 LOWXI 0. H.. R O S t R R O C G H N. 1.. FARRA. L. & RANDALL R. J. (1951) J . h i d . Chcrn. 193, 165-175. NACHBAL'R J.. COLBEA~: A . & VIGNAIS P. (1972) Biochim. hiopli!.\. .Acrd 274, 4 2 W 6 . NEWKIRKJ. D & Wai-rE M. (1971) Biochim. hiophys. Acrlr 198. 562-576.
NLWKIKKJ . D. & WAITEM. (1973) Biodiirn hr~)p/ir.s..Actti 225. 224 ~233. PAPHADJ01'01'LOS D. (1973) In Fornr irnd F,irrctroit ill Phil\pho/ipid\ (AVSILI G. B.. DA\VWYR. M. C. & HAWIHORW J N. eds.) 2nd re\iwd ed.. p. 143. Else\ier. Plmsrerdam. SiFbtL L. M & Mol;ri- K . J . (1966) Biodiim. hroph!.s. Arru 112. 346-363. SMlni M. H. (19631 Bioclten~J . 89. 45P. Ssiim M. H. (1970) in Nuridhook o/ BiochviiistrT (SEER H. ,A. ed.) 2nd. ed.. p C3. The Chemical Rubber Co.. Cle\eland. OH. U.S.A. S w n i J. B.. SILVER M. J. & WERSTERG . R. (1973) Biochrm. .I. 131. 61.5-618.
620
J. A. Rmw and G. R. WEBSTER
TORQVEBIAI'-COLARD 0.. PAYSAST M.. WALD R. & P O L O W V S ~ I J. (1970) Bull. SOL.. Chifn. hid. 52, 1 Oh1 -1 07 1. VAS DEENFN L. L. M.. & DE HAASG. H. (1966) A . R F ~ Biochrm. 35. 157-191. V A N GOLDE L. M. G.. FLEISCHER B. & FLEISCHER S. (1971) B i n c h i . htoplixs. Acto 249, 318-330. VOGEL W. C. & Z I ~ L. T (1964) J . Lipid Rrs. 5, 177-180. WAITEM. & VAKDEENEY L. L. M. (1967) Bincliini. h1ophr.s. 4cto 137. 498-517. WAITEM. & SISSONP. (19730) J. hiol. Climi. 248, 7201-7306.
WAITE M. & SISWN P. (19736) J . hiol. Cheni. 248. 7985-7992. WAIE M . & SISSON P. (1974) J . hiof. Chrrn. 249, 64014405. H . & DEBVCH H. (1972) H o p p e - S r ~ l e r ' . ~ . WOELKH., FLRNISS Z.phjsiol. Chrm. 353, 11 11-I 119. WOELKH. & PORCELLATI G. (1973) Hoppr-Srder's Z . p h j siol. Cliern. 354, 9&100. ZIEVEF.J. & ZIEW L. (1972) Biochrni. hiopfir.v. Rcs. Cotilrnufi. 47. 148C1485.
Abstract-A novel phospholipase A , . present in human cerebral cortex and active at pH 9.25 upon ultrasonicated phosphatidylethanolamine is described. It has been purified 39-fold from acetone-dried powders of cortex by ammonium sulphate fractionation at 250.; saturation. followed by Sephadex GI50 gel filtration. A molecular weight of approx 500,OOO has been found by Sepharose 6B gel filtration. The enzyme was slightly stimulated by 2 mM-Ca*’ and was inhibited by 1 mM-HgZ+ and by all detergents tested. The enzyme hydrolysed phosphatidylcholine at I8”, of the rate for phosphatidylethanolamine, lysophospholipids to a lesser degree and neutral lipids not at all. There appeared to be a preference for fluid phosphatidylethanolamine substrates. The similarities of the enzyme to other phorpholipases A , are discussed.
THE PRESENCE of positionally-specific phosphatide I-acyl and 2-acyl hydrolases (EC 3.1.1.32 and 3.1.1.4) is now well established in most mammalian tissues including brain (VAN DEE” & DE HAAS. 1966). GATT(1968) first reported the isolation of purified preparations of phospholipase A, from rat and calf brain which were active at p H 4.0. Purified preparations of a similar phospholipase A, and of a phospholipase A2. also optimally active at low pH, were obtained from human nervous tissue in this laboratory (COOPER & WEBSTER,1970, 1972). Both of these enzymes with acid pH optima hydrolysed phosphatidylcholine (PC) more actively than phosphatidylethanolamine (PE). During this earlier work, some evidence was also found for phospholipase A , activity in the alkaline pH range, which appeared t o be due to a separate enzyme. Other experiments (MOORE& WEBSTERunpublished) confirmed this finding with rat brain and showed that for the alkaline phospholipase A , activity in this tissue. PE was the preferred substrate. The present study reports the isolation in soluble form from human cerebral cortex of an alkaline phospholipase A , activity, its partial purification and characterization. The enzyme differs appreciably in its properties from the phospholipase A, active at acid pH in human brain (COOPER & WERSTER.1970) and resembles the phospholipase A , in rat liver described & WAITE(1971. 1973). which is optiby NEWKIRK mally active at p H 9.0 with P E as preferred substrate. I
Deceased 4 November. 1974.
