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Covalent binding of intermediates formed during the metabolism of arachidonic acid by human platelet subcellular fractions
PROSTAGLAN DIN S
COVALENT BINDING OF INTERMEDIATES FORMED DURING THE METABOLISM OF ARACHIDONIC ACID BY HUMAN PLATELET SUBCELLULAR FRACTIONS A. G. E. Wilson, H. C. Kung, M. W. Anderson and T. E. Eling National I n s t i t u t e of Environmental Health Sciences Laboratory of Pharmacokinetics and Laboratory of Pulmonary Function and Toxicology (Prostaglandin Group) Research Triangle Park, North Carolina 27709
SEPTEMBER 1979 VOL. 18 NO. 3
409
PROSTAGLANDINS INTRODUCTION The metabolism of 14C-arachidonic acid (AA) by the f a t t y acid cyclo-oxygenase pathway of guinea pig lung microsomes ( I ) and a number of other tissues (2) results in r a d i o a c t i v i t y becoming covalently bound to tissue macromolecules. This r a d i o a c t i v i t y is due to i n t e r mediates formed by the cyclo-oxygenase pathway, most probably the endoperoxides PGGp and PGHp (1,2). In t h i s study we have investigated whether covalentl~ bound intermediates would also be produced during the metabolism of AA by the human p l a t e l e t f a t t y acid cyclo-oxygenase system (cyclo-oxygenase pathway). In addition to the cyclo-oxygenase pathway, p l a t e l e t s have also been shown to contain a lipoxygenase enzyme capable of converting AA to the hydroxy acid, 12L-hydroxy-8 cis, I0 trans, 14 c i s - e i c o s t r i e n o i c acid (HETE) (3,4). The p o s s i b l i ty that metabolism of AA by the lipoxygenase pathway may also produce covalently bound intermediates was investigated. During the course of this study i t became necessary, for reasons discussed in the t e x t , to investigate the possible presence of a microsomal lipoxygenase, and the p o s s i b i l i t y that intermediates formed by t h i s pathway may also covalently bind to protein. MATERIALS AND METHODS Materials 1-14C-AA (sp. act. = 61 mCi/mm~le) was purchased from Amersham Corp., (Arlington ~eights, I I I ) ; 9- H-PGF~ (sp. act. = 15 Ci/mmole), 5,6,8,11,~2,14,15-~H-PGE2 (sp. act. = 200:~Ci/mmole), and 5,6,8,9,11, 12,14,15- H-AA (sp. act. = I00 Ci/mmole) were purchased from New England Nuclear (Boston, Mass.). Arachidonic acid was obtained from Nu Chek Prep (Elysian, Minn.). Indomethacin was a g i f t from Merck (Rahway, N.J.) and N-0164 was a g i f t from Dr. Nelson, Nelson Research and Development Company ( I r v i n , C a l i f . ) . Silica gel G t h i n - l a y e r chromatography plates (250 N) were purchased from Analabs Inc. (Wilmington, Del.). Radioactivity determinations were made using Aquasol s c i n t i l l a t i o n f l u i d (New England Nuclear). Expired (24 hours) human p l a t e l e t - r i c h plasma concentrate was obtained from the Red Cross, Durham, North Carolina. Arachidonic acid was p u r i f i e d by column chromatography as described by Parkes and Eling (5) and the p u r i t y rechecked before each experiment by t h i n - l a y e r chromatography. Methods The p l a t e l e t - r i c h plasma was centrifuged at 2,000 x g for 15 minutes at 4°C, and the r e s u l t i n g p e l l e t suspended in O.IM Tris buffer, pH 7.6 and sonicated for 30 seconds at 70 watts (Sonifer Cell Disruptor, Ultrasonices, Inc., Plainview, New York). The microsomal f r a c t i o n was then prepared as described by Needleman et al. (6) and washed and resuspended twice in ice cold 0.25M sucrose. The
410
SEPTEMBER 1979 VOL. 18 NO. 3
PROSTAGLANDINS I00,000 x g supernatant (soluble) fraction obtained by the centrifugation procedure was used without further purification. The incubation mixtures consisted of the following: 1-14C-AA (555,000 dpm, 4 nmoles evaporated to dryness), O. IM HEPESbuffer, pH 7.8, 0.25M sucrose, and 4 mg microsomal or soluble fraction protein, all in a total volume of 2.0 ml. Certain incubations contained heatdenatured microsomal or soluble fraction protein used to assess the extent of nonenzymatic conversion and binding of AA. The mixtures were incubated for 8 minutes at 37°C, the reaction stopped by adjusting the pH to 3.5 with 0.2 N HCI and extracted four times with 5 volumes of ethyl acetate. Following evaporation of the ethyl acetate extracts, the residues were redissolved in methanol and a small volume applied to s i l i c a gel G plates and developed in either solvent system C (chloroform: methanol:acetic acid:water = 90:8:1:0.8 v/v) described by Nugteren and Hazelhof (7), or light petroleum:diethyl ether:acetic acid (50:50:I v/v) described by Nugteren (4). The former system permits good separation of PG's and TXB~, whereas the l a t t e r gives excellent separation of 12-hydroxyhepta~ecatrienoic acid (HHT), HETE and AA. Plates were scraped (4mm sections) into Aquasol liquid s c i n t i l l a t i o n f l u i d (New England Nuclear) and the radioactivity was determined by liquid s c i n t i l l a t i o n counting. Metabolites were tentat i v e l y identified by co-chromatography with authentic standards. The protein in the aqueous layer was precipitated with I0% TCA, washed with TCA, and exhaustively extracted with methanol-water (80:20 v/v) and chloroform-methanol (2:1 v/v) until no more radioactivity could be extracted. The protein was digested in N NaOH and aliquots were taken for radioactivity and protein determination (8). The radioactivity associated with the protein was designated as protein-bound or covalently-bound metabolites. The radioactivity remaining in the protein-free water layer was determined and designated water-soluble metabolites. Total recovery of radioactivity was greater than 97%. Data has been presented as mean ~ S.E.M. and the significance evaluated by student's t - t e s t . RESULTS When the metabolism of 14C-AA by human platelet microsomal fraction was studied, the majority of the metabolized AA was distributed between the cyclo-oxygenase products, TXBp, PG's and HHT. (See Table l ) However, a significant amount (approximately 15% of the AA metabolized) chromatographed as the hydroxy acid, HETE. In addition approximately 5% of the AA metabolized was bound to protein. The radioactivity associated with the protein could not be dissociated even by such treatments as exhaustive solvent extraction, hot methanol reflux, or digestion with guanidine-HCl followed by Sephadex-column chromatography. Thus the radioactivity was strongly associated with the protein and most probably covalently bound (9). The amount of radioactivity associated with the platelet microsomal protein was of a
SEPTEMBER 1979 VOL 18 NO. 3
411
