Jun 16, 1980 - triene A, 5-trans-5,6-oxido-7,9,11,14-icosatetraenoic acid; SRS-GSH, ..... acteristic of conjugated triene chromophores. Exposure of SRS.
Proc. Nati. Acad. Sci. USA
Vol. 77, No. 10, pp. 5688-5692, October 1980 Biochemistry
Identification of the slow reacting substances from cat paws (arylsulfatase/cysteinylglycine/guinea pig ileum/aminopeptidase/cysteine)
JOEL HOUGLUM, JIN-KEON PAI, VINCE ATRACHE, DAi-EUN SOK, AND CHARLES J. SIH* School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706
Communicated by Henry A. Lardy, June 16, 1980
Perfusion of cat paws with compound 48/80 ABSTRACT released two slow reacting substances (SRSs) which were isolated and characterized as 5-hydroxy-S-cysteinylglycyl-7,9,11,14-icosatetraenoic acid (SRS I) and -hydroxy-6S-cysteinyl7,9,11,14-icosatetraenoic acid (SRS II) on the basis of chemical degradations, amino acid analyses, spectroscopic and enzymic experiments, and comparison with synthetic samples. The smooth muscle-contractile activities of synthetic 5-hydroxy-6 y-glutamylcysteinylglycyl-7,9,11,14-icosatetraenoic acid, synthetic5-hydroxy--S-cysteinyl-7,9,11,14-icosatetraenoic acid, and SRS II were not inactivated by arylsulfatase. On the other hand, the spasmogenic activities produced by synthetic 5-hydroxy-6S-cysteinylglycyl-7,9,11,14-icosatetraenoic acid and SRS I were destroyed at the same rate by the arylsulfatase. This mode of inactivation was attributed to an aminopeptidase activity in the arylsulfatase preparation because 5-ydroxyl-6-Scysteinyl-7,9,11,14-icosatetraenoic acid was isolated and identified as the reaction end product. Because the properties of SRS from cat paws closely resemble those of SRS generated by immunological stimulation of human tissues (SRS-A) and because all known SRS-A are inactivated by arylsulfatases, we contend that 5-hydroxy-6S-cysteinylglycyl-7,9,11,14-icosatetraenoic acid (SRS I) corresponds to SRS-A.
In 1938, Feldberg and Kellaway (1) first described a substance that caused the guinea pig ileum to contract more slowly and with more sustained action than did histamine; they named the compound "slow reacting substance (SRS)." Two years later, Kellaway and Trethewie (2) reported that slow reacting substance of anaphylaxis (SRS-A) was released from sensitized tissue by a specific antigen. Subsequently, it was shown (3) that SRS-A was one of several pharmacologic mediators that may be important in human asthma. SRS has since been found to be released from many types of tissues and cell suspensions including guinea pig, rabbit, monkey, bovine, and human lungs (4-6), cat paws (7, 8), isolated rat mast cells (9), human leukocytes (10) and nasal polyps (11), human and rat leukemic basophils (12, 13), and rat peritoneal fluid (14, 15). Here we present our studies on the purification and identification of two SRSs from cat paws after perfusion with Compound 48/80 (16) and biochemical evidence supporting the contention that SRS I corresponds to SRS-A.