’ Present address: Department of
Biochemistry. Aberdeen University. Marischal College, Aberdeen. AB9 1AS. U.K .Ahhrei~ioriomi t r t d PC. phosphatidylcholine: IysoPC. Iq’sophosphatidylcholine: PE. phosphatidylethanolamine: IysoPE. lysophosphatidylethanolamine: GGIG: glycylglycine:glycine buffer (see Methods).
MATERIALS AND METHODS Chemicals. All chemicals were of Analar or best available grade. Methanol and CHCI, were redistilled before use. CHCI, being immediately restabilized with 20” (viv) methanol. Diethyl ether was dried over sodium metal and acetone over anhydrous Na,SO,. Radioactive fatty acids [l-tJC]arachidonic, linoleic. palmitic and [9.10(nt3H]stearic acids). glyceryl tri-[ 1 -‘*C]oleate. glyceryl tri-[l-”C]stearate and cholesteryl [l-”C]oleate were purchased from the Radiochemical Centre. Amersham. Bucks.. U.K. Pure unlabelled phospholipids for use as carriers were obtained from Lipid Products. Epsom. Surrey. U.K. and triolein. tristearin and cholesteryl oleate from Sigma Biochemical Co. (London), Ltd., Kingston-upon-Thames, Surrey. U.K. Bufer. Throughout this work. a buffer consisting of equimolar amounts of glycine and glycylglycine was used. Addition of NaOH to this mixture gave smooth buffering over the range pH 7.&10.5. The abbreviation G G G refers to the above mixture, the concentration stated being the total concentration of glycine plus glycylglycine. E.xrractioit of tvi;wie from bruiit. Human brain material was obtained at post mortem. usually within 24 h of death from cases of either sex showing no evidence of neurological disease. Samples of cortex were treated with acetone. butan-1-01 and diethyl ether as described by GALLAI-HATCHARD rt a/. (1962). The resulting acetone-dried powders were stored over silica gel at -70 C and yielded 108.3 mg powder!@ fresh weight 1s.D. = 5 . 2 : 10 preparations). The enzymic actibity was extracted by stirring the acetonedried powder with 0.5 w-NaCI buffered with 0.02 M-GGG . pH 8.0. at a concentratioii of 50 mg powder ml buffer. for I h at W C with magnetic stirring. The mixture was then centrifuged at 100.ooO g for I h and the resulting clear, amber-coloured supernatant fraction either used directly or further purified by (NH,),SO, fractionation and gel filtration. 4nttwntiint s t t l p h i r e /rwtiomirio)i. Solid (NH4)>S0, (0.141 g;ml). was added to the saline extract and after dissolution of the (NH&SO,. allowed to stand for 45 min at 0 4 C and then centrifuged at 3000 g for 20 mtn. The
613
J. A. RWKF and G. R. WEBSTER
614
supernatant fluid was discarded and the precipitate was redissolbed in 0.5 hi-NaC1. 002 hf-GG.G.. pH 8.0. usually in I 10 original volume of ths extract. The amount of (NH4)2S0, used corresponded to 25",, saturation on the nomogram of D I X O& ~ WEBB11958). Gel .filrrution. The redissolbed (NH,),SOd precipitate was applied to a 2.5 x 30 cm column containing Sephadex GI50 (Pharmacia. Sweden) equilibrated with 0.5 M-NaCI. 0.02 M-GG G. pH 8.0. and eluted with the same buffer. Molrcular . s i x . Crude NaCl extracts o r redissolved 25",, (NH,),SO, precipitates were filtered through a 2.6 x 82 cm column containing Sepharose 6B (Pharmacia. Sweden). equilibrated with 0.5 M-NaCI. 0.02 M-GGIG. pH 8.0. The column was calibrated with the following proteins: porcine thyroglobulin. bovine catalase, equine heart cytochrome c (Sigma Biochemical Co.(London). Ltd.) and bovine plasma albumin (Armour Pharmaceutical Co., Ltd. Eastbourne. Sussex. U.K.). Stokes radii (r.) for the markers were calculated from diffusion coefficients given by EDSALL(1953) and SMITH(1970). Blue Dextran 2ooO (Pharmacia. Sweden) was used as a void-volume marker. K , values for the marker proteins and for phospholipase A, were calculated from their elution volumes ( K , = ( V , - K)/(Y - V,) where V, = elution volume of the protein. V, = void volume. and = total bed volume). and K ; , , was plotted against Stokes radius according to the correlation of Porath as described by SIEGEL& MONTY(1966). The molecular weight of phospholipase A, was calculated from the determined Stokes radius using the equation (2) cited by SIEGEL & MONTY(1966), and assuming a partial specific volume of 0.73 cm3/g and a frictional ratio of 1.25. characteristic of gobular proteins (SMITH.1963). Siihsrrures. I-[9.10(~i)-'H]Stcaro~l. 2-acyl PC and PE. I-[I-'4C]palmitoyl. 2 acyl PE. I-acyl. 2-[l-"C]linoleoyl and I-acyl. 2-[1-'*C]arachidonyl PEs were prepared as described by SMITHrt n / . (1973). except that for the 2-acyl labelled PEs, I-acyl IysoPE (egg) replaced 1-acyl IysoPC as the acyl-acceptor group. l-[9.l0(nt['H]Stearoyl IysoPE and IysoPC were prepared by the action of the phospholipase A, of Agkistrodon p f s u r o r u s pisciooriis venom (Ross Allen's Reptile Institute. Silver Springs. Florida, U S A . ) on the appropriate labelled diacyl substrates and were freed from contaminating traces of free fatty acid on small silicic acid columns. The intramolecular distribution of the radioactive fatty acids in. the biosynthetic phospholipids was determined (SMITHer a/.. 1973) and at least 98",, of the label was in the C1 position of the 1-acyl-labelled lipids and in the C2 position of the 2-acyl labelled lipids. Specific
TABLE 1. SPECIFICRADIOACTIVITIESOF
Specific radioactivity (mCi/mmol)
Substrate I-[3H]Stearoyl, 2-acyl PE
LIPID SUBSTRATES
(I)
(ii) l-[14C]Palmitoyl, 2-acyl PE 1,2-[i4CIDipalmitoyl PE I-Acyl. 2-['4Cllinoleoyl PE 1-Acyl, 2-[i4C]arachidonyl PE I-['HIStearoyl, 2-acyl PC 1-['HIStearoyl lyu, PE I-['HIStearoyl lyso P C Glyceryl tri-[ 14CJoleate Glyceryl tri-CL4C]stearate Cholesteryl [I4CJoleate
