FORMATION BY CATHEPTIC ENZYME IN RAT STOMACH

Motoki KOBAYASHI and Katsuya OHATA Departmentof Pharmacology,Kyoto College of Pharmacy, Yamashina,Kyoto 607, Japan Accepted July 6, 1982

Abstract-Four uterine-contractile substances (a, b, c and d) were extracted from procedures with acetic acid, n-butanol, distilled water, and methanol from the reaction mixture obtained by incubating rat plasma with the kininforming enzyme in the rat stomach. Gradient and equilibrium chromatography on SP-Sephadex C-25 columns were carried out with the extract containing these materials (a, b, c and d). The materials could not be separated by chromatography on SP-Sephadex C-25, but were separated by high performance liquid chromatography (HPLC) with a Zorbax ODS reversed-phase column. All the materials (a, b, c and d) contracted the rat uterus and relaxed the rat duodenum. The uterine-contractile activity of the materials was abolished by chymotrypsin, but not by trypsin treat ment. The retention times of materials a, c and d on H PLC were different from that of kallidin, MLBK, (BK) and ; but the retention time of material b was equal to that of BK. The content ratio of a: b: c: d was 4: 4: 67: 25, calculated from the uterine-contractile activity of these materials. The apparent molecular weight of the major material c, estimated by gel chromatography, was 1650. Material c contracted the rat uterus (1.2X10-1° g/ml), relaxed the rat duodenum (2X10''° g/ml), and produced a fall in rabbit blood pressure (4.4X10-8 g/kg). Material c was classified as a biologically BK-like which is distinct from kallidin, MLBK, BK and neurotensin.

In a previous paper, we reported that the stomach (KFE) and kininogen of rat plasma kinin-forming enzyme (KFE), which has a (KGN) were prepared by the method pH optimum of 4.8, existed in the rat stomach described previously (2). (1). We showed that KFE had characteristics Generation of from KGN by KFE: that were similar to those of cathepsin D (2). The incubation was carried out using a The subtance from rat plasma produced by mixture of 140 ml of KGN solution (50 mg/ KFE was classified as a biologically brady ml) dissolved in 0.05 M phosphate buffer (pH kinin (BK)-like substance (1). This report 4.8) and 140 ml of the KFE preparation (5 describes the isolation, purification, and mg/ml) dissolved in the same buffer for 16 hr identification of the BK-like substance at 37'C. The reaction was terminated by generated by KFE. boiling for 5 min. A part of the resultant solution was adjusted to pH 8.0 with 3 N MATERIALSAND METHODS NaOH and assayed with the isolated rat Preparation of kinin-forming enzyme and uterus. kininogen: Kinin-forming enzyme from rat Extraction procedure: The reaction mixture containing uterine-contractile activity, ob above was applied to the column; the column tained from the incubation described above, was washed with 500 ml of distilled water and was extracted according to the method then developed with a linear gradient of described previously (3). The reaction mixture ammonium formate solution, pH 5.4 (0 (240 ml) was adjusted to pH 2.0 with 3 N 0.4 M). The fractions of 20 ml each were HCI and saturated with NaCl. After addition collected, lyophilized, and a part of each of an equal volume of n-butanol, the mixture dried residue was dissolved in 0.9% saline. was shaken for 10 min and centrifuged at The resultant solution was assayed with 700 g for 10 min. The n-butanol phase was the isolated rat uterus. pipetted to another tube. The extraction with The lyophilized preparation containing n-butanol was then repeated. After addition uteri ne-contractile activity, obtained from of 10 g of anhydrous sodium sulfate per 40 ml the gradient chromatography, and standard of the combined n-butanol phase, the solution materials were dissolved in 0.05 M Tris-HCI was stored overnight at -20°C. After centri Buffer, pH 8.0, containing 0.08 M NaCI. fugation at 700 g for 10 min, the supernatant The resultant solutions were submitted to fraction was transferred to another tube and equilibrium chromatography on a SP 2 volume of petroleum ether were added. Sephadex C-25 column, 1.3x14 cm, by the The mixture was extracted with a one tenth method described previously (3). Fractions volume of distilled water by shaking for 10 of 6 ml each were collected and assayed with min, centrifuged at 700 g for 10 min, and the the isolated rat uterus. aqueous phase was pipetted to another tube. The fractions containing uterine-contractile The extraction with distilled water was activity, obtained from the