MATERIALS AND METHODS Materials. Type V sulfatase (Patella vulgata), type I soybean lipoxygenase (155,300 units/mg), and Compound 48/80 were products of Sigma. The SRS antagonist FPL 55712 was supplied by Fisons Limited (Longborough, England). The chromatographic adsorbents were Amberlite XAD-7 (Mallinckrodt) and Waters silica gel Sep-Pak cartridges (disposable columns). A
model M-6000 pump equipped with an U6K injector and a model 77 double-beam UV (254 nm) detector (Waters Associates) was used for high-pressure liquid chromatography (HPLC). A microparticulate reverse phase (,uC18) preparative column (0.94 X 50 cm) of Partisil-10 M9 ODS (Whatman) was used for SRS purification. The radial compression separation system consisted of a Waters radial compression module (RCM-100) with a radial-Pak C18 cartridge (0.8 X 10 cm). SRS Assay. SRS bioactivity was assayed on guinea pig ileum segments by a modification of the procedure reported by Chakravarty (17). Histamine was assayed in the presence of atropine (1 MM) and SRS was assayed in the presence of atropine (1 ,uM) and mepyramine (1 ,M). One unit of SRS was defined as that amount of SRS which caused a contraction with a peak height equal to that induced by 5 ng of histamine base. After the log-dose-response curve for histamine was determined, a similar curve of an in-house standard of SRS was determined and from this the other SRS samples were quantitated. Only aqueous samples were bioassayed. Samples that had been evaporated to dryness were redissolved in 0.1% NH40H because water did not completely dissolve the sample. Large quantities (0.5 ml) of NH40H at this concentration had no effect on the ileum response. Samples of SRS were assayed in the presence of 20-40 nM FPL 55712, a reversible SRS inhibitor (18). Cat Paw Perfusion. Cat paws were perfused by a modification of the procedure reported by Hogberg et al. (19). Cats (1.5-5 kg) of either sex were anesthetized with sodium pentobarbital (50 mg/kg; intraperitoneally) and subsequently killed with an intravenous injection of 1 ml of saturated KCl. Immediately after the death of the animal, the main artery of each paw was cannulated with polyethylene tubing (0.6 mm inside diameter). The paws were cut off above the ankle joint and hung from a hook in a water-jacketed jar which was maintained at 25°C by a water circulating pump. The paws were perfused with a 154 mM NaCl/0.9 mM CaCl2/2.7 mM KCl containing 10% (vol/vol) S6rensen phosphate buffer (Na2HPO4 + KH2PO4, 67 mM) at pH 7.0. This buffered salt solution was maintained at 25°C and was simultaneously perfused through the set of four paws from each cat with a four-channel peristaltic pump. After the paws were perfused for 45 min to remove most of the blood, 50 Mg of the releasing factor (Compound 48/80) was administered and the perfusion was conAbbreviations: SRS, slow-reacting substance; SRS-A, SRS of anaphylaxis; HPLC, high-pressure liquid chromatography; 5-HPETE, 5hydroperoxy-6-trans-8,11,14-cis-icosatetraenoic acid; SRS-Cys, 5hydroxy-6-S-cysteinyl-7,9,11,14-icosatetraenoic acid; SRS-Cys-Gly, 5-hydroxy-6-S-cysteinylglycyl-7,9,11,14-icosatetraenoic acid; leukotriene A, 5-trans-5,6-oxido-7,9,11,14-icosatetraenoic acid; SRS-GSH,
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate
5 -
acid.
this fact.
*
5688
hydroxy-6-'-glutamylcysteinylglycyl-7,9,11,14-icosatetraenoic
To whom reprint requests should be addressed.
Biochemistry: Houglum et al. tinued for an additional 180 min. During this period, the effluents were collected by gravity drainage into flasks- cooled to 40C.