312 449 1.6 0.03 6.6 1.0 405 449 72 13 37 10
radioactivities of the lipid substrates used are giien in Table 1. The [l-'T]dipalmitoyl PE was a gift from Dr. J. D. BILLIMORIA. Dept. of Chemical Pathology. Westminster Hospital. London, U.K. The lipids were stored at -15:C in CHCI,. For each experiment an appropriate volume of solution was pipetted into a test tube and the solvent removed at 50 C under N:. The lipid was dispersed in 0.01 M - G G G .pH 9.25 at 0 4 C by exposure for 2 niin in a 100 Watt ultrasonic disintegrator (Measuring and Scientific Equipment Ltd.. Horshum. Sussex. U . K . ) tuned to maximum amplitude. The dispersion contained I p i o l lipid,'ml and approx 10' c.p.m. ml. . 4 , w i ~ Incubations . were carried out in glass-stoppered tubes. Normally 1 ml contained 30 pmol GG,G. pH Y.25. 2 jimol CaCI,, an appropriate amount of lipid aubstrate (usually 100 nmol) and enzyme. The reaction was started by the addition of ice-cold enzyme to the tubes at 37 C and incubated for appropriate times. normally 10 min. The enzymic reaction was halted by adding 2.5 ml of methanol and the lipids were extracted according to BLIGH & DYFR (1959) by adding 1.5 ml of CHCI, (containing 2 p o l of carrier fatty acid). I.5 ml of water and 0. I ml of 1 wsodium acetate/acetic acid buFler. pH 4.0. Assays were carried out in duplicate and control tubes without enzyme were included in each experiment. Chromatographic separation of the radioactive products and their measurement were as described by COOPFR & WEBSTER (1970). Protein was estimated by the modification of the method of LOWRYer a / . (1951) described by HARTREF (1972). using bovine plasma albumin as standard. Absorption at ZSO nm was used to monitor the elution of protein from gelfiltration columns.
RESULTS Initial experiments were carried o u t with crude brain extracts using l-[3H]stearoyl, 2-acyl a n d I-acyl. 2-[14C]linoleoyl PEs as substrates to detine the types of phospholipase A activity detectable a t alkaline p H . In Table 2 a r e shown the results of three experiments each carried o u t with a different extract. In every case the principal radioactive products were free C3H]stearic acid a n d 2-[iJC]linoleoyl lyso P E ; relatively small amounts of free [iJC]linoleic acid a n d l-[3H]stearoyl IysoPE were formed. T h e major phospholipase A activity is quite clearly of the A, type; A, as assessed by formation of either [iJC]linoleic acid or l-[3H]stearoyl IysoPE averaged lo",, of the A , activity as shown in t h e last two columns of Table L.
In two further experiments (conditions as in Table 2), the hydrolysis of I-[3H]stearoyl IysoPE was compared with that of l-[3H]stearoyl, 2-acylPE. T h e amounts of C3H]stearic acid released from the IysoPE were 22";, a n d 470: of the fatty acid released from PE. These findings indicate that appreciable lysophospholipase activity (EC 3.1.1.5) at p H 9.25 also exists in the crude extracts. Whether this is d u e to an enzyme different from the A,-type activity cannot be determined from these results. In each of the three experiments in Table 2, the extent of hydrolysis of the fatty acid from the I-acyl position, a s expressed
Phospholipase A , activity at alkaline pH in human brain TABLE 2. HYOK~LYSIS OF ACYL-LABELLED PE Substrate
( i ) I-[3Hlstearoyl.2-acyl PE
['Hlfatty acid Product
BY CRUDE EXTRACTS OF HUMAN CEREBRAL CORTEX
lii) 1-acyl. 2-[14C]linoleoylPE
[3H]lyso PE (b)
(a)
615
['Qfatty-acid
['4C]lyso PE
(C)
Id)
Phospholipase A, activity as a percentage of total activity 100b/a lmjd
~
Experiment 7
0 58 1 49
3
0 90
1
0 08 004 0 03
0 09 0 07
0 47 0 92 0 61
0 06
194 78 10 1
144 28 28
Products are given in mU/mg protein. The amounts of protein assayed in experiments I. 2 and 3 were 725, 650 and X70 p p respectively. by 2-['4C]linoleoyl IysoPE formation. is consistently less than that estimated from [3H]stearic acid release. Although it is possible that some lysophospholipase action on [lJC]linoleoyl IysoPE may have occurred. the free [ ' 4 ~ l i n o l e i cacid formed is too small to account for this deficit. The protein content of the 0.5 wNaCI extracts of acetone-dried human cerebral cortex was 1Cl5 mg/ml. However the specific activity of the phospholipase A , enzyme was more variable: most values were in the range 0.42-1.00 mU/mg protein, which corresponds to 1&27 mU/g of fresh tissue. Substantial purification of the crude saline extracts of human cerebral cortex was achieved by precipitation with (NH,),SO, at 25':, saturation followed by gel tiltration of the redissolved (NH&SO, precipitate through Sephadex (3150 (Table 3). The increase in 0.7 (7 expts)] of the total phospholiactivity [ I S pase A , activity that occurs after the (NH&SO, fractionation appears to be due to removal of bulk protein which interferes with the assay system. Readdition of equivalent amounts of dialysed supernatant from the 25'; (NH,),SOJ fractionation reversed this increase in activity. However, subfractionation of the 25",, (NH,),SO, supernatant, by increasing (NH,),SO, saturation, did not produce any subfraction w i h a significantly increased inhibitory ability. The W , ,(NH,)2S0, precipitate was redissolved in one-tenth of the original volume of extract and applied to a Sephadex (3150 column. from which the activity was quantitatively recovered at the void volume of the gel bed. TABLE 3.