PROSTAGLANDINS
s i m i l a r magnitude to that found with the microsomal f r a c t i o n s prepared from guinea pig lungl4ram and bovine seminal v e s i c l e s , and r a t kidney (2). Incubation of C-AA with denatured ( b o i l e d ) microsomal protein produced no detectable l e v e l s of AA metabolites and only i n s i g n i f i c a n t amounts in the water layer or associated with the protein. The addition of indomethacin to the incubation mixture s i g n i f i c a n t l y i n h i b i t e d the production of PGE^, TXB~, and HHT, whereas the production of HETE was s i g n i f i c a n t l y inc~easedL(See Table I ) . The amount of covalently bound r a d i o a c t i v i t y appeared to be s l i g h t l y increased by the presence of indomethacin, whereas the quantity of water-soluble metabolites formed was unaffected. This was in sharp contrast to the finding with both guinea pig lung and bovine seminal Vesicle microsomes, in which the presence of indomethacin completely i n h i b i t e d the covalent binding
(1,2). The chemical N-0164 has been shown to p r e f e r e n t i a l l y i n h i b i t the thromboxane synthetase pathway at low concentrations, whereas at higher concentrations the cyclo-oxygenase is also i n h i b i t e d ( I 0 ) . As shown in Table 2, the presence of N-0164 markedly decreased the amounts of TXB2 and HHT formed from AA by p l a t e l e t microsomes, without a f f e c t i n g the amount of r a d i o a c t i v i t y bound to protein. The t o t a l amount of AA metabolized was also increased (approximately 23%), which was a r e f l e c t i o n of the large increase seen in HETE. Detectable l e v e l s of PGEp were only produced in p l a t e l e t microsomes when incubated in the pregence of N-0164. Since both TXBp and HHT l e v e l s were i n h i b i t e d , i t would appear t h a t , at the concentration of N-0164 used, both the thromboxane synthetase and to some extent the cyclo-oxygenase pathway, were i n h i b i t e d . These results would suggest that in addition to the cyclo-oxygenase pathway another enzyme may be present in the p l a t e l e t microsomal fract i o n which also produces intermediate(s) capable of c o v a l e n t l y binding to protein. This pathway apparently metabolizes AA to HETE and would appear to be analogous to the previously reported cytoplasmic l i p o x y genase system (3,4). To determine whether the s o l u b l e - f r a c t i o n lipoxygenase was capable of producing reactive intermediates that could covalently bind to protein, the metabolism of AA by the I00,000 x g supernatant f r a c t i o n of human p l a t e l e t s was investigated. As seen in Table 3, HETE was the only product r o u t i n e l y detected in s i g n i f i c a n t amounts and contamination by cyclo-oxygenase products was i n s i g n i f i c a n t . Approximately 510% of the AA metabolized was found to be c o v a l e n t l y bound to protein, while 2% was in the form of water-soluble products. The addition of 50 NM indomethacin to the incubation appears to have stimulated the production of HETE, and produced increased amounts of r a d i o a c t i v i t y covalently bound to protein and in the water layer (see Table 3). This would appear to support our hypothesis that the lipoxygenase pathway also converts AA to reactive intermediates capable of covalently binding to tissue protein.
412
SEPTEMBER 1979 VOL. 18 NO. 3
PROSTAGLANDINS Eicosatetraynoic acid (TYA) is a known i n h i b i t o r of both the cyclo-oxygenase and lipoxygenase pathways of AA metabolism (3,11). TYA should therefore i n h i b i t the formation of covalently bound radioa c t i v i t y in both the soluble and microsomal f r a c t i o n from p l a t e l e t s . This was found to be the case, as the presence of TYA (150 pM) almost completely i n h i b i t e d both the covalent binding and metabolism of AA (Tables 4 and 5). 3The covalent binding was not due to end-products since addition of H-PG's, TXBp and HETE resulted i % o n l y i n s i g n i f i cant amounts of binding. Furthermore, addition of "~C-AA to boiled microsomes resulted in only minimal levels of binding. DISCUSSION This study demonstrates that during the metabolism of AA by p l a t e l e t microsomes, intermediate(s) are produced that become coval e n t l y bound to microsomal protein. We previously reported ( I ) that during the metabolism of AA by guinea pig lung and bovine seminal vesicle microsomes, covalently bound intermediate(s) were also formed. We postulated that the intermediate(s) most l i k e l y responsible for the binding were the cyclo-oxygenase metabolites, endoperoxides PGG~ and/or PGH~ (2,12). I t was therefore expected that a similar f~nding would be observed during the metabolism of AA by p l a t e l e t microsomes. However, i t was both surprising and c o n f l i c t i n g that i n h i b i t i o n of the cyclo-oxygenase pathway did not reduce the covalent binding. The mechanism(s) involved in the production of covalently bound intermediates in p l a t e l e t microsomes is apparently more complex than for the tissues examined. The binding in p l a t e l e t microsomes is apparently not solely due to the endoperoxides, but other metabol i t e s are produced by p l a t e l e t microsomes which also covalently bind. A potential explanation of the observed findings is that a pathway in addition to the cyclo-oxygenase pathway could also produce reactive intermediates from AA which covalently bind. An essential feature, i f t h i s is the case, is that in the presence of indomethacin more products, and thus covalently bound intermediates, should be produced by t h i s pathway ( i . e . , i t is not i n h i b i t e d by indomethacin). This could possibly be the other enzyme shown to be associated with p l a t e l e t s , namely the lipoxygenase pathway (4). That such a lipo×ygenase pathway is present in p l a t e l e t microsomes is suggested by the large amounts of a metabolite produced during the metabolism of AA, which chromatographed as HETE. Furthermore, in the presence of indomethacin, increased amounts of HETE were produced by AA in both p l a t e l e t microsomal and soluble fractions. There exists some discrepancy in the l i t e r a t u r e as to whether the lipoxygenase, which metabolizes AA to HETE, is associated with the
SEPTEMBER 1979 VOL. 18 NO. 3
413