Purification. The ice-cold perfusate (4-4.5 liters) from two cats was passed over an XAD-7 column (2.2 X 17 cm). After the column was washed with 100 ml of distilled water followed by
20 ml of methanol, SRS was eluted with 125 ml of methanol. The eluate was evaporated to dryness and stored at -250C. Crude SRS concentrate from 10 cats was pooled and dissolved in 50 ml of doubly distilled water, acidified to pH 2.5 with 0.1 M HC1, and extracted four times with equal volumes of ethyl acetate, which was evaporated to dryness under reduced pressure. The residue was dissolved in 0.7 ml of NH40H solution (pH 9.5), evaporated to dryness, and redissolved in 0.2 ml of CH2Cl2/MeOH/H20, 65:2010 (vol/vol), before application onto a disposable silica gel column. The column was washed with 3 ml of CH2Cl2/MeOH/H20, 65:20:10 (vol/vol), and 2.5 ml of CH2Cl2/MeOH/H20, 65:30:10. SRS was then eluted with 4 ml of CH2Cl2/MeOH/H20, 65:40:10. After evaporation of the eluate to dryness, the residue was dissolved in 0.2 ml of MeOH/H20, 1:1, and chromatographed over a MC18 reversephase preparative column of Partisil-10 (10 Mm) M9 ODS (Whatman), which was protected by a CO:PELL ODS preco; lumn (Whatman). The solvent used for elution was MeOH/ H20, 1:1, at a flow rate of 3 ml/min. Inactivation of SRS by Soybean Lipoxygenase. The reaction mixture contained 5 nmol (based on UV at 280 nm, e 40,000) of SRS and 50 ug of soybean lipoxygenase in 1 ml of Tyrode's buffer at pH 7.8. Spectra were recorded at various time intervals with a Cary model 118 spectrophotometer. Inactivation of SRS by Arylsulfatase. The system contained 5 units of arylsulfatase and varying amounts of SRS [500 units of SRS I or synthetic 5-hydroxy-6-S-cysteinylglycyl7,9,11,14-icosatetraenoic acid (SRS-Cys-Gly) or 5-hydroxy6-'y-glutamylcysteinylglycyl-7,9,11,14-icosatetraenoic acid (SRS-GSH); 50 units of SRS II or synthetic 5-hydroxy-6-S-cysteinyl-7,9,11,14-icosatetraenoic acid (SRS-cys)] in 1 ml of 0.1 M sodium acetate buffer at pH 5.7. Incubation was at 370C. At the indicated time intervals an aliquot was removed from the reaction mixture and frozen at -780C. This aliquot was dissolved in 1 ml of Tyrode buffer and the residual SRS activity was assayed. Control values were obtained by incubation of boiled enzyme with SRS for the time intervals indicated. Desulfurization of SRS I and SRS II. Samples of purified SRS I and SRS II [15-20 ,g, estimated by UV at 280 nm (e = 40,000)] were separately treated with 10 mg of Raney nickel (W-2) in refluxing ethanol for 30 min. The reaction mixtures were then filtered and concentrated under reduced pressure. The crude desulfurized products were first methylated with CH2N2, trimethylsilylated with Tri-Sil/BSA (Pierce), and subjected to gas chromatography-mass spectral analysis (3% OV-1, 6-ft column, 150-2400C at 80//min) on a Finnigan model 4021-T instrument. Spectra were recorded at 70 eV with an ionizing current of 0.35 mA. Leukotriene A Methyl Ester. To a solution of (d)-5-hydroperoxy-6-trans-8,11,14-cts-icosatetraenoic acid [(-)-5HPETE] methyl ester (20) (40 mg, 0.12 mmol) in 1 ml of CH2Gl2, cooled to -780C, was added methyldicyclohexylamine (0.48 mmol) followed by methanesulfonyl chloride (14 mg, 0.12 mmol). After the reaction mixture was stirred for 40 min at -780C, water was added and the mixture was extracted with hexane containing 1% triethylamine. The combined hexane extracts were washed with water, dried over Na2SO4, and evaporated to dryness. Preparative thin-layer chromatography of the crude product on silica gel plates [prewashed with hexane/triethylamine, 10:1 (vol/vol)] with ether/hexane, 1:2