To assess the purity of the phospholipase A, preparations after gel filtration, samples of the purified preparations were incubated with 1-['Hlstearoyl. 2-acyI PE. I-acyl. 2-['*C]Enoleoyl PE and I-['Hlstearoyl IysoPE as in Table 2. Both the phosphobpase A z and phospholipase activities were substantially reduced (Table 4). The phospholipase Az activity was reduced to an average of 1"; of the phospholipase A , activity: a 9-fold reduction as compared with the crude extract. The lysophospholipase activity was reduced to an average of So/,; a 4 to 5-fold reduction from the crude extract. Indeed when the lysophospholipase activity was measured in the presence of unlabelled PE (10nmol of 1-['Hlstearoyl IysoPE + 90 nmol of egg PE), the lysophospholipase activities in experiments 1 and 2 (Table 4) were 0.13 and 0.23 mU/mg protein, 0.2 and 1.1", respectively of the activity towards l-[3H]stearoyl, 2-acyl PE. The [L"C]linoleic acid production in Table 4. then probably represents the combined phospholipase A2 and lysophospholipase activities in purified preparations. As in Table 2, the ['"C]Iinoleoyl IysoPE formed was consistently less than the ['Hlstearic acid released. Smbility. Acetone-dried powders of human cortex were routinely stored at -20-C over silica gel. The phospholipase A, activity of freshly prepared extracts made from any powder remained constant during 4 months storage. Thus the enzyme appears to be stable in the powder form. In solution. however. the phospholipase A , was unstable. Crude saline extracts lost 1-20", activity per week at 0 or -2OC. Purified preparations following gel chromatography were
F'URIFICATION OF PHOSPHOLIPASE
A,
ACTIVITY
Protein Amount
Cnncen-
asayed
tration
Total
(mg!ml)
h.) Crude extract
Enzymic activity Specific activity
540-760
(rnUimg
Img)
Recovery V'J
Recovery
protein
r")
12.6 k 1.4
16@261
100
0.5-1.2
100
+ 2.9
14.5-_'6.9
Purification (X)
1
Redissolved (NH,),SO,
precipitate Sephadex GI50 peak fraction
75-170 35-70
11 2
0.7k0.1
Results are expressed. where applicable. as the mean in Methods.
1.3-2.7 S.D.
8.8
1.6
0.9+0.1
6.4-23.3 13.9-38.9
k 21
18 ? 6.2
35k7.7
39+11.1
151
from 7 experiments The method of purification is described
J . A. R ~ K and E G. R. WEBSTER
616 TABLr 4
H \ D R O L \ S I S O C ACYL-LABELLED
Subatrate
(I)
Products
PE
AND
LYSOPE BY
I-['Hlstearoyl. 2-acyl PE
(11)
PURIFIED EXTRACTS OF HUMAN CEREBRAL CORTEX
I-acyl, 2-[14CllinoleoylPE
(111)
l-['H]stearoyl lyso PE
['Hlfatty acid
['Hllyso PE
['4C]fatty acid
['4C]lyso PE
['Hllatty acid
59.6 21.2
0 0
0.9 0.1
19.3 7.6
2.8 2.5
Experiment I 7
Products are given in mU!mg protein. The amounts of protein assayed in experiments I and 2 were 34 and 44 lip respectively.
more unstable, losing 8"" per day at -20°C. At -8O~C. however, this loss could be reduced to 2-37,; per day. Purified preparations were therefore stored in sealed plastic ampoules at -8OC. As might be expected. the enzyme was heat-labile. Crude saline extracts were inactivated by heating at 60°C for 5 min. The phospholipase A, was also sensitive to acid pH. Dialysis of a crude extract at W ' C , overnight against 0.5 M-NaCI, 0.02 M-Tris-malate buffer, pH 5.0, reduced the activity by 929,. This is in marked contrast to the acid phospholipase A, previously described (COOPER & WEBSTER,1970), which was stable to dialysis at pH 4.0. Drprndencr on p H . The hydrolysis of 1-C3H] stearoyl. 2-acyl PE by purified preparations of phospholipase A, was greatest in the range, pH 9.2-9.6. Very little activity could be detected at pH 8.0 or below (Fig. I). Subsequent studies were carried out at pH 9.25. cfecr of biralenr carioris. The effect of Ca2+ upon phospholipase A activities has been one of the chief tests by which they are differentiated. The phospholip a x A, activity of human cortex does not seem to have an absolute requirement for Caz+, since treatment with EDTA does not affect the enzymic activity.
