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The action of prostaglandin synthetase on 2-arachidonyl-lecithin
SHORT COMMUNICATIONS
430 BRA 53I90
The action of prostaglandin synthetase on 2-arachidonyl-lecithin In studying the specificity of the enzyme system present in the particulate fraction of sheep vesicular glands, it was found earlier’ that free essential fatty acids are converted in high yield into prostaglandins, whereas their methyl esters, as demonstrated with dihomo-y-linolenic acid (all-cis-S,rr,I4-eicosatrienoic acid) and arachidonic acid (all-cis-5,8,II,I4-eicosatetraenoic acid)2, give only very small conversions into free prostaglandins. From these observations it was concluded that the free carboxyl group was necessary for the conversion. Obviously the esters are first split by an esterase or by hydrolysis and the free acids converted subsequently. As in the animal the polyunsaturated fatty acids are mainly present in phospholipids, I-stearoyl-2-[3H]arachidonyl-lecithin was used as substrate prostaglandin synthesis from the bound precursor acid.
to study a possible direct
The results presented here indicate that conversion took place only after addition of lecithinase A to the incubation mixture, which is another proof of the free carboxyl group being essential to the enzymic reaction. PHIAll-cis-5,8,rI,I4-eicosatetraenoic acid was prepared by reduction of the corresponding tetraynoic acid with 3H, (ref. 3), purity >gg.o o/0(gas-liquid chromatography), thin-layer chromatography on silica/AgNO, showed that 98.0 “/bof the radioactivity
was present in the arachidonic
acid spot; Dr. H. v. D. BOSCH (Organic Chem-
istry Laboratory, University of Utrecht, The Netherlands) prepared the abovementioned lecithin by biosynthesis 4. It was extracted by the method of BLIGH ANI) DYERS and purified by thin-layer chromatography. More than ggsi of the [3H]arachidonic acid was attached to the z-position, as checked with lecithinase A. The specific radioactivity was 2.2 mC/mmole (1.2.106 counts/min per mg). The lecithinase preparation Crotalus adamanteus venom (ex Sigma Chemical Co., lot No. 53B-2160), when incubated with [I-‘Klarachidonic acid, did not convert into prostaglandin E,, suggesting that it did not contain any prostaglandin synthetase activity. The particulate fraction of sheep vesicular glands was prepared as described beforee. Thinlayer chromatography with o.5-mm layers. system diethyl
was performed on Silica gel-G-HR (ex Machery Nagel and Co.) The solvent systems used were: for prostaglandins as such:
described by GREEN AND SAMUELSSON~ (System A); for prostaglandin B: ether-acetic acid (g8:2, v/v) (System B) ; for lipids: chloroform-methanol-
acetic acid-water
(go: 6:
I
:0.75,
by vol.) (System
ether (g:I, v/v) (System D). A lipid extract from the particulate
C) ; for fatty acids: benzene-diethyl
fraction
and the seminal
vesicles
was
prepared using the method of FOLCH, LEES AND SLOANE STANLEY~ to compare the fatty acid compositions in a total hydrolysate and in the free fatty acid and phospholipid fractions. The two latter fractions were isolated via preparative thin-layer chromatography (System C), the fatty acids from the phospholipids being isolated after hydrolysis. Methyl esters were prepared with diazomethane and analysed by gas-liquid chromatography on 2 columns (Column I : polyethyleneglycol adipate (PEGA) 5 %, length 140 cm, diameter 3 mm, temp. 185”; Column 2 : Apiezon 3.6%, length 200 cm, followed by comdiameter 4 mm, temp. 210’; support Diatoport S). Identification Biochim.
Biophys.
Acta,
164 (1968)
430-432
SHORT
431
COMMUNICATIONS
paring their retention volumes with those of the reference fatty acids. In one fraction the fatty acids were also separated by thin-layer chromatography on silica gel-AgNO, (System D). Fractions containing 20:3 w6 and 20:4 c06+20:5 ~3 were isolated and again subjected to gas-liquid chromatographic analysis. Furthermore, part of these fractions were hydrogenated in glacial acetic acid with Pd on carbon. The retention volumes of these reduced acids indicated that C,, acids were actually present. Table I shows that 20:3 occurs in a higher concentration than 20: 4 in all fractions. The ratio ot 20:3 to 20:4 is about 6, except in the free fatty acid fraction. TABLE
I
FATTY ACID COMPOSITION OF VARIOUS FRACTIONS OF THOSE
OF
ITS
PARTICULATE
WHOLE
VESICULAR
~___~
Fat&
acid
__~~. --
Particulate
Whole gland Total lipid
Free fatty
I6:o
21.6
7.8
16:1
1.7 20.7
2.0
18:o
18:1 18:2
"js;:
acid
Phospholifiid 23.1 1.6
2.1
20:306 ro:4w6 20:4 w 3* 20:5w3
8.2
* Tentatively
I.3
OF
SHEEP
AND
OF
Free fattv acid
Phospholipid
25.4
11.8
27.8
__2.1
1g.0
18.8
34.5 4.’