Proc. Natl. Acad. Sci. USA 77 (1980)
5689
(vol/vol), containing 1% triethylamine as developing solvent afforded 6 mg of leukotriene A methyl ester, whose ultraviolet spectrum showed Xm (ethanol) 279 with shoulders at 269 and 290 nm; PMR (90 MHz, CDCI3) 5 0.9 (t, 3H, CH2CH3); 1.3 (i,
0 0 3.2 CH-CH-CH2); (m, CH-CH-CH2); 2.8 (m, 8H, CH2); 3.67 (s, 3H, COOCH3); 5.2-5.6 (m, CH2-HC=CH-CH2); 5.75-6.3 [m, 2H (-CH=CH-)3]; mass spectrum (m/e) 332 (M+), 301 (M-31), 231 (M-101), 189 (M-143), 143 (C7H1103), 101 (C5H902). The reactions of leukotriene A methyl ester with glutathione, cysteinylglycine, and cysteine were carried out by the method described by Corey et al. (21). The desired SRSs were purified by HPLC on a Radial Pak C18 cartridge (0.8 X 10 cm) in a
Radial compression separation system (RCM-100). SRS-GSH was eluted at 13 min (flow rate, 2 ml/min) with a mobile phase of MeOH/H20, 55:45 (vol/vol), containing 0.05% acetic acid buffered to pH 5.4 (NH40H). SRS-Cys-Gly and SRS-Cys had retention times of 7.5 and 11 min, respectively, with MeOH/ H20, 7:3, containing 0.05% acetic acid buffered to pH 5.4 (NH40H) (flow rate, 2.5 ml/min). RESULTS Using a purification procedure consisting of XAD-7 column chromatography, ethyl acetate extraction, and reverse-phase HPLC (preparative MC18), we have purified two SRSs from cat paw perfusates with an overall recovery of 20% (Table 1). The elution profiles of SRS I and SRS II from the reverse-phase HPLC column are shown in Fig. 1. SRS I was eluted after 13 min and had a specific activity of 4400 units/nmol (based on e 40,000 at 280 nm). SRS II was eluted from the column at 15.4 mm and had a specific activity of 65 units/nmol. SRS I and SRS II had identical UV absorption spectra with absorption maxima at 280 nm and shoulders at 270 and 292 nm (Fig. 2). Exposure of either SRS I or SRS II to soybean lipoxygenase resulted in a rapid loss of bioactivity accompaiiied by a shift of uv absorption maximum from 280 to 308 nm (Fig. 3). Hydrogenation of either SRS I or SRS II over Raney nickel afforded 5-hydroxyicosanoic and icosanoic acids. 5-Hydroxyicosanoic acid was identified by gas chromatography/mass spectrometric analyses of the trimethylsilyl ether and methyl ester derivatives which showed prominent peaks at m/e 399 (M-15); 383 (M-31); 313 (M-101, loss of -CH2(CH2)2CO2CH3) and a base peak at 203 [(CH3)sSiO+=CH(CH2)3CO2CH3]. The same derivative of authentic 5-hydroxyicosanoic acid afforded a mass speetrum virtuilly identical to the spectra of desulfurized SRS I and SRS II derivatives. Amino acid analyses after acid hydrolysis of SRS I gave cysteine and glycine in a ratio of 0.5:1; SRS II afforded cysteine Table 1. Purification of SRS I and II % Specific activity. Yield Units mg recovery Procedure units/,ug 0.044 533,000 12,000 Pbrfusate 1.325 75 XAD-7 400,000 302 Ethyl acetate 13.333 24 80 extraction 320,000 Disposable column 272 85 1 272,000 silica gel Preparative $C18 41 HPLC 0.012* 8900 SRS I 106,800 150 0.023* 3,450 SRS II * The amounts of SRS I and SRS II were calculated from the absorbance at 280 nm, assuming e = 40,000.
5690
Proc. Natl. Acad. Sci. USA 77 (1980)
Biochemistry: Houglum et al. 8
SRS 11
/
Histamine
6 CJ
W 0
4
2
0
to w0.
x
..1 10
w
uO
SRS-CysGly
8
100 SRS-GSII
.
/.
.
1
1000 SRS-Cys Histamine
co 6
-6.
c:.0
o
48'
4
/
CU
//
2
SRS I /, sRS II
.
1
0 3 6 9 12 15 18 2L 24 27 30
Time, min FIG. 1. Reverse-phase HPLC of SRS I and SRS II from cat paw perfusates. Stationary phase (preparative ,UC18), M9 ODS; mobile phase, methanol/water, 1:1 (vol/vol); flow rate, 3 m/min.