-
Molecular size. The Sepharose 6B column was calibrated with a mixture of proteins as described in the Methods section. The elution volume of the phospholipase A , peak corresponds to a mean K d value of
100 -
60
However, the enzyme is always slightly and variably stimulated by 2 m - C a 2 + (2.5-fold; S.D. = 0.8: 7 expts) in purified preparations. Above this concentration the effect of CaZ+is variable. Sometimes there is further activation. sometimes no increase or even inhibition. Thus. 2 mM-Ca2+ was routinely used. No other bivalent cation had a greater stimulatory effect than Ca2+, although Mg2+ and Co2+ gave a slight activation. However, Cu2', Zn" and Hgz+ all exhibited some inhibitory activity, 1 mM-HgZ+ giving almost total inhibition of the phospholipase A, activity. This suggested the presence of a sulphydryl group which might be essential for enzymic activity. This idea is supported by the observation that 10 mMp-hydroxymercuribenzoate completely inhibits the phospholipase A, activity. EfjPct o j detergerits. Much work on phospholipase A activities has been carried out using phospholipiddetergent emulsions as substrates. It was important, therefore to determine the effect of various detergents on the alkaline phospholipase A,. The anionic delergents, sodium deoxycholate, sodium taurocholate and sodium dodecylsulphate; nonionic detergents. Tween 20 and Triton X-100and the cationic detergent. cetyl trimethylammonium bromide were tested at a con-
-
20 -
i /:
80
10-
I
I
90
00
FIG. 1. The effect of pH on phospholipase A, activity. Results are the means from 3 experiments. The activity at each pH was calculated as a percentage of the activity at pH 9.25. Activities corresponding to lOOo< were from 14.4 to 30 rnU/mg protein and 2 8 4 3 pg protein were assayed in individual experiments.
r,
, 4
FIG. 2. Calibration of Sepharose 68 column. Calculation of I(d and rs are described in Methods. The proteins arc: 1. Horse heart cytochrome c ; 2. Bovine serum albumin: 3. Bovine catalase; 4. Phospholipase A, or human cerebral cortex; 5 . Porcine thyroglobulin.
Phospholipase A , activity at alkaline pH in human brain TABLE
617
5. HYDROLYSIS OF ACYL-LABELLED
Activity P")
Phospholipid l-[3H]StearoyL 1-['HIStearoyl, l-[3H]Stearoyl, l-['H]Stearoyl,
PHOSPHOLIPIDS BY
A,
PHOSPHOLIPASE
100
2-acyl PE 2-acyl PC lyso PE lyso PC
17.6 & 2.1 9.1 f 1.8 1.4 I 0.5 ~
l/s,
pM-'
FIG. 3. Lineweaver-Burk plot for hydrolysis of PE by phospholipase A,. Assays. volume 1 ml, contained 30 pnol of GG/G, pH 9.25, 2 pmol of Ca" and appropriate amounts of 1-['Hlstearoyl, 2-acyl PE and enzyme. Incubation was for I min at 37 C. I/v Values are the mean values f S.D. from 6 experiments. with the hydrolysis at 50 ~ M - P set E at unity to normalize the data from indivi-
OF DIFFERENT TABLE6. HYDROLYSIS
4
0.31 1
5
I . Human cerebral cortex 7. Human cerebral cortex 3.
Human brain
4.
Rat brain Rat brain microsomes
5.
?"
I-['H]Stearoyl. 2-acyl PE I-[3H]Palmitoyl. 2-acyl PE 1, 2-[1JClDipalmitoyl PE I-Acvl. 2-r'4Cllinoleovl PE I-Acyl. 2-['4Clarachidonvl . PE L
>
82.4 f 7.0
DISCUSSION
OF NERVOUS TISSUE
optimum
500,000 48,300
9.25 4.60
Ultrasonicated PE Taurocholate emulsified PC
75.000
4.30
-
4.00
TaurocholateiTriton X-100 emulsified PC Triton X-100 emulsified PC Taurocholate! Deoxycholate emulsified PC
6.80
51.9 f 11.0
The present work describes a phospholipase A, activity not previously found in nervous tissue. It is distinct from the phospholipase A, described in several earlier reports (Table 7). The probable reasons why such an activity has not been detected before are the inhibitory action of detergents on this enzyme. which occurs at concentrations that other workers have used t o emulsify their phospholipid substrates. and the alkaline pH optimum. Another feature which has proved useful in the purfication procedure is the large molecular size of the phospholipase A,, which
Mol wt
~
LOO I03 6 f 8.1 0.2 f 0.1
The I-acyl hydrolysis was estimated as formation of free fatty acid for nos I . 2. 3 and of IysoPE for nos 4 and 5. Results are expressed as a percentage of the hydrolysis of substrate no. 1 with mean and S.E.M. from 3 experiments. The activities corresponding to 100", activity were 2C-35 mU/mg protein and the amounts of protein assayed. 3C-55 p g protein in individual experiments.