equilibrium chro repeated. The combined aqueous phase was matography, were pooled and submitted to adjusted to pH 7.0 with 1 N NaOH and gradient chromatography as described above evaporated to dryness at below 35'C. The for desalting. dried residue was extracted twice with 240 Gel chromatography: The preparation con ml of methanol by shaking for 10 min. After taining uterine-contractile activity (600 t g centrifugation at 700 g for 10 min, the BK equivalent), obtained from chromato supernatant fraction was evaporated to dry graphy on SP-Sephadex C-25, was dissolved ness at below 35°C. The dried residue was in 0.2 N acetic acid and chromatographed on chromatographed on SP-Sephadex C-25. a Sephadex G-25 column, 2.6x89 cm. The Chromatography on SP-Sephadex C-25: flow rate was 14 ml/hr. The fractions of The preparation containing uterine-con 10 ml each were collected, lyophilized, and a tractile activity (800 i g BK equivalent on part of each fraction was dissolved in 0.9% assay with the isolated rat uterus in the saline. The resultant solutions were tested presence of atropine and dibenamine), with the isolated rat uterus. obtained from the extraction procedure, was For estimation of the molecular weight of dissolved in 300 ml of distilled water and the test material, a 0.9x97 cm column of submitted to gradient chromatography on a Sephadex G-25 (suspended in 0.2 N acetic SP-Sephadex C-25 column, 2.5x46 cm, by acid) was utilized. The flow rate was 7.0 ml/ the method described previously (3). The hr. Fractions of 2.0 ml each were collected column resin was suspended in 0.5 M and tested with the isolated rat uterus ammonium formate for 24 hr and washed according to the same method described with distilled water before use. The pre above. The following substances were used paration dissolved in distilled water described as standard materials: kallidin, neurotensin and Ieu5-enkephalin. as described above. Fractions of 0.7 ml each The above two columns were also utilized were collected, adjusted to pH 7.0 with 3 N for desalting after high performance liquid NaOH, lyophilized, and a part of each chromatography (HPLC). fraction was dissolved in 0.9% saline. The High voltage paper electrophoresis resultant solutions were assayed with the (HVPE): HVPE was performed on Whatman isolated rat uterus. The fractions containing No. 1 filter paper (Whatman Ltd., England) uterine-contractile activity were combined, using a Model HPE-V electrophoresis ap lyophilized and desalted by gel chromato paratus (Toyo Kagaku Sangyo Co., Ltd., graphy. HPLC was repeated with the Osaka, Japan) at 50 volts per cm with n desalted preparation. hexane as an inert cooling medium. The Enzymatic treatment: Digestions with tryp buffer used in this study consisted of 0.6 N sin and chymotrypsin were carried out by formic acid-2 N acetic acid (1 :1), pH 1.9. incubating 1 ml of the preparation containing Densitometry was carried out with a Model the uterine-contractile activity (0.25 i g BK CS-910 dual wave-length TLC scanner equivalent) with 0.2 ml of enzyme solution (Shimadzu Scientific Instruments, Inc., Kyoto, (1 mg/ml in distilled water) at 37'C, pH 8.0, Japan). for 20 min. The reactions were terminated by HPLC: A Model LC-3A high performance boiling for 5 min. After centrifugation at 700 g liquid chromatography (Shimadzu Scientific for 10 min, the supernatant fractions were Instruments, Inc., Kyoto, Japan) with a assayed with the isolated rat uterus. Model UVIDEC 100-III ultraviolet spectro Biological procedure: The bioassay was photometer (Japan Spectroscopic Co., Ltd. carried out by the method described (JASCO), Tokyo, Japan) and a Model SIL previously (3). De Jalon's solution containing 1A loop injector (JASCO) were utilized. atropine (10-6 g/ml) and dibenamine (10"' Chromatograms were recorded with a Model g/ml) was used to suspend the isolated rat U-125 MN single-pen recorder (Shimadzu uterus, and a solution containing additional Scientific Instruments, Inc., Kyoto, Japan). diphenhydramine (10'8 g/ml) and pro The column used in this study was a Zorbax pranolol (10'8 g/ml) was used to suspend ODS reversed-phase column (Du Pont Instru the isolated rat duodenum. ments, U.S.A.) of dimensions 0.46x25 cm. The blood pressure was recorded from the The mobile phase was 0.05 M potassium carotid artery of urethanized (1.5-2.0 g/kg, dihydrogen phosphate (pH 2.5)-acetonitrile s.c.) rabbits. The test sample was adminis (8:2). The flow rate was 0.1 ml/min, and the tered into the cannule tied into the vena separation was performed at ambient temper