the sole amino acid (Table 2). To determine the NH2-terminal amino acid in SRS I, the sample was dansylated and hydrolyzed and the dansyl derivatives were analyzed by thin-layer chromatography;'no dansyl-glycine was detected. To confirm the chemical identities of SRS I and SRS II from cat paws, we synthesized these substances using a biomimetic approach. Treatment of (±)-5-HPETE methyl ester with methanesulfonyl chloride and methyldicyclohexylamine at -780C yielded the desired common intermediate, leukotriene A methyl ester, accompanied by a small quantity of the 1,2as
0.2
280
280
SRS I
@
S
I
-~~~~~~~~~~~~I
5 I /
i
°X 0 /.S
0.10
~~~~~~~0.1
250
270
I
I
290
310
330
250
270
1000
290
310
330
elimination product, 5-oxo-icosa-6-trans-8,11,14-cis-tetraenoic acid methyl ester. SN2 displacement of the epoxide in leukotriene A by the respective nucleophiles (21) (cysteine, cysteinylglycine, and glutathione) gave the corresponding adducts as pairs of diastereomers which were not separable by HPLC under various conditions. All three synthetic products (two diastereomers) (SRS-CysGiy, SRS-Cys, and SRS-GSH) had UV absorption maxima at 280 nm with characteristic shoulders at 270 and 292 nm. The rate of inactivation and the associated spectral change observed upon treatment with soybean lipoxygenase and the retention times on HPLC [preparative jaC18, MeOH/H20, 1:1 (vol/vol); and Radial-Pak C18, MeOH/H20, 7:3 buffered at pH 5.4] of synthetic SRS-Cys-Gly and SRS-Cys were indistinguishable from the corresponding properties of SRS I and SRS II, respectively. Also, the slopes of the log-dose-contractile response curves (Fig. 3) of synthetic SRS-cys and SRS-Cys-Gly coincided with those of SRS II and SRS I, respectively. In addition, the patterns of reversal of the contractile activity by FPL 55712 for SRS II and SRS I were found to be indistinguishable from those of corresponding synthetic derivatives. On the other hand, synthetic SRS-GSH had a specific activity of 650 units/nmol but its pattern of contractile response was only partially reversed by FPL 55712; it was only slowly attacked by soybean lipoxygenase. The spasmogenic activities of synthetic SRS-Cys-Gly and SRS I were inactivated at approximately the same rate by a limpet arylsulfatase preparation whereas the bioactivities of synthetic SRS-GSH, synthetic SRS-Cys, and SRS II were not diminished (Fig. 4). The small loss of bioactivity by SRS II is due to the contamination of a trace quantity of SRS I in the SRS II
sample.
2
I%\-, I
100
pmol FIG. 3. Dose-response curves for SRS I, SRS II, synthetic SRS-GSH, SRS-Cys-Gly, and SRS-Cys, and histamine on isolated guinea pig ileum. The amount of SRS was calculated from the absorbance at 280 rim, assuming e = 40,000.
lI
SRS I A 0.2~~~~~~~~.
10
350
Wavelength, hm FIG. 2. UV absorbance spectra of SRS I and SRS II. (A) Spectra were recorded in methanol. (B) Spectra of SRS I and SRS IT before (O min) and after treatment with soybean lipoxygenase (5 min). Spectra were recorded in Tyrodes buffer.
Table 2. Amino acid composition of SRS I, SRS II, and synthetic SRSs Glutamic acid, Glycine, 1/2 cystine, nmol nmol nmol Glutathione 5.37 5.63 5.06 SRS I 0.26 2.24 4.50 SRS II 0.37 0.98 3.05 6.00 6.38 3.30 Synthetic SRS-GSH * Each compound (10 nmol) was hydrolyzed with 6 M HC1/0.5% phenol at 110°C in vacuo for 24 hr. The results are based on the absorbance it 280 nm (E = 40,000). SRS I and SRS II from the preparative 1C1s reverse-phase column was further purified by using the radial compression module (RCM-100) [Radial-Pak C1s; MeOH/H20 70:30 (vol/vol), for SRS I and SRS II, pH 5.4].
Proc. Natl. Acad. Sci. USA 77 (1980)
Biochemistry: Houg'lum et al. 120 100 80
3\
60 40 ¢,20
SRS-II
_
__
\
.
60
30
-1 120
90
0
g 120
SRS-GSH
100 SRS-Cys
80 \ 60 40\ 20-
SRS-Cys-Gly
120
90 60 30 Incubation time, min
FIG. 4. Inactivation of SRS bioactivity by arylsulfatase.