7. PHOSPHOLIPASES A , TABLE
Source
PE's
Hydrolysis (46)
PE
No.
1 2 3
(s.D. = 0.02) in 5 determinations giving a mean Stokes radius of 65.3A (Fig. 2.). From these data a molecular weight of about 0.5 x lo6 was calculated. K , t d u e . An estimate of the K , for phospholipase A, with PE as substrate was made. A value of 51 phi (s.D. = 21 ~ I M in ) 5 experiments was obtained (Fig. 3). Enzyme protein. For the normal range of enzyme protein concentrations (2C-lo0 p g protein) used with purified phospholipase A , preparations the rate of hydrolysis was essentially linear. Substrate spec#ificity. Various lipids were tested in the standard assay conditions. Table 5 shows that the specificity is directed towards phospholipids with PE as the optimal substrate. Neutral lipids (glyceryl trioleate, tristearate and cholesteryl oleate) were also tested in the same conditions. N o activity was observed using these substrates. Differently acyllabelled PEs were also examined (Table 6). IrStearoyl and palmitoyl, 2-acyl PEs were hydrolysed at similar rates. However, the nature of the C2 fatty acyl ester appeared to affect the hydrolysis of the I-acyl group. The most unsaturated fatty acid, arachidonic acid, was the preferred C2 ester group.
ACYL-LABELLED
BY PHOSPHOLIPASE A,
dual experiments.
NO.
~~
Results are means from 6 experiments f S.D. The activity is given as a percentage of the hydrolysis with diacyl PE as substrate. The activities equivalent to 100°,, were 18.3-31.7 mU/mg protein and the amount of protein assayed 3C-50 pg in individual experiments.
Preferred substrate
Reference Present work COOPER(1971). COOPER& WEBSTER (1970) WOELKer a / . (1972) G A(1968) ~ WOELK& PORCELLATi (1973)
J. A. RWKF and G. R. WFRSTER
61x
suggests that i t exist^ in an aggregated form. By using Sephadei G 150. which completely excludes molecules of this size. the phospholipase A , could be eluted quickly at the void volume of the gel bed during purification. This prevented loss in activity due to the instabilit) of the enzyme at 0 C and allowed a quantitative recovery of the activity from Sephadex G150. The total exclusion of phospholipase A , from Sephadex G150 also enabled a complete separation from the other phospholipases A , in human brain of molecular weights 49,500 and 76.000 reported by COOPER (1971) and by WOELK('I t i / . (1972) respectively. Crude saline extracts of acetone-dried brain powders also contained a lysophospholipase activity. This was partially removed during the purification of phospholipase A, and i t i s possible that this activity may be due to two different enzymes. The actibity lost on purification could be attributed to the lqsophospholipase enzyme. molecular weight approv 30.000 (COOPER 6r WEBSTER.unpublished). found by COOPER& WEBSER (1970) in their 0.9,, NaCl extracts of acetone-dried brain powders. This lysophospholipase may be analogous to that described & GATT(1968) which also exhibited a by LELBOMTZ neutral pH optimum and low molecular weight (1S,Oo(t-20.000). The activity which remained associated with the phospholipase A , was probably due to the phospholipase A , itself. .since the ratio of lysophospholipase activities on IysoPC compared with IysoPE (0.16)is similar to the ratio of phospholipase A , activities on PC compared with PE (0.181. The 39-fold purification of the phospholipase A , obtained was related to the activity present in 0.5 wNaCI extracts of acetone-dried powders. ANSELL 11961) estimated that the protein content of human cerebral cortex was 75 mg pr0tein.g fresh weight. Since the protein content of the crude saline extracts ( I 1-14 m g h l ) corresponded to 25-30 mg protein extracted/g fresh weight (I ml extract i s equivalent to 0.46 g fresh weight). an additional purification factor of 2.S3-fold can be added to give an overall purification or 100-120-fold for the phospholipase A , activity when related 10 fresh weight. BAZAN(1971). in the only previous work which demonstrated the existence of brain phospholipase A activity using ultrasonicated phospholipids as substrates, showed that this phospholipase A, activity
,
TABLE 8 COMPARISON OF ALKALINE ~~
was localized i n the microsomal fraction of rat brain. This finding i s consistent with data that hake been obtained with rat liver. Phospholipase A , activities which are similar to the present enzyme were first described in microsomes b) WAITE6r VAN DEENEN (19671 and in Golgi bodies by VA": GOLDEvr trl. (1971). The existence of these activities was confirmed by NACHRAURul. (1972) and extended by NE\VKIRK & WAiTE (1973). N E W K I R 8K 2. WAITE (1971. 1973) found that the phospholipaw A , activities in microsomes and plasma membranes were almo5t identical and the alkaline phospholipase A , in brain described here resembles them in several respects (Table 8). Furthermore. both TORQUELIIAU-COLAKD PI t r l . (19701 and NACHBACIR Y I ( I / . (1973) showed that the alkaline phospholipase A , activity in rat liver could be extracted by NaCl and KCI from the membrane, a property that the cortical phospholipase A , showed on extraction from acetone-dried powders WAITE& Sissor; (1973~1)have also solubilized the phospholipase A, from rat liver plasma membrane with heparin. a property not shared by the phospholipase A , from rat-liver microsomes. They showed that the postulate of ZIEVE& Z l E V E (1972) that the plasma membrane phospholipase A , is identical with postheparin serum phospholipase A , (VOGEL& ZIEM. 1964) is correct. However. this heparin-solubilized phospholipase A , differs in several respects from the phospholipase A , described in this study; it appears to be smaller and heterogeneous in size (mol wt 1OO,OOO1 it is inhibited b l free fatty acid. and hydrolyses IysoPE at the same rate as PE (WAI: & S~SSCIX, 1973h). Whether the phospholipase A , of human brain has any transacylating activity similar to the heparin-solubilized plasma membrane activity (WAITE & SISSON. 