PROSTAGLANDINS microsomal or soluble f r a c t i o n . The presence of the enzyme in both bovine and human p l a t e l e t soluble fractions has been reported (4 and 13). However, Ho et al. (14) recently suggested that this enzyme was only located in the p a r t i c u l a t e f r a c t i o n and found no evidence of a s o l u b l e - f r a c t i o n lipoxygenase. Our data would suggest that both membrane and cytoplasmic lipoxygenases, which metabolize AA to HETE, are present in human p l a t e l e t s . That the lipoxygenase pathway could r e s u l t in the production of covalently bound intermediates from AA was seen in the studies using the soluble f r a c t i o n . That the covalent binding was not d i r e c t l y due to HETE was shown when radiolabeled HETE was added to protein, since a l l of the r a d i o a c t i v i t y could be extracted with organic solvents. Nugteren (4) has isolated an intermediate which has a hydroperoxy group at C-12 ( i . e . , 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid, HPETE) and is formed during the metabolism of AA by the lipoxygenase pathway. We have previously shown that the e l e c t r o p h i l i c i n t e r mediates formed by the cyclo-oxygenase pathway ( i . e . , PGG~/PGH~) can covalently bind to protein (2,12). I t may therefore be t~at t~e 12hydroperoxy group in HPETE is s u f f i c i e n t l y e l e c t r o p h i l i c to react with nucleophilic sites on protein, and with other nucleophiles, e . g . , glutathione. I f this is the case, then the water-soluble metabolites may well be glutathione conjugates of HPETE, especially since high glutathione concentrations e x i s t in the cytoplasm (15 and 16). The i d e n t i t y of the covalently bound intermediate produced by the lipoxygenase pathway is at present uncertain. However, i t cannot be overlooked that the binding may be due to an e l e c t r o p h i l i c intermediate other than HPETE. In summary, i t would appear that in p l a t e l e t microsomes two pathways are present ( i . e . , cyclo-oxygenase and lipoxygenase) that produce reactive intermediates from AA which covalently bound to tissue protein. The r e l a t i v e k i n e t i c s of the production of reactive intermediates by both these pathways remains to be determined. Whether this binding occurs in the i n t a c t p l a t e l e t and what role, i f any, this binding has in normal p l a t e l e t function and aggregation remains to be determined. The endoperoxides are known to play an important role in p l a t e l e t aggregation (17,18). I t is i n t e r e s t i n g to speculate that such binding, i f i t occurs, could have a regulatory role in the control of the ' f r e e ' endoperoxide concentration and thereby a role in p l a t e l e t aggregation. At present l i t t l e is known about the role of the lipoxygenase enzyme and i t s metabolites of AA, i . e . , HETE and HPETE. Until more is known about the importance of these metabolites, i t is d i f f i c u l t to assess the significance of the covalent binding. The physiological and t o x i c o l o g i c a l significance of the covalent binding of intermediates formed from AA by the cyclo-oxygenase and lipoxygenase pathways, remains to be established.
414
SEPTEMBER 1979 VOL 18 NO. 3
PROSTAGLANDINS
ACKNOWLEDGEMENTS We wish to thank Ms. Sandra Smith and Mr. Claude Raines f o r t h e i r e x c e l l e n t technical assistance, and Kathleen Conant and Frances Holloway f o r t h e i r assistance in the preparation of t h i s manuscript.
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PROSTAGLANDINS
REFERENCES I.
Eling, T. E., Wilson, A. G. E., Chaudhari, A. and Anderson, M. W. Covalent binding of intermediate(s) in prostaglandin biosynthesis to guinea pig lung microsomal protein. Life Sci. 21:245-252, 1977.
2.
Anderson, M. W., Crutchley, D. J., Chaudhari, A., Wilson, A.G.E. and Eling, T. E.: Studies on the covalent binding of an i n t e r mediate(s) in prostaglandin biosynthesis to tissue macromolecules. Biochim. Biophys. Acta 573:40-50, 1979.
3.
Hamberg, M. and Samuelsson, B.: Prostaglandin endoperoxides: Novel transformation of arachidonic acid in human platelets. Proc. Natl. Acad. Sci. 71:3400-3404, 1974.
4.
Nugteren, D. H.: Arachidonate lipoxygenase in blood platelets. Biochem. Biophys. Acta 380:299-307, 1975.
5.
Parkes, D. G. and Eling, T. E.: Characterization of prostaglandin synthetase in guinea pig lung. Isolation of a new prostaglandin derivative from arachidonic acid. Biochemistry 13:2598-2604, 1974.
6.
Needleman, P., Moncada, S., Bunting, S., Vane, J. R., Hamberg, M. and Samuelsson, B.: I d e n t i f i c a t i o n - o f an enzyme in p l a t e l e t microsomes which generates thromboxane A~ from prostaglandin endoperoxides. Nature (London), 261:558~560, 1976.
7.
Nugteren, D. H. and Hazelhof, E.: Isolation and properties of intermediates in prostaglandin biosynthesis. Biochim. Biophys. Acta 326:448-461, 1973.
8.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275, 1951.
9.
Gillette, J. R. and Pohl, L. R.: A prospective on covalent binding and toxicity. J. Toxicol. Env. Hlth. 2:849-871, 1977.
I0.
Kulkarni, P. S. and Eakins, K. E.: N-0164 i n h i b i t s generation of thromboxane-Ap-like a c t i v i t y from prostaglandin endoperoxides by human p l a t e l e t microsomes. Prostaglandins 12:465-469, 1976.
II.
Falardeau, P., Hamberg, M. and Samuelsson, B.: Metabolism of 8,11,14-eicosatrienoic acid in human platelets. Biochim. Biophys. Acta 441:193-200, 1976.
416
SEPTEMBER 1979 VOL. 18 NO. 3
PROSTAGLANDINS
12.
Crutchley, D. J., Hawkins, H. J., Eling, T. E. and Anderson, M. W.: Covalent binding of prostaglandins G9 and H2 to components of ram seminal vesicle microsoma? fractTons. Biochem. Pharmacol. 28:1519-1523, 1979.
13.
Turner, S. R., Tainer, J. A. and Lynn, W. S.: Biogenesis of chemotactic molecules by the arachidonate lipoxygenase system of platelets. Nature (London) 257:680-681, 1975.
14.
Ho, P. P. K., Walters, P. and Sullivan, H. R.: A particulate arachidonate lipoxygenase in human blood platelets. Biochim. Biophys. Acta 76:398-405, 1977.
15.
Gardner, H. W.: Decomposition of l i n o l e i c acid hydroperoxides. Enzymic reactions compared with non-enzymic. J. Agricult. Food Chem. 23:129-136, 1975.
16.
Flohe, L. and Gunzler, W. A. in Glutathione: Metabolism and Function (Arias, I. M. and Jakoby, W. B., eds.) Raven Press, New York, 1975, pp 17-34.
17.
Hamberg, M., Svensson, J., Wakabayashi, TI, and Samuelsson, B.: Isolation and structure of two prostaglandin endoperoxides that cause p l a t e l e t aggregation. Proc. Natl. Acad. Sci. 71:345-349, 1974.
18.
Svensson, J . , Hamberg, M., and Samuelsson, B.: Prostaglandin endoperoxides IX. Characterizatioa of rabbit aorta contracting substance (RCS) from guinea pig lung and human platelets. Acta Physiol. Scand. 94:222-228, 1975.