marginalis after intravenous administration ature. Ultraviolet detection was carried out of atropine (2 mg/kg, 1 min pretreated), at 220 and 280 nm. The preparation contain dibenamine (5 mg/kg, 40 min pretreated), ing uterine-contractile activity (540 ,ig BK and propranolol (0.3 mg/kg, 1 min pre equivalent), obtained from gel chromato treated). graphy, or the standard materials: kallidin, Recovery: The recovery of standard BK MLBK, BK and neurotensin (160 fig/ml was about 75% in the extraction step, and each) were dissolved in distilled water. A that in the SP-Sephadex, Sephadex G-25 and 0.05 ml aliquot of each of the resultant HPLC steps was about 90%. solutions was injected into the two columns Materials: The following reagents were (each 25 cm long and joined in series) used: SP-Sephadex C-25 Fine and Sephadex through a 5 cm long pre-column and eluted G-25 Fine (Pharmacia Fine Chemicals, Sweden); kallidin, MLBK, BK, neurotensin and leu5-enkephalin (Protein Research Foun dation, Osaka, Japan); trypsin from bovine pancreas Type III and chymotrypsin from bovine pancreas (Sigma Chemical Co., U.S.A.); atropine sulfate (E. Merck AG, Germany); dibenamine HCI (Nakarai Chemi cals Ltd., Kyoto, Japan); propranolol HCI (ICI Pharmaceutical Division, England): diphenhydramine HCI (Tanabe Seiyaku Co., Ltd., Osaka, Japan) and acetonitrile (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

RESULTS Fig. 1. Gradient chromatography of the extract. The lyophilized preparation (800 fcg BK Generation and extraction of the uterine equivalent on assay with the isolated rat uterus contractile substance: The uterine-contractile in the presence of 10-6 g/ml atropine and 10-7 g/ ml dibenamine) obtained from the extraction substance was generated by the incubation of procedure was dissolved in distilled water and KGN with KFE. Seven grams of the KGN applied to a 2.5x46 cm SP-Sephadex C-25 preparation were capable of yielding 1,400 column. The column was washed with 1 500 ml ,ag of BK equivalent on assay with the isolated of distilled water and then developed with a linear gradient of ammonium formate solution, rat uterus in the presence of atropine and pH 5.4 (0-0.4 M). The flow rate was 45 ml/hr. dibenamine. The reaction mixture was sub Ultraviolet detection was carried out at 280 rim. mitted to extraction with n-butanol, distilled Fractions of 20 ml each were collected, and a water and methanol. The resultant extract part of each fraction was dissolved in 0.9% saline. The resultant solution was tested with the contracted the isolated rat uterus in the isolated rat uterus in the presence of 10-6 g/ml presence of atropine and dibenamine, and it atropine and 10-7 g/ml diberiamine. relaxed the isolated rat duodenum in the additional presence of diphenhydramine and elution pattern of the preparation, obtained propranolol. The extract produced a fall in from gradient chromatography, assayed with rabbit blood pressure after intravenous admin the isolated rat uterus in the presence of istration of atropine, dibenamine and pro atropine and dibenamine. Panel B shows the pranolol. elution pattern of the standard materials: BK, Gradient chromatography on SP-Sephadex MLBK and kallidin detected by assay with C-25: Linear gradient chromatography was the isolated rat uterus. The retention time of carried out with the lyophilized preparation the preparation differed from those of the (800 ,tg BK equivalent) obtained from the standard materials. extraction procedure. The uterine-contractile Gel chromatography on Sephadex G-25: activity (700 ag BK equivalent) was detected The preparation obtained from equilibrium in fractions No. 21 to 25 in the presence of chromatography was applied to gel chromato atropine and dibenamine (Fig. 1). These graphy. The uterine-contractile activity was fractions also relaxed the isolated rat detected in fractions No. 29 to 31 in the duodenum in the additional presence of presence of atropine and dibenamine (Fig. 3). diphenhydramine and propranolol. Analytical HVPE: The preparation obtained Equilibrium chromatography on SP from gel chromatography was submitted to Sephadex C-25: In Fig. 2, panel A shows the HYPE. Two peaks and one shoulder were Fig. 3. Gel chromatography of the preparation obtained from equilibrium chromatography. Fig. 2. Equilibrium chromatography of the pre The preparation (3 ,tg BK equivalent on assay paration obtained from gradient chromato with the isolated rat uterus in the presence of graphy. The lyophilized preparation (120 Pg 10-6 g/ml atropine and 10-7 g/ml dibenamine) BK equivalent on