1973h. 1974) has yet to be determined. Thus. on balance. the phospholipase A , of human cerebral cortex seems to resemble in particular the NaCI-solubilized phospholipase A of rat liver mrcrosomes and plasma membranes. The phospholipase A , described here would appear from Tables 5 and 6 to be specific for phospholipids and to possess some selectivity towards certain molecular species of PE. The discrepancy between phospholipase A , activities measured by free ratty acid formation from I-acyl labelled PE and IysoPE from 2-acyl labelled PE is real. It i s not caused by contaminating lysophospholipase or phospholipase A 2 (71
,
,
PHOSPHOLIPASE A OF CEREBRAL CORTEX wim THE PHOSPHOLIPASES A , 01 R A T L I V E R LLICROSOMES AND PL4SMA MEMBRANES ~
~
~
~~~
PH optimum
Preferred substrate
Mol wt
Human cerebral cortex
9.25
Ultrasonicated PE
500.000
51 11x1 Present work
Rat liver microsomes and plasma membranes
9.25
Uitraaonicated PE
5M).000
30
Enzyme
Reference
K,,
~
I'M
NEUklRL;
& WAITI; (1971.
IY73)
Rat liver microsomes and plasma membranes
8.5CL9.00
Ultrasonicated PE
~-
40 )IM NACHRAIIR 1'1 O f . (1977)
Phosphohpase A , activity at alkaline pH in human brain activities. For example, when 1 -acyl, 2-[1JC]linoleoyl PE is hydrolysed (Table 4). the contribution from lysophospholipase and phospholipase A2 activities as measured by ['4C]linoleic acid formation. would account lor less than 5"" of this discrepancy which must therefore be reflected in the substrate composition. Except for the pure dipdlmitoyl PE, the substrates in Table 6 consist of a mixture of molecular species of differing acyl composition. All the substrates have largely saturated fatty acids at the C1 position since substrates 4 and 5 (unlabelled i n the C1 position) are senthesized from hen egg I-acyl IysoPE whose composition is a mixture of palmitic and stearic acids. Therefore the differing rates of hydrolysis must be a reflection of the C2 acyl-composition. The data for substrates 3-5 (Table 6) suggest that the more unsaturated the C2 fatty acid, the greater the hydrolysis. The I-acyl labelled substrates (nos I and 2) were synthesized using rat liver homogenates and endogenous 2-acyl IysoPE as the acyl acceptor. So these PEs would presumably have the 2-acyl composition of rat liver PE. HOPKINSer [ I / . (1968) gave the 2-acyl composition of rat liver PE as arachidonic acid, 47"b docosahexaenoic acid, 33"; and linoleic acid, 10"; and these acids occur as tetraenoic, hexaenoic and dienoic species, respectively (KANOH,1969). Therefore the higher hydrolysis rates observed with the I-stearoyl and palmitoyl labelled PEs may have been due to the high content of docosahexaenoic acid in these substrates. However, the conclusion that the phospholipase A , possesses a greater specificity for PE species with polyunsaturated C2 acyl group needs substantiation by further experimental work using more clearly defined substrates. At this stage. a more likely explanation is that the polyunsaturated fatty acids affect the micellar substrates in such a way as to facilitate hydrolysis. Increasingly unsaturated phospholipids occupy greater cross-sectional areas at air-water interfaces (PAPAHADJOPOIJLOS, 1973): 'melt' at lower temperatures (CHAPMAN. 1973) and give more permeable synthetic bilayer membranes (DE GIFRef d..1966). This increase in fluidity of phospholipid structure accompanying increasing unsaturation in acyl composition could explain the apparent specificity of the phospholipase A , for polyunsaturated molecular species of PE. as micelles which contain these unsaturated species might be more easily bound by the enzyme o r else the ester linkage may be more accessible to the phospholiprrse A , . Physiologically. the function of this new phospholipase A, must remain obscure until more is known about its subcellular site in nervous tissues. By analogy with the phospholipase A, active in the microsomes and plasma membrane fractions of rat liter, i t is tempting to assign a similar site in ncrvous tissue and to suggest that the phospholipase A , may have a role in the modification of phospholipids. synthesized t l r 11o1u in the microsomes of nervous tissue. rici the deacylation reacylation cycle postulatcd by
619
LANDS& HART(1964) and thus play a role in defining and maintaining the characteristics of the nerve cell membrane. Adriou'leti~ri~ierits-Thiswork was made possible by facilities provided in the Department by the Wellcome Trust and The Medical Research Council. J.R. is grateful to Professor C . LONG.Institute of Basic Medical Sciences, Royal College of Surgeons, London. for invaluable advice is preparing the text and to Miss GLYNIS LAYTON for technical assistance during this work.