SEPTEMBER 1979 VOL. 18 NO. 3
417
0
G~
0
m:
m~
m~
m~
4~ Oo
672.9 +
34.7 +
8.8 +
86.0
3.5
0.4
9.3*
1.2+
5.7*
4.2 +
0.4+
732.4 + 58.5
47.5 +
7.8 +
524.3 + 37.7*
90.5 +
5.0 ~
57.3 ~
C ' S i g n i f i c a n t l y d i f f e r e n t from control P
bValues reported are Mean ~ S.E.M. picomoles per mg. protein N=4.
Incubation
I08.9
135.9
86.6
500.3
25.8
51.0
35.2
INDOMETHACIN-TREATED (50uM) PICOMOLES % CONTROL
a I n i t i a l concentration of AA in the incubation was 2NM ( i . e . , 4 nmoles per incubation). conditions and analysis of metabolites are described in the Methods section.
TOTAL
Protein Bound
Water-Soluble
I04.8 + 20.8
HETE
1.5
351.0 + 41.5
9.8 ~
163.8 ~ 18.3
CONTROL PICOMOLES
HHT
PGE2
TXB2
AA METABOLITES
Effect of Indomethacin
The Covalent Binding of AA Intermediates to Human Platelet Microsomal
Protein and Formation of Water-Soluble Metabolites:
TABLE I.
Z
>
©
0
z
O0
0
m~ m:
m~
m~
AA METABOLITES
732.9 + 31.8
1.0
0.4
3.4*
2.9
1.3 *
0 . 5 N.S.
0.2 +
892.4 + 45.6
20.5 +
m
4.8 +
718.0 + 3 7 . 3 *
70.8 +
39.5 ~
38.8 ~
ND = Not detectable ( i . e . < 0.5 picomole)
* S i g n i f i c a n t l y d i f f e r e n t from control P
Values reported are Mean + S.E.M. picomoles per mg. protein N=4
Incubation
123
98.0
73.4
298.0
27.0
-
19.2
N-O164-TREATED (237 uM) PICOMOLES % CONTROL
I n i t i a l concentration of AA in the incubation was 2pM ( i . e . , 4 nmoles per incubation. conditions and analysis of metabol~tes are described in the Methods section.
TOTAL
20.8 +
P r o t e i n Bound
--
6,8 +
Water-Soluble
6.2
241.0 +
HETE
9.3
262.3 + 14.9
ND
202.0 ~
CONTROL PICOMOLES
HHT
PGE2
TXB2
The Effect of Thromboxane Synthetase I n h i t i t o r N-0164 on
the Covalent Binding of AA Intermediates to Human Platelet Microsomal Protein.
TABLE 2.
Z
0
0
P
Z
cm
m~ m:
m~
m~
:b
4~
0.7
0.3
5.4
0.8 ~
376.5 + 33.1
47.5 +
10.5 +
318.5 + 26.9 ~
Cyclo-oxgenase products represented < 5% of total AA metabolized
~Significantly different from control P
Values reported are Mean ~ S.E.M. picomoles per mg. protein N=4
Incubation
131.5
161.0
131.3
128.0
INDOMETHAClN-TREATED (50uM) PICOMOLES % CONTROL
I n i t i a l c o n c e n t r a t i o n o f AA i n the i n c u b a t i o n was 2NM ( i . e . , 4 nmoles per i n c u b a t i o n . c o n d i t i o n s and a n a l y s i s o f m e t a b o l i t e s are described in the Methods s e c t i o n .
286.3 + 14.9
29.5 +
P r o t e i n Bound
TOTAL
8.0 +
248.8 + 13.9
CONTROL PICOMOLES
Water-Soluble
HETE
AA METABOLITES
E f f e c t o f Indomethacin.
Covalent Binding of AA I n t e r m e d i a t e s to Human P l a t e l e t Soluble
F r a c t i o n P r o t e i n and the Formation o f Water-Soluble M e t a b o l i t e s :
TABLE 3.
Z
Z
0 c~
Z 0
o~
~N
< 0
m~
m~
m~
2.0
0.8
5.5 + 0.34
1.0 + 0.042
ND
4.5 + 0.32
PICOMOLES
.
I00.0
17.8
.
82.2 .
.
.
.
ND = Not detectable ( i . e . < 0.5 picomole)
Cyclo-oxgenase products represented < 5% of total AA metabolized
*Significantly different from control P
Values reported are Mean ~ S.E.M. picomoles per mg. protein N=4
.
Incubation
1.6
4.6
1.4
TYA-TREATED (150uM) % AA CONVERTED % CONTROL
Effect of
I n i t i a l concentration of AA in the incubation was 2 pM ( i . e . , 4 nmoles per incubation). conditions and analysis of metabolites are described in the Methods section.
349.1 + 29.3
21.3 +
P r o t e i n Bound
TOTAL
8.5 +
319.3 + 26.5
CONTROL PICOMOLES
TYA (5,8,11,14-Eicosatetraynoic Acid).
Protein and Formation of Water-Soluble Metabolites:
The Covalent Binding of AA Intermediates to Human Platelet Soluble Fraction
Water-Soluble
HETE
AA METABOLITES
TABLE 4.
Z
r"
0
c)
m:
m~
m~
t~
216.8 + 34.1
HETE
I.I
863.2 + 78.5
30.8 +
*
23.4 + 3.0
0.9 + 0. I *
1.2 + 0.2*
ND
13.5 + 1.5 ~
1.8 + 0.2 ~
6.0÷I.0
different
ND = Not d e t e c t a b l e ( i . e . ,
*Significantly < 0.5 picomole)
from c o n t r o l P
Values reported are Mean + S.E.M. picomoles per mg. p r o t e i n N=4
Incubation
2.7
2.9
10.9
---
3.9
7.6
2.5
TYA-TREATED (150uM) PICOMOLES % CONTROL
I n i t i a l c o n c e n t r a t i o n of AA i n the i n c u b a t i o n was 2NM ( i . e . , 4 nmoles per i n c u b a t i o n ) . c o n d i t i o n s and a n a l y s i s of m e t a b o l i t e s are described i n the Methods s e c t i o n .
TOTAL
P r o t e i n Bound
0.8
346.5 + 30.8
HHT
10.8 +
23.3 +_ 1.0
PGE2
Water-Woluble
235.0 +_ 10.7
CONTROL PICOMOLES
TXB2
AA METABOLITES
TYA ( 5 , 8 , 1 1 , 1 4 - E i c o s a t e t r a y n o i c A c i d ) .