30.2
6.0 23.0 6.6
1.6 8.3 1.6
2.5 II.5
2.1
8.2
0.9
6.0
5.0 1.2
fraction
Total lipid
5.3 29.4 5.7
20:1 20:2
GLAND
FRACTION
4.8 -
4.6
4.3 53.8 6.9 I.9 6.7 7.9
tr. z.1
I.9 I.8
1.0
16.2 29.2
4.3 3.4 12.7 2.1 2.1 I.2
1.0
identified.
Mixtures were prepared in the following way: to 2 ml 0.1 M phosphate buffer, pH 7.5, containing 0.001 M EDTA and O.OOIM cysteine, 200 ,ug desoxycholic acid was added to facilitate the solution of the added lecithin (IOO ,ug) in order to render the latter more accessible to the lecithinase. Incubations I and II (Table II) were performed with 2 mg protein of the vesicular gland preparation and 0.7 puglecithinase for 15 min at 37’. In III and IV the mixtures were first kept for 15 min at 37’ after the addition of the lecithinase (0.7 ,ug) and then for 5 min at 80” to destroy lipase activity before the addition of vesicular gland protein (2 mg) and subsequent incubation for 15 min at 37”. The blank, without lecithinase, was treated as I and II. All incubations were stopped by the addition of citric acid until pH < 3.500 pg prostaglandin E, was added to facilitate recovery of radioactive prostaglandin formed. Lipids were extracted with diethyl ether; the extracts were washed with water, carefully taken to dryness in vacua, and purified by thin-layer chromatography (System A). Since it was shown in previous experiments with 14C-labelled precursors that part of the radioactivity found in the spot corresponding with prostaglandin E was invariably lost upon further purification, the prostaglandin E isolated from the thin-layer chromatographic plates was dehydrated with 0.5 M KOH. The prostaglandins B thus produced were purified by thin-layer chromatography (System B) until constant specific radioactivity. The amount of prostaglandin B was determined from the ultraviolet absorbance at 278 nm (ref. 9), that of endogenous prostaglandin E was 3 ,ug/mg protein. The total radioactivity originally present in prostaglandin can be calculated from the specific activity, the amount of prostaglan~iOd?im.
Biophys.
Acta,
164 (1968)
430-432
432
SHORT COMMUNICATIONS
din E added, and the amount of endogenous prostaglandin E (Table II). In the biosynthesis of prostaglandin E, from iaH]arachidonic acid, the tritium at C-9 is lostlo, while in the dehydration, the tritium atoms at C-8 and C-12 are also lost. Thus only 5 out of 8 tritium atoms are retained in the prostaglandin B. To calculate the conversion percentage, the radioactivity must therefore be multiplied by S/5 to compensate for these losses. As the amount of radioactivity incubated was IZOOOO countsjmin, the conversion can be given as a percentage of the arachidonic acid originally present (last column in Table II). TABLE
II
RESULTS Kpt. No.
_ .--_ .^~__. Pvostaglandirz N Comzts/min Coztntsjnztw (Ml Per iig ._
i
20.3
II III IV Blank
136 104 184 2Q
--.
--. 276 156 III 250 -
Counts/min
totid
pvostagEandin 3 708 582 557 708 0 _~_ ..____
‘4 I.15 I.1 1.4
Counts/mi?z
totLdx s/s ___-
.-
1134 931 893 ‘135 0 ~.___.
0.95 0.78
_._
O-73 0.95 0.0 ._~ --
The results of a pre-incubation with lecithinase (III and IV) were identical with those obtained in Expts. I and II. Because the blank experiment (without lecithinase) did not result in the formation of radioactive prostaglandin, we may conclude that the enzyme system in the particulate fraction of sheep vesicular glands is able to produce prostaglandin only from free fatty acid precursors. This is in agreement with the results of many experiments carried out during the production of prostaglandins in which we never could detect any prostaglandin in the phospholipid fraction. Similar results were obtained independently by LANDS AND SAMUELSSON’~, who used the lipase action of the high speed supernatant of a homogenate of sheep vesicular glands or rat liver. BARTELS, VOGT AND WILLE*~ found that injection of phospholipase glandin
A into perfused
release,
which
frog intestine
is in agreement
Unileuer Research Laboratory, Vlaardirgen (The Netherlands)
with
preparations
led to an increase
of prosta-
these findings.