When synthetic- SRS-Cys-Gly was incubated with arylsulfatase for 30 min, a less-polar product was formed (Fig. 5). Its retention time on the radial compression Radial-Pak C18 column (11 min; MeOH/H20, 7:3, pH 4.5), coincided with that of SRS-Cys. Amino acid analysis of this product showed only cysteine, substantiating that the glycine residue was cleaved off the substrate. Its uv absorption spectrum, the contractile response of the ileum and pattern of reversal by FPL 55712, and the rate of spectral change after exposure to soybean lipoxygenase were indistinguishable from those properties of SRSCys.
DISCUSSION Our results show that perfusion of cat paws with Compound 48/80 produced SRS I and SRS II whose UV absorption spectra were identical to the spectrum of a leukotriene (22) a-d characteristic of conjugated triene chromophores. Exposure of SRS I and SRS II to soybean lipoxygenase shifted the absorption maximum from 280 to 308 nm, suggesting the presence of a cis,cis-1,4-diene within these molecules. 5-Hydroxyicosanoic acid was identified as the main product obtained by hydrogenation of either SRS I and SRS II with Raney nickel which is known to cleave thioether bonds (23, 24). These results are in
t = 30
(...
03 6
91215
...
min
.
036921518
Time, min FIG. 5.
HPLC elution
profile of SRS-Cys-Gly after exposure to
arylsulfatase. HPLC was carried out on a Radial-Pak C18 liquid chromatography cartridge (0.8 x 10 cm) using a radial compression separation system (Waters Associates module RCM-100). The mobile phase consisted of methanol/water, 7:3 (vol/vol), containing 0.05% acetic acid buffered to pH 5.4 (NH40H); flow rate, 2.5 ml/min.
5691
agreement with the supposition that SRS I and SRS II contain 5-hydroxy-7,9,11,14-icbsatetraenoic acid as the fatty acid portion of the molecule but differ in the thioether substituent at C-6. Anmino acid and NH2-terminal sequence analyses revealed that the anoint acid portion of the molecule in SRS I is cysteinylglycine and in SRS II is cysteine. Although the mechanism for the generation of cysteine after strong acid hydrolysis is not understood, it was apparent that the thioether linkages in these molecules were cleaved because synthetic SRS-GSH likewise afforded cysteine upon acidic hydrolysis (Table 2). These results strongly suggest that the structure of SRS I is 5-hydroxy-6-S-cysteinylglycyl-7,9,11,14-icosatetraenoic acid and that of SRS II is 5-hydroxy-6-S-cysteinyl-7,9,11,14icosatetraenoic acid. The presence of a sulfate group in SRS-A had been repeatedly proposed on the basis of arylsulfitase inactivation experiments (25,26). Because SRS I also is readily inactivated by arylsulfatase (Fig. 4) and there existed the possibility that some labile group in SRS I and SRS II was lost dining chemical degradative studies, we decided to verify their identities by chemical synthesis. The transformation of (+)-5-HPETE methyl ester into leukotriene A methyl ester requires the selective abstraction of a C-10 proton and activation of the hydroperoxyl group at C-5. After many unsuccessful attempts, it was found that reaction of (+)-5-HPETE methyl ester with methanesulfonyl chloride and a hindered base such as methylcyclohexylamine (to minimize 1,2-elimination) at lbw temperatures afforded, in 15% yield, the desired leukotriene A methyl ester whose spectral properties were in good agreement with those derived from independent chemical syntheses (27, 28). Introduction of the thio subsfituents was effected by reaction of leukotriene A with the respective nucleophiles. Although we have been unable to separate the two diastereomners of the synthetic derivatives, it is noteworthy that the properties of the diastereomeric mixtures of synthetic SRS-Cys and SRS-Cys-Gly agreed well with the corresponding properties of SRS II and SRS I, lending further support to our structural assignment. The inactivation of SRS-A by arylsulfatase had been a longstanding enigma. The availability of synthetic substances of known chemical identities allowed us to define this mode of inactivation. Although the bioactivities of synthetic SRS-GSH, SRS-Cys, and SRS II were not inactivated by the Limpet arylsulfatase preparation, the contractile activities of synthetic SRS-Cys-Gly and SRS I were destroyed at approximately the same rate by the arylsulfatase. Because SRS-Cys was identified as the reaction product after exposure of SRS-Cys-Gly to arylsulfatase, one can now conclude that this inactivation was the result of the cleavage of the cysteinyl-glycine peptide bond and not the result of the hydrolysis of a sulfate ester. At the present time, it is not known whether arylsulfatase per se possesses both esteratic and peptidatic activities similar to chymotrypsin. Highly purified arylsulfatase (29) was reported to inactivate SRS-A, and very low concentrations of SRS-A served as a competitive inhibitor during the hydrolyses of arylsulfate esters catalyzed by arylsuifatase. Alternatively, it is also possible that aryfsulfatase preparations are contaminated with an aminopeptidase responsible for the cleavage of S-Cys-Gly derivatives, similar to those found in the kidney (g9). It is noteworthy that y-glutamyl transpeptidase and leukotrienes were also reported to be present in kidney tissues (30, 31). During the preparation of this manuscript, two groups reported the isolation of 5-hydroxy-6-S-cysteinylglycyl7,9,11,14-icosatetraenoic acid from rat basophil leukemic (RBL-1) cells (32, 33). Although at the Fourth International Prostaglandin Conference in 1979 (34), Samuelsson reported
Proc. Natl. Acad. Sci. USA 77 (1980)
Biochemistry: Houglum et al.