E f f e c t of
The Covalent Binding o f AA Intermediates to Human P l a t e l e t Microsomal
P r o t e i n and the Formation of Water-Soluble M e t a b o l i t e s :
TABLE 5.
m=
>
0
COVALENT BINDING OF INTERMEDIATES FORMED DURING THE METABOLISM OF ARACHIDONIC ACID BY HUMAN PLATELET SUBCELLULAR FRACTIONS A. G. E. Wilson, H. C. Kung, M. W. Anderson and T. E. Eling National I n s t i t u t e of Environmental Health Sciences Laboratory of Pharmacokinetics and Laboratory of Pulmonary Function and Toxicology (Prostaglandin Group) Research Triangle Park, North Carolina 27709
SEPTEMBER 1979 VOL. 18 NO. 3
409
PROSTAGLANDINS INTRODUCTION The metabolism of 14C-arachidonic acid (AA) by the f a t t y acid cyclo-oxygenase pathway of guinea pig lung microsomes ( I ) and a number of other tissues (2) results in r a d i o a c t i v i t y becoming covalently bound to tissue macromolecules. This r a d i o a c t i v i t y is due to i n t e r mediates formed by the cyclo-oxygenase pathway, most probably the endoperoxides PGGp and PGHp (1,2). In t h i s study we have investigated whether covalentl~ bound intermediates would also be produced during the metabolism of AA by the human p l a t e l e t f a t t y acid cyclo-oxygenase system (cyclo-oxygenase pathway). In addition to the cyclo-oxygenase pathway, p l a t e l e t s have also been shown to contain a lipoxygenase enzyme capable of converting AA to the hydroxy acid, 12L-hydroxy-8 cis, I0 trans, 14 c i s - e i c o s t r i e n o i c acid (HETE) (3,4). The p o s s i b l i ty that metabolism of AA by the lipoxygenase pathway may also produce covalently bound intermediates was investigated. During the course of this study i t became necessary, for reasons discussed in the t e x t , to investigate the possible presence of a microsomal lipoxygenase, and the p o s s i b i l i t y that intermediates formed by t h i s pathway may also covalently bind to protein. MATERIALS AND METHODS Materials 1-14C-AA (sp. act. = 61 mCi/mm~le) was purchased from Amersham Corp., (Arlington ~eights, I I I ) ; 9- H-PGF~ (sp. act. = 15 Ci/mmole), 5,6,8,11,~2,14,15-~H-PGE2 (sp. act. = 200:~Ci/mmole), and 5,6,8,9,11, 12,14,15- H-AA (sp. act. = I00 Ci/mmole) were purchased from New England Nuclear (Boston, Mass.). Arachidonic acid was obtained from Nu Chek Prep (Elysian, Minn.). Indomethacin was a g i f t from Merck (Rahway, N.J.) and N-0164 was a g i f t from Dr. Nelson, Nelson Research and Development Company ( I r v i n , C a l i f . ) . Silica gel G t h i n - l a y e r chromatography plates (250 N) were purchased from Analabs Inc. (Wilmington, Del.). Radioactivity determinations were made using Aquasol s c i n t i l l a t i o n f l u i d (New England Nuclear). Expired (24 hours) human p l a t e l e t - r i c h plasma concentrate was obtained from the Red Cross, Durham, North Carolina. Arachidonic acid was p u r i f i e d by column chromatography as described by Parkes and Eling (5) and the p u r i t y rechecked before each experiment by t h i n - l a y e r chromatography. Methods The p l a t e l e t - r i c h plasma was centrifuged at 2,000 x g for 15 minutes at 4°C, and the r e s u l t i n g p e l l e t suspended in O.IM Tris buffer, pH 7.6 and sonicated for 30 seconds at 70 watts (Sonifer Cell Disruptor, Ultrasonices, Inc., Plainview, New York). The microsomal f r a c t i o n was then prepared as described by Needleman et al. (6) and washed and resuspended twice in ice cold 0.25M sucrose. The
410
SEPTEMBER 1979 VOL. 18 NO. 3
PROSTAGLANDINS I00,000 x g supernatant (soluble) fraction obtained by the centrifugation procedure was used without further purification. The incubation mixtures consisted of the following: 1-14C-AA (555,000 dpm, 4 nmoles evaporated to dryness), O. IM HEPESbuffer, pH 7.8, 0.25M sucrose, and 4 mg microsomal or soluble fraction protein, all in a total volume of 2.0 ml. Certain incubations contained heatdenatured microsomal or soluble fraction protein used to assess the extent of nonenzymatic conversion and binding of AA. The mixtures were incubated for 8 minutes at 37°C, the reaction stopped by adjusting the pH to 3.5 with 0.2 N HCI and extracted four times with 5 volumes of ethyl acetate. Following evaporation of the ethyl acetate extracts, the residues were redissolved in methanol and a small volume applied to s i l i c a gel G plates and developed in either solvent system C (chloroform: methanol:acetic acid:water = 90:8:1:0.8 v/v) described by Nugteren and Hazelhof (7), or light petroleum:diethyl ether:acetic acid (50:50:I v/v) described by Nugteren (4). The former system permits good separation of PG's and TXB~, whereas the l a t t e r gives excellent separation of 12-hydroxyhepta~ecatrienoic acid (HHT), HETE and AA. Plates were scraped (4mm sections) into Aquasol liquid s c i n t i l l a t i o n f l u i d (New England Nuclear) and the radioactivity was determined by liquid s c i n t i l l a t i o n counting. Metabolites were tentat i v e l y identified by co-chromatography with authentic standards. The protein in the aqueous layer was precipitated with I0% TCA, washed with TCA, and exhaustively extracted with methanol-water (80:20 v/v) and chloroform-methanol (2:1 v/v) until no more radioactivity could be extracted. The protein was digested in N NaOH and aliquots were taken for radioactivity and protein determination (8). The radioactivity associated with the protein was designated as protein-bound or covalently-bound metabolites. The radioactivity remaining in the protein-free water layer was determined and designated water-soluble metabolites. Total recovery of radioactivity was greater than 97%. Data has been presented as mean ~ S.E.M. and the significance evaluated by student's t - t e s t . RESULTS When the metabolism of 14C-AA by human platelet microsomal fraction was studied, the majority of the metabolized AA was distributed between the cyclo-oxygenase products, TXBp, PG's and HHT. (See Table l ) However, a significant amount (approximately 15% of the AA metabolized) chromatographed as the hydroxy acid, HETE. In addition approximately 5% of the AA metabolized was bound to protein. The radioactivity associated with the protein could not be dissociated even by such treatments as exhaustive solvent extraction, hot methanol reflux, or digestion with guanidine-HCl followed by Sephadex-column chromatography. Thus the radioactivity was strongly associated with the protein and most probably covalently bound (9). The amount of radioactivity associated with the platelet microsomal protein was of a
SEPTEMBER 1979 VOL 18 NO. 3
411
