Identification of the slow reacting substances from cat paws.

Abstract

Perfusion of cat paws with compound 48/80 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-cysteinyl-7,9,11,14-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) (,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 determin...

Characterization of slow reacting substances (SRSs) of rat basophilic leukemia (RBL-1) cells: effect of cysteine on SRS profile.

“…Peak la was characterized as SRS-GSH on the basis of the following observations: Its ultraviolet absorption spectrum showed a maximum at 280 nm with shoulders at 270 and 292 nm; its retention time was identical to that of synthetic SRS- (11); and amino acid analysis (Table 1) indicated that the molecule contained glutamic acid, cysteine, and glycine. Calculated on the basis of the ultraviolet absorbance at 280 nm, approximately 1 mol of glutamic acid and 1 mol of glycine per mol of SRS were obtained.…”

“…The system contained: 0.8 unit of y-glutamyl transpeptidase (bovine); 55 nmol of synthetic SRS-GSH (11), and 10 mM MgCl2 in 1 ml of 0.05 M Tris-HCl buffer, pH 8.5. Incubation was carried out with and without 10 mM L-cysteine. After 30 min, 4 ml of cold ethanol was added to the reaction mixture, which was allowed to stand for 30 min at SoC.…”

Enzymatic study to characterize the slow reacting substance of anaphylaxis (SRS-A) and leukotrienes.

“…Therefore, it has been suspected that the inactivation of SRS-A by the arylsulfatase preparation is due to a contaminating protease rather than to the arylsulfatase itself. This enzyme preparation cleaves the peptide bond in highly bioactive LTD4 (SRS-cystinyl glycyl form) to form much less bioactive LTE4 (SRS-cystinyl form) (8,20,21). In our experiments, limpet arylsulfatase type V which was contaminated by aminopeptidase activity markedly inactivated LTD4 and inactivated guinea pig SRS-A and rat SRS to a lesser extent, but did not inactivate LTC4 and human SRS at all (Fig.…”

“…This transformation increases the biological activity on the guinea pig ileum (8,9). In this study, the activity of LTD4 was about 10 times more potent than that of LTC4 in the isolated guinea pig ileum (Fig.…”

“…SRS-A or SRS has been found to be released from many types of tissues and cells in human, guinea pig and other animal lungs (3)(4)(5), human leucocytes (6, 7), rat peritoneal cells (4), cat paws (8), and leukemia basophils of rats and humans (9, 10) with immunological stimulations or ionophore A231 87.…”