5692
0
ON
H~02
C~~~C
0
2
K 5H
1
Glutathione transferase
Leukotriene A
I?* Iys-Gly
enzympe
H Cc2 5HI- S-CH2
CHCONHCH2COOH I NHCO(CH2 )2 NCOON Leukotriene C NH2
+
OH
'y-Glutamyl
*/transferase
-HC02H C5H
1
SC4H2 CHCONHCH2COOH NH2
SRS-I (SRS-A)
Aminopep tidase (scarylsulfatase ") OH
=HCo2H C5H
I
SCH2
CHCOOH
I
NH2
SRS-II
FIG. 6. Proposed scheme of formation and degradation of SRS I (SRS-A).
the structure of an SRS from murine mastocytoma cells to be 5-hydroxy-6-S-cysteinyl-7,9,11,14-icosatetraenoic acid and termed it "leukotriene C," this structural assignment was later revised and 5-hydroxy-6-'y-glutamylcysteinylglycyl7,9,11,14-icosatetraenoic acid was named "leukotriene C' (22). Because SRS from cat paws closely resembles SRS formed by immunological stimulation of human tissues (13) and because all known SRS-A have been reported to be inactivated by arylsulfatases (25, 26, 29, 35), we contend that 5-hydroxy-6-Scysteinylglycyl-7,9,11,14-icosatetraenoic acid (SRS I) corresponds to SRS-A. One may envisage that SRS I (SRS-A) could arise via an enzyme-catalyzed SN2 attack of cysteinylglycine on the epoxide in leukotriene A, or it could be derived from the precursor, leukotriene C, by the action of y-glutamyl transpeptidase (3). In turn, leukotriene C could originate from leukotriene A via the action of glutathione transferase (36). The cleavage of the cysteinylglycine peptide bond converts the highly bioactive SRS I (SRS-A) into a much less bioactive SRS II (Fig. 6). This inactivation mechanism may play an important role in the regulation of the effective levels of SRS-A within the tissues. We thank Dr. Daniel Omilianowski for the amino acid and NHr terminal sequence analyses. This work was supported in pgrt by Grant AM 09688 from the National Institutes of Health. 1. Feldberg, W. & Kellaway, C. H. (1938) J. Physiol. (London) 94, 187-226. 2. Kellaway, C. H. & Trethewie, E. R. (1940) Q. J. Exp. Physsol. 30, 121-145. 3. Brocklehurst, W. E. (1962) Prog. Allergy 6,539-558.
4. Burka, J. F. & Eyre, P. (1974) Can J. Physiol. Pharmacol. 52, 1201-1204. 5. Orange, R. P., Austen, W. G. & Austen, K. F. (1971) J. Exp. Med. 134, 136s-148s. 6. Brocklehurst, W. E. (1960) J. Physiol. (London) 151, 416435. 7. Strandberg, K. & Uvnis, B. (1971) Acta Physiol. Scand. 82,
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