H. VONKEMAN D. A. VAN DORP
I D. H. NUGTEREN, R. K. BEERTHUIS AND D. A.VAN DORP, Rec. Tmv.Chint., 85 (1966) 405. 2 C. B. STRLIIJK,It.K. BEERTHUIS, H. J. J. PABDN AND D. A. VAN DORP, Rec. Trav.Chim.,85 (1966) 1233. 3 U.A.VAN DORP,R.K.BEERTHL'IS,D.H.NUGTEREN AND H.VONKEMAN,B~OCJZ~?~. Bio$h_vs. i3cta. 90 IIQh4)204. 4 A. F. ko&&&o~ XNIJ W. E. hf.LANDS, Biochenzistvy, I (1962) 804. 5 E. G. BLIGH AND W. 1. DYER,&=. f.Biochem. Pkysiol., 37 (1959) 9x1. b C. B. STRUIJK, R.Ii. ~~EERTHUISANDD.A.VAN DO~P,~~S~B‘ER~~TRBMANDB.SAMUELSSON, Pposta~Za~d~ns, PYOC. 2nd Nobel Sywp., Stockkol~, Almqvist and Wiksell, Stockholm, 1967. 7 K. GREEN AND B. SAMUELSSON, J.LipidRes., 5 (1964) 117. Y J. FOLCH, M. LEES AND G. H. SLOANE STANLEY, J. BioE. Chew., 226 (1957) 497. 9 S.BERGSTR~M,R. RYHAGE,B.SAMUELSSON AND J.S~~v~~~,f.Riot.che~., r#(1963)3555. IO D. KLENBERG AND B.~AMUELSSON, ActaChem. Stand., 19(1965) 534. II W. E. M. LANDS AND B. SAMuELSSoN,~iochi~~.~io~~t~s.Acfa, 164 (1968) 426. 12 J. BARTELS, W. VOGT AND G. WILLE, Arch. Exptl. Pat/ml. Pharmakol., 259 (1968) 152
Received
August
rqth,
1968
Biochim. Biophys. Acta, 164 (1968) 430-432
430 BRA 53I90
The action of prostaglandin synthetase on 2-arachidonyl-lecithin In studying the specificity of the enzyme system present in the particulate fraction of sheep vesicular glands, it was found earlier’ that free essential fatty acids are converted in high yield into prostaglandins, whereas their methyl esters, as demonstrated with dihomo-y-linolenic acid (all-cis-S,rr,I4-eicosatrienoic acid) and arachidonic acid (all-cis-5,8,II,I4-eicosatetraenoic acid)2, give only very small conversions into free prostaglandins. From these observations it was concluded that the free carboxyl group was necessary for the conversion. Obviously the esters are first split by an esterase or by hydrolysis and the free acids converted subsequently. As in the animal the polyunsaturated fatty acids are mainly present in phospholipids, I-stearoyl-2-[3H]arachidonyl-lecithin was used as substrate prostaglandin synthesis from the bound precursor acid.
to study a possible direct
The results presented here indicate that conversion took place only after addition of lecithinase A to the incubation mixture, which is another proof of the free carboxyl group being essential to the enzymic reaction. PHIAll-cis-5,8,rI,I4-eicosatetraenoic acid was prepared by reduction of the corresponding tetraynoic acid with 3H, (ref. 3), purity >gg.o o/0(gas-liquid chromatography), thin-layer chromatography on silica/AgNO, showed that 98.0 “/bof the radioactivity
was present in the arachidonic
acid spot; Dr. H. v. D. BOSCH (Organic Chem-
istry Laboratory, University of Utrecht, The Netherlands) prepared the abovementioned lecithin by biosynthesis 4. It was extracted by the method of BLIGH ANI) DYERS and purified by thin-layer chromatography. More than ggsi of the [3H]arachidonic acid was attached to the z-position, as checked with lecithinase A. The specific radioactivity was 2.2 mC/mmole (1.2.106 counts/min per mg). The lecithinase preparation Crotalus adamanteus venom (ex Sigma Chemical Co., lot No. 53B-2160), when incubated with [I-‘Klarachidonic acid, did not convert into prostaglandin E,, suggesting that it did not contain any prostaglandin synthetase activity. The particulate fraction of sheep vesicular glands was prepared as described beforee. Thinlayer chromatography with o.5-mm layers. system diethyl
was performed on Silica gel-G-HR (ex Machery Nagel and Co.) The solvent systems used were: for prostaglandins as such:
described by GREEN AND SAMUELSSON~ (System A); for prostaglandin B: ether-acetic acid (g8:2, v/v) (System B) ; for lipids: chloroform-methanol-
acetic acid-water
(go: 6:
I
:0.75,
by vol.) (System
ether (g:I, v/v) (System D). A lipid extract from the particulate
C) ; for fatty acids: benzene-diethyl
fraction
and the seminal
vesicles
was
prepared using the method of FOLCH, LEES AND SLOANE STANLEY~ to compare the fatty acid compositions in a total hydrolysate and in the free fatty acid and phospholipid fractions. The two latter fractions were isolated via preparative thin-layer chromatography (System C), the fatty acids from the phospholipids being isolated after hydrolysis. Methyl esters were prepared with diazomethane and analysed by gas-liquid chromatography on 2 columns (Column I : polyethyleneglycol adipate (PEGA) 5 %, length 140 cm, diameter 3 mm, temp. 185”; Column 2 : Apiezon 3.6%, length 200 cm, followed by comdiameter 4 mm, temp. 210’; support Diatoport S). Identification Biochim.