PROSTAGLANDINS
s i m i l a r magnitude to that found with the microsomal f r a c t i o n s prepared from guinea pig lungl4ram and bovine seminal v e s i c l e s , and r a t kidney (2). Incubation of C-AA with denatured ( b o i l e d ) microsomal protein produced no detectable l e v e l s of AA metabolites and only i n s i g n i f i c a n t amounts in the water layer or associated with the protein. The addition of indomethacin to the incubation mixture s i g n i f i c a n t l y i n h i b i t e d the production of PGE^, TXB~, and HHT, whereas the production of HETE was s i g n i f i c a n t l y inc~easedL(See Table I ) . The amount of covalently bound r a d i o a c t i v i t y appeared to be s l i g h t l y increased by the presence of indomethacin, whereas the quantity of water-soluble metabolites formed was unaffected. This was in sharp contrast to the finding with both guinea pig lung and bovine seminal Vesicle microsomes, in which the presence of indomethacin completely i n h i b i t e d the covalent binding
(1,2). The chemical N-0164 has been shown to p r e f e r e n t i a l l y i n h i b i t the thromboxane synthetase pathway at low concentrations, whereas at higher concentrations the cyclo-oxygenase is also i n h i b i t e d ( I 0 ) . As shown in Table 2, the presence of N-0164 markedly decreased the amounts of TXB2 and HHT formed from AA by p l a t e l e t microsomes, without a f f e c t i n g the amount of r a d i o a c t i v i t y bound to protein. The t o t a l amount of AA metabolized was also increased (approximately 23%), which was a r e f l e c t i o n of the large increase seen in HETE. Detectable l e v e l s of PGEp were only produced in p l a t e l e t microsomes when incubated in the pregence of N-0164. Since both TXBp and HHT l e v e l s were i n h i b i t e d , i t would appear t h a t , at the concentration of N-0164 used, both the thromboxane synthetase and to some extent the cyclo-oxygenase pathway, were i n h i b i t e d . These results would suggest that in addition to the cyclo-oxygenase pathway another enzyme may be present in the p l a t e l e t microsomal fract i o n which also produces intermediate(s) capable of c o v a l e n t l y binding to protein. This pathway apparently metabolizes AA to HETE and would appear to be analogous to the previously reported cytoplasmic l i p o x y genase system (3,4). To determine whether the s o l u b l e - f r a c t i o n lipoxygenase was capable of producing reactive intermediates that could covalently bind to protein, the metabolism of AA by the I00,000 x g supernatant f r a c t i o n of human p l a t e l e t s was investigated. As seen in Table 3, HETE was the only product r o u t i n e l y detected in s i g n i f i c a n t amounts and contamination by cyclo-oxygenase products was i n s i g n i f i c a n t . Approximately 510% of the AA metabolized was found to be c o v a l e n t l y bound to protein, while 2% was in the form of water-soluble products. The addition of 50 NM indomethacin to the incubation appears to have stimulated the production of HETE, and produced increased amounts of r a d i o a c t i v i t y covalently bound to protein and in the water layer (see Table 3). This would appear to support our hypothesis that the lipoxygenase pathway also converts AA to reactive intermediates capable of covalently binding to tissue protein.
412
SEPTEMBER 1979 VOL. 18 NO. 3
PROSTAGLANDINS Eicosatetraynoic acid (TYA) is a known i n h i b i t o r of both the cyclo-oxygenase and lipoxygenase pathways of AA metabolism (3,11). TYA should therefore i n h i b i t the formation of covalently bound radioa c t i v i t y in both the soluble and microsomal f r a c t i o n from p l a t e l e t s . This was found to be the case, as the presence of TYA (150 pM) almost completely i n h i b i t e d both the covalent binding and metabolism of AA (Tables 4 and 5). 3The covalent binding was not due to end-products since addition of H-PG's, TXBp and HETE resulted i % o n l y i n s i g n i f i cant amounts of binding. Furthermore, addition of "~C-AA to boiled microsomes resulted in only minimal levels of binding. DISCUSSION This study demonstrates that during the metabolism of AA by p l a t e l e t microsomes, intermediate(s) are produced that become coval e n t l y bound to microsomal protein. We previously reported ( I ) that during the metabolism of AA by guinea pig lung and bovine seminal vesicle microsomes, covalently bound intermediate(s) were also formed. We postulated that the intermediate(s) most l i k e l y responsible for the binding were the cyclo-oxygenase metabolites, endoperoxides PGG~ and/or PGH~ (2,12). I t was therefore expected that a similar f~nding would be observed during the metabolism of AA by p l a t e l e t microsomes. However, i t was both surprising and c o n f l i c t i n g that i n h i b i t i o n of the cyclo-oxygenase pathway did not reduce the covalent binding. The mechanism(s) involved in the production of covalently bound intermediates in p l a t e l e t microsomes is apparently more complex than for the tissues examined. The binding in p l a t e l e t microsomes is apparently not solely due to the endoperoxides, but other metabol i t e s are produced by p l a t e l e t microsomes which also covalently bind. A potential explanation of the observed findings is that a pathway in addition to the cyclo-oxygenase pathway could also produce reactive intermediates from AA which covalently bind. An essential feature, i f t h i s is the case, is that in the presence of indomethacin more products, and thus covalently bound intermediates, should be produced by t h i s pathway ( i . e . , i t is not i n h i b i t e d by indomethacin). This could possibly be the other enzyme shown to be associated with p l a t e l e t s , namely the lipoxygenase pathway (4). That such a lipo×ygenase pathway is present in p l a t e l e t microsomes is suggested by the large amounts of a metabolite produced during the metabolism of AA, which chromatographed as HETE. Furthermore, in the presence of indomethacin, increased amounts of HETE were produced by AA in both p l a t e l e t microsomal and soluble fractions. There exists some discrepancy in the l i t e r a t u r e as to whether the lipoxygenase, which metabolizes AA to HETE, is associated with the
SEPTEMBER 1979 VOL. 18 NO. 3
413