REFERENCES ANSELLG. B. (1961) in Biochrmisrs' Handbook (LONGC., ed.) p. 641. Spon, London. BAZAN N. G.. JR. (1971) Actn Pliysiol. lor. omrr. 21, 101 106. -
BLIGH E. G . & DYER W. J. (1959) Cur,. J. Bioclirm. Physiol. 37,911-917.
CHAPMAN D. (1973) in Fomi (ind F L I I I C I ~ofO IP/imp/io/ipidS I (AZISELLG. B.. DAWSOY R. M. C. & HAWTHORNE J. N., eds.). 2nd revised ed. pp. 117-123. Elsevier. Amsterdam. COOPER M. F. 11971) Ph.D. Tlieris, Drptrrrnirnr of Chemical Parholoyj. Guy's Hospital Medical School, London. COOPER M. F. & WEBSTER G. R. (1970) J. Nruroclterti. 17, 1543-1554.
COOPER M. F. & WERSTERG. R. (1972) J . Nrrfrochmi. 19, 333-340. Dt GIERJ., MANDERSLWT J. G. & VAN DEEKEN L. L. M. 11966) Biuc~lrt~~t. hiophj.\. rlrrci 1% 666675. Dixox M. & WEBBE. C . (19.58)EnrTnirs p. 49. Longmans. Green. London. EDSALLJ. T. (I9531 in 'The Proreiris (NEURATH H. & BAILEY K., eds.) Vol. I. p. 549. Academic Press. New York. GALLAI-HATCHARD J.. MACEEW. L.. THOMPWN R. H. S. & WEBSTERG. R. (1962) J . Neirrochmi. 9, 545-554. GATTS . (1968) Bioch~m.hioplij,. Artf7 159, 3M-316. HARTREE E. F. (1972) .4rio/j.r. Biocheni 48, 4 2 2 4 2 7 . Homius S. M.. SHEEHAK G . & LYMANR. L. (1968) Biochim. hrophys. .Acre 164. 272--277. KAWH H (1969) Bioc/iefri. hiiipli!.s. Acrlc 176, 75663. L.ANDSW C M. 6: HARTP. (1964) J . Lipid Res. 5. 81-87. Lclllo\lrL Z . & G ~ T s. T (1968) B f o d ~ i n fhtuphi.s. . .4m 164. 439 441 LOWXI 0. H.. R O S t R R O C G H N. 1.. FARRA. L. & RANDALL R. J. (1951) J . h i d . Chcrn. 193, 165-175. NACHBAL'R J.. COLBEA~: A . & VIGNAIS P. (1972) Biochim. hiopli!.\. .Acrd 274, 4 2 W 6 . NEWKIRKJ. D & Wai-rE M. (1971) Biochim. hiophys. Acrlr 198. 562-576.
NLWKIKKJ . D. & WAITEM. (1973) Biodiirn hr~)p/ir.s..Actti 225. 224 ~233. PAPHADJ01'01'LOS D. (1973) In Fornr irnd F,irrctroit ill Phil\pho/ipid\ (AVSILI G. B.. DA\VWYR. M. C. & HAWIHORW J N. eds.) 2nd re\iwd ed.. p. 143. Else\ier. Plmsrerdam. SiFbtL L. M & Mol;ri- K . J . (1966) Biodiim. hroph!.s. Arru 112. 346-363. SMlni M. H. (19631 Bioclten~J . 89. 45P. Ssiim M. H. (1970) in Nuridhook o/ BiochviiistrT (SEER H. ,A. ed.) 2nd. ed.. p C3. The Chemical Rubber Co.. Cle\eland. OH. U.S.A. S w n i J. B.. SILVER M. J. & WERSTERG . R. (1973) Biochrm. .I. 131. 61.5-618.
620
J. A. Rmw and G. R. WEBSTER
TORQVEBIAI'-COLARD 0.. PAYSAST M.. WALD R. & P O L O W V S ~ I J. (1970) Bull. SOL.. Chifn. hid. 52, 1 Oh1 -1 07 1. VAS DEENFN L. L. M.. & DE HAASG. H. (1966) A . R F ~ Biochrm. 35. 157-191. V A N GOLDE L. M. G.. FLEISCHER B. & FLEISCHER S. (1971) B i n c h i . htoplixs. Acto 249, 318-330. VOGEL W. C. & Z I ~ L. T (1964) J . Lipid Rrs. 5, 177-180. WAITEM. & VAKDEENEY L. L. M. (1967) Bincliini. h1ophr.s. 4cto 137. 498-517. WAITEM. & SISSONP. (19730) J. hiol. Climi. 248, 7201-7306.
WAITE M. & SISWN P. (19736) J . hiol. Cheni. 248. 7985-7992. WAIE M . & SISSON P. (1974) J . hiof. Chrrn. 249, 64014405. H . & DEBVCH H. (1972) H o p p e - S r ~ l e r ' . ~ . WOELKH., FLRNISS Z.phjsiol. Chrm. 353, 11 11-I 119. WOELKH. & PORCELLATI G. (1973) Hoppr-Srder's Z . p h j siol. Cliern. 354, 9&100. ZIEVEF.J. & ZIEW L. (1972) Biochrni. hiopfir.v. Rcs. Cotilrnufi. 47. 148C1485.