Biophys.
Acta,
164 (1968)
430-432
SHORT
431
COMMUNICATIONS
paring their retention volumes with those of the reference fatty acids. In one fraction the fatty acids were also separated by thin-layer chromatography on silica gel-AgNO, (System D). Fractions containing 20:3 w6 and 20:4 c06+20:5 ~3 were isolated and again subjected to gas-liquid chromatographic analysis. Furthermore, part of these fractions were hydrogenated in glacial acetic acid with Pd on carbon. The retention volumes of these reduced acids indicated that C,, acids were actually present. Table I shows that 20:3 occurs in a higher concentration than 20: 4 in all fractions. The ratio ot 20:3 to 20:4 is about 6, except in the free fatty acid fraction. TABLE
I
FATTY ACID COMPOSITION OF VARIOUS FRACTIONS OF THOSE
OF
ITS
PARTICULATE
WHOLE
VESICULAR
~___~
Fat&
acid
__~~. --
Particulate
Whole gland Total lipid
Free fatty
I6:o
21.6
7.8
16:1
1.7 20.7
2.0
18:o
18:1 18:2
"js;:
acid
Phospholifiid 23.1 1.6
2.1
20:306 ro:4w6 20:4 w 3* 20:5w3
8.2
* Tentatively
I.3
OF
SHEEP
AND
OF
Free fattv acid
Phospholipid
25.4
11.8
27.8
__2.1
1g.0
18.8
34.5 4.’
30.2
6.0 23.0 6.6
1.6 8.3 1.6
2.5 II.5
2.1
8.2
0.9
6.0
5.0 1.2
fraction
Total lipid
5.3 29.4 5.7
20:1 20:2
GLAND
FRACTION
4.8 -
4.6
4.3 53.8 6.9 I.9 6.7 7.9
tr. z.1
I.9 I.8
1.0
16.2 29.2
4.3 3.4 12.7 2.1 2.1 I.2
1.0
identified.
Mixtures were prepared in the following way: to 2 ml 0.1 M phosphate buffer, pH 7.5, containing 0.001 M EDTA and O.OOIM cysteine, 200 ,ug desoxycholic acid was added to facilitate the solution of the added lecithin (IOO ,ug) in order to render the latter more accessible to the lecithinase. Incubations I and II (Table II) were performed with 2 mg protein of the vesicular gland preparation and 0.7 puglecithinase for 15 min at 37’. In III and IV the mixtures were first kept for 15 min at 37’ after the addition of the lecithinase (0.7 ,ug) and then for 5 min at 80” to destroy lipase activity before the addition of vesicular gland protein (2 mg) and subsequent incubation for 15 min at 37”. The blank, without lecithinase, was treated as I and II. All incubations were stopped by the addition of citric acid until pH < 3.500 pg prostaglandin E, was added to facilitate recovery of radioactive prostaglandin formed. Lipids were extracted with diethyl ether; the extracts were washed with water, carefully taken to dryness in vacua, and purified by thin-layer chromatography (System A). Since it was shown in previous experiments with 14C-labelled precursors that part of the radioactivity found in the spot corresponding with prostaglandin E was invariably lost upon further purification, the prostaglandin E isolated from the thin-layer chromatographic plates was dehydrated with 0.5 M KOH. The prostaglandins B thus produced were purified by thin-layer chromatography (System B) until constant specific radioactivity. The amount of prostaglandin B was determined from the ultraviolet absorbance at 278 nm (ref. 9), that of endogenous prostaglandin E was 3 ,ug/mg protein. The total radioactivity originally present in prostaglandin can be calculated from the specific activity, the amount of prostaglan~iOd?im.