PROSTAGLANDINS microsomal or soluble f r a c t i o n . The presence of the enzyme in both bovine and human p l a t e l e t soluble fractions has been reported (4 and 13). However, Ho et al. (14) recently suggested that this enzyme was only located in the p a r t i c u l a t e f r a c t i o n and found no evidence of a s o l u b l e - f r a c t i o n lipoxygenase. Our data would suggest that both membrane and cytoplasmic lipoxygenases, which metabolize AA to HETE, are present in human p l a t e l e t s . That the lipoxygenase pathway could r e s u l t in the production of covalently bound intermediates from AA was seen in the studies using the soluble f r a c t i o n . That the covalent binding was not d i r e c t l y due to HETE was shown when radiolabeled HETE was added to protein, since a l l of the r a d i o a c t i v i t y could be extracted with organic solvents. Nugteren (4) has isolated an intermediate which has a hydroperoxy group at C-12 ( i . e . , 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid, HPETE) and is formed during the metabolism of AA by the lipoxygenase pathway. We have previously shown that the e l e c t r o p h i l i c i n t e r mediates formed by the cyclo-oxygenase pathway ( i . e . , PGG~/PGH~) can covalently bind to protein (2,12). I t may therefore be t~at t~e 12hydroperoxy group in HPETE is s u f f i c i e n t l y e l e c t r o p h i l i c to react with nucleophilic sites on protein, and with other nucleophiles, e . g . , glutathione. I f this is the case, then the water-soluble metabolites may well be glutathione conjugates of HPETE, especially since high glutathione concentrations e x i s t in the cytoplasm (15 and 16). The i d e n t i t y of the covalently bound intermediate produced by the lipoxygenase pathway is at present uncertain. However, i t cannot be overlooked that the binding may be due to an e l e c t r o p h i l i c intermediate other than HPETE. In summary, i t would appear that in p l a t e l e t microsomes two pathways are present ( i . e . , cyclo-oxygenase and lipoxygenase) that produce reactive intermediates from AA which covalently bound to tissue protein. The r e l a t i v e k i n e t i c s of the production of reactive intermediates by both these pathways remains to be determined. Whether this binding occurs in the i n t a c t p l a t e l e t and what role, i f any, this binding has in normal p l a t e l e t function and aggregation remains to be determined. The endoperoxides are known to play an important role in p l a t e l e t aggregation (17,18). I t is i n t e r e s t i n g to speculate that such binding, i f i t occurs, could have a regulatory role in the control of the ' f r e e ' endoperoxide concentration and thereby a role in p l a t e l e t aggregation. At present l i t t l e is known about the role of the lipoxygenase enzyme and i t s metabolites of AA, i . e . , HETE and HPETE. Until more is known about the importance of these metabolites, i t is d i f f i c u l t to assess the significance of the covalent binding. The physiological and t o x i c o l o g i c a l significance of the covalent binding of intermediates formed from AA by the cyclo-oxygenase and lipoxygenase pathways, remains to be established.
414
SEPTEMBER 1979 VOL 18 NO. 3
PROSTAGLANDINS
ACKNOWLEDGEMENTS We wish to thank Ms. Sandra Smith and Mr. Claude Raines f o r t h e i r e x c e l l e n t technical assistance, and Kathleen Conant and Frances Holloway f o r t h e i r assistance in the preparation of t h i s manuscript.
SEPTEMBER 1979 VOL. 18 NO. 3
415
PROSTAGLANDINS
REFERENCES I.
Eling, T. E., Wilson, A. G. E., Chaudhari, A. and Anderson, M. W. Covalent binding of intermediate(s) in prostaglandin biosynthesis to guinea pig lung microsomal protein. Life Sci. 21:245-252, 1977.
2.
Anderson, M. W., Crutchley, D. J., Chaudhari, A., Wilson, A.G.E. and Eling, T. E.: Studies on the covalent binding of an i n t e r mediate(s) in prostaglandin biosynthesis to tissue macromolecules. Biochim. Biophys. Acta 573:40-50, 1979.
3.
Hamberg, M. and Samuelsson, B.: Prostaglandin endoperoxides: Novel transformation of arachidonic acid in human platelets. Proc. Natl. Acad. Sci. 71:3400-3404, 1974.
4.
Nugteren, D. H.: Arachidonate lipoxygenase in blood platelets. Biochem. Biophys. Acta 380:299-307, 1975.
5.
Parkes, D. G. and Eling, T. E.: Characterization of prostaglandin synthetase in guinea pig lung. Isolation of a new prostaglandin derivative from arachidonic acid. Biochemistry 13:2598-2604, 1974.
6.
Needleman, P., Moncada, S., Bunting, S., Vane, J. R., Hamberg, M. and Samuelsson, B.: I d e n t i f i c a t i o n - o f an enzyme in p l a t e l e t microsomes which generates thromboxane A~ from prostaglandin endoperoxides. Nature (London), 261:558~560, 1976.
7.
Nugteren, D. H. and Hazelhof, E.: Isolation and properties of intermediates in prostaglandin biosynthesis. Biochim. Biophys. Acta 326:448-461, 1973.
8.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275, 1951.
9.
Gillette, J. R. and Pohl, L. R.: A prospective on covalent binding and toxicity. J. Toxicol. Env. Hlth. 2:849-871, 1977.
I0.
Kulkarni, P. S. and Eakins, K. E.: N-0164 i n h i b i t s generation of thromboxane-Ap-like a c t i v i t y from prostaglandin endoperoxides by human p l a t e l e t microsomes. Prostaglandins 12:465-469, 1976.
II.
Falardeau, P., Hamberg, M. and Samuelsson, B.: Metabolism of 8,11,14-eicosatrienoic acid in human platelets. Biochim. Biophys. Acta 441:193-200, 1976.
416
SEPTEMBER 1979 VOL. 18 NO. 3
PROSTAGLANDINS
12.
Crutchley, D. J., Hawkins, H. J., Eling, T. E. and Anderson, M. W.: Covalent binding of prostaglandins G9 and H2 to components of ram seminal vesicle microsoma? fractTons. Biochem. Pharmacol. 28:1519-1523, 1979.
13.
Turner, S. R., Tainer, J. A. and Lynn, W. S.: Biogenesis of chemotactic molecules by the arachidonate lipoxygenase system of platelets. Nature (London) 257:680-681, 1975.
14.
Ho, P. P. K., Walters, P. and Sullivan, H. R.: A particulate arachidonate lipoxygenase in human blood platelets. Biochim. Biophys. Acta 76:398-405, 1977.
15.
Gardner, H. W.: Decomposition of l i n o l e i c acid hydroperoxides. Enzymic reactions compared with non-enzymic. J. Agricult. Food Chem. 23:129-136, 1975.
16.
Flohe, L. and Gunzler, W. A. in Glutathione: Metabolism and Function (Arias, I. M. and Jakoby, W. B., eds.) Raven Press, New York, 1975, pp 17-34.
17.
Hamberg, M., Svensson, J., Wakabayashi, TI, and Samuelsson, B.: Isolation and structure of two prostaglandin endoperoxides that cause p l a t e l e t aggregation. Proc. Natl. Acad. Sci. 71:345-349, 1974.
18.
Svensson, J . , Hamberg, M., and Samuelsson, B.: Prostaglandin endoperoxides IX. Characterizatioa of rabbit aorta contracting substance (RCS) from guinea pig lung and human platelets. Acta Physiol. Scand. 94:222-228, 1975.
SEPTEMBER 1979 VOL. 18 NO. 3
417
0
G~
0
m:
m~
m~
m~
4~ Oo
672.9 +
34.7 +
8.8 +
86.0
3.5
0.4
9.3*
1.2+
5.7*
4.2 +
0.4+
732.4 + 58.5
47.5 +
7.8 +
524.3 + 37.7*
90.5 +
5.0 ~
57.3 ~
C ' S i g n i f i c a n t l y d i f f e r e n t from control P
bValues reported are Mean ~ S.E.M. picomoles per mg. protein N=4.
Incubation
I08.9
135.9
86.6
500.3
25.8
51.0
35.2
INDOMETHACIN-TREATED (50uM) PICOMOLES % CONTROL
a I n i t i a l concentration of AA in the incubation was 2NM ( i . e . , 4 nmoles per incubation). conditions and analysis of metabolites are described in the Methods section.
TOTAL
Protein Bound
Water-Soluble
I04.8 + 20.8
HETE
1.5
351.0 + 41.5
9.8 ~
163.8 ~ 18.3
CONTROL PICOMOLES
HHT
PGE2
TXB2
AA METABOLITES
Effect of Indomethacin
The Covalent Binding of AA Intermediates to Human Platelet Microsomal
Protein and Formation of Water-Soluble Metabolites:
TABLE I.
Z
>
©
0
z
O0
0
m~ m:
m~
m~
AA METABOLITES
732.9 + 31.8
1.0
0.4
3.4*
2.9
1.3 *
0 . 5 N.S.
0.2 +
892.4 + 45.6
20.5 +
m
4.8 +
718.0 + 3 7 . 3 *
70.8 +
39.5 ~
38.8 ~
ND = Not detectable ( i . e . < 0.5 picomole)
* S i g n i f i c a n t l y d i f f e r e n t from control P
Values reported are Mean + S.E.M. picomoles per mg. protein N=4
Incubation
123
98.0
73.4
298.0
27.0
-
19.2
N-O164-TREATED (237 uM) PICOMOLES % CONTROL
I n i t i a l concentration of AA in the incubation was 2pM ( i . e . , 4 nmoles per incubation. conditions and analysis of metabol~tes are described in the Methods section.
TOTAL
20.8 +
P r o t e i n Bound
--
6,8 +
Water-Soluble
6.2
241.0 +
HETE
9.3
262.3 + 14.9
ND
202.0 ~
CONTROL PICOMOLES
HHT
PGE2
TXB2
The Effect of Thromboxane Synthetase I n h i t i t o r N-0164 on
the Covalent Binding of AA Intermediates to Human Platelet Microsomal Protein.
TABLE 2.
Z
0
0
P
Z
cm
m~ m:
m~
m~
:b
4~
0.7
0.3
5.4
0.8 ~
376.5 + 33.1
47.5 +
10.5 +
318.5 + 26.9 ~
Cyclo-oxgenase products represented < 5% of total AA metabolized
~Significantly different from control P
Values reported are Mean ~ S.E.M. picomoles per mg. protein N=4
Incubation
131.5
161.0
131.3
128.0
INDOMETHAClN-TREATED (50uM) PICOMOLES % CONTROL
I n i t i a l c o n c e n t r a t i o n o f AA i n the i n c u b a t i o n was 2NM ( i . e . , 4 nmoles per i n c u b a t i o n . c o n d i t i o n s and a n a l y s i s o f m e t a b o l i t e s are described in the Methods s e c t i o n .
286.3 + 14.9
29.5 +
P r o t e i n Bound
TOTAL
8.0 +
248.8 + 13.9
CONTROL PICOMOLES
Water-Soluble
HETE
AA METABOLITES
E f f e c t o f Indomethacin.
Covalent Binding of AA I n t e r m e d i a t e s to Human P l a t e l e t Soluble
F r a c t i o n P r o t e i n and the Formation o f Water-Soluble M e t a b o l i t e s :
TABLE 3.
Z
Z
0 c~
Z 0
o~
~N
< 0
m~
m~
m~
2.0
0.8
5.5 + 0.34
1.0 + 0.042
ND
4.5 + 0.32
PICOMOLES
.
I00.0
17.8
.
82.2 .
.
.
.
ND = Not detectable ( i . e . < 0.5 picomole)
Cyclo-oxgenase products represented < 5% of total AA metabolized
*Significantly different from control P
Values reported are Mean ~ S.E.M. picomoles per mg. protein N=4
.
Incubation
1.6
4.6
1.4
TYA-TREATED (150uM) % AA CONVERTED % CONTROL
Effect of
I n i t i a l concentration of AA in the incubation was 2 pM ( i . e . , 4 nmoles per incubation). conditions and analysis of metabolites are described in the Methods section.
349.1 + 29.3
21.3 +
P r o t e i n Bound
TOTAL
8.5 +
319.3 + 26.5
CONTROL PICOMOLES
TYA (5,8,11,14-Eicosatetraynoic Acid).
Protein and Formation of Water-Soluble Metabolites:
The Covalent Binding of AA Intermediates to Human Platelet Soluble Fraction
Water-Soluble
HETE
AA METABOLITES
TABLE 4.
Z
r"
0
c)
m:
m~
m~
t~
216.8 + 34.1
HETE
I.I
863.2 + 78.5
30.8 +
*
23.4 + 3.0
0.9 + 0. I *
1.2 + 0.2*
ND
13.5 + 1.5 ~
1.8 + 0.2 ~
6.0÷I.0
different
ND = Not d e t e c t a b l e ( i . e . ,
*Significantly < 0.5 picomole)
from c o n t r o l P
Values reported are Mean + S.E.M. picomoles per mg. p r o t e i n N=4
Incubation
2.7
2.9
10.9
---
3.9
7.6
2.5
TYA-TREATED (150uM) PICOMOLES % CONTROL
I n i t i a l c o n c e n t r a t i o n of AA i n the i n c u b a t i o n was 2NM ( i . e . , 4 nmoles per i n c u b a t i o n ) . c o n d i t i o n s and a n a l y s i s of m e t a b o l i t e s are described i n the Methods s e c t i o n .
TOTAL
P r o t e i n Bound
0.8
346.5 + 30.8
HHT
10.8 +
23.3 +_ 1.0
PGE2
Water-Woluble
235.0 +_ 10.7
CONTROL PICOMOLES
TXB2
AA METABOLITES
TYA ( 5 , 8 , 1 1 , 1 4 - E i c o s a t e t r a y n o i c A c i d ) .
E f f e c t of
The Covalent Binding o f AA Intermediates to Human P l a t e l e t Microsomal
P r o t e i n and the Formation of Water-Soluble M e t a b o l i t e s :
TABLE 5.
m=
>
0