Biophys.
Acta,
164 (1968)
430-432
432
SHORT COMMUNICATIONS
din E added, and the amount of endogenous prostaglandin E (Table II). In the biosynthesis of prostaglandin E, from iaH]arachidonic acid, the tritium at C-9 is lostlo, while in the dehydration, the tritium atoms at C-8 and C-12 are also lost. Thus only 5 out of 8 tritium atoms are retained in the prostaglandin B. To calculate the conversion percentage, the radioactivity must therefore be multiplied by S/5 to compensate for these losses. As the amount of radioactivity incubated was IZOOOO countsjmin, the conversion can be given as a percentage of the arachidonic acid originally present (last column in Table II). TABLE
II
RESULTS Kpt. No.
_ .--_ .^~__. Pvostaglandirz N Comzts/min Coztntsjnztw (Ml Per iig ._
i
20.3
II III IV Blank
136 104 184 2Q
--.
--. 276 156 III 250 -
Counts/min
totid
pvostagEandin 3 708 582 557 708 0 _~_ ..____
‘4 I.15 I.1 1.4
Counts/mi?z
totLdx s/s ___-
.-
1134 931 893 ‘135 0 ~.___.
0.95 0.78
_._
O-73 0.95 0.0 ._~ --
The results of a pre-incubation with lecithinase (III and IV) were identical with those obtained in Expts. I and II. Because the blank experiment (without lecithinase) did not result in the formation of radioactive prostaglandin, we may conclude that the enzyme system in the particulate fraction of sheep vesicular glands is able to produce prostaglandin only from free fatty acid precursors. This is in agreement with the results of many experiments carried out during the production of prostaglandins in which we never could detect any prostaglandin in the phospholipid fraction. Similar results were obtained independently by LANDS AND SAMUELSSON’~, who used the lipase action of the high speed supernatant of a homogenate of sheep vesicular glands or rat liver. BARTELS, VOGT AND WILLE*~ found that injection of phospholipase glandin
A into perfused
release,
which
frog intestine
is in agreement
Unileuer Research Laboratory, Vlaardirgen (The Netherlands)
with
preparations
led to an increase
of prosta-
these findings.
H. VONKEMAN D. A. VAN DORP
I D. H. NUGTEREN, R. K. BEERTHUIS AND D. A.VAN DORP, Rec. Tmv.Chint., 85 (1966) 405. 2 C. B. STRLIIJK,It.K. BEERTHUIS, H. J. J. PABDN AND D. A. VAN DORP, Rec. Trav.Chim.,85 (1966) 1233. 3 U.A.VAN DORP,R.K.BEERTHL'IS,D.H.NUGTEREN AND H.VONKEMAN,B~OCJZ~?~. Bio$h_vs. i3cta. 90 IIQh4)204. 4 A. F. ko&&&o~ XNIJ W. E. hf.LANDS, Biochenzistvy, I (1962) 804. 5 E. G. BLIGH AND W. 1. DYER,&=. f.Biochem. Pkysiol., 37 (1959) 9x1. b C. B. STRUIJK, R.Ii. ~~EERTHUISANDD.A.VAN DO~P,~~S~B‘ER~~TRBMANDB.SAMUELSSON, Pposta~Za~d~ns, PYOC. 2nd Nobel Sywp., Stockkol~, Almqvist and Wiksell, Stockholm, 1967. 7 K. GREEN AND B. SAMUELSSON, J.LipidRes., 5 (1964) 117. Y J. FOLCH, M. LEES AND G. H. SLOANE STANLEY, J. BioE. Chew., 226 (1957) 497. 9 S.BERGSTR~M,R. RYHAGE,B.SAMUELSSON AND J.S~~v~~~,f.Riot.che~., r#(1963)3555. IO D. KLENBERG AND B.~AMUELSSON, ActaChem. Stand., 19(1965) 534. II W. E. M. LANDS AND B. SAMuELSSoN,~iochi~~.~io~~t~s.Acfa, 164 (1968) 426. 12 J. BARTELS, W. VOGT AND G. WILLE, Arch. Exptl. Pat/ml. Pharmakol., 259 (1968) 152
Received
August
rqth,
1968
Biochim. Biophys. Acta, 164 (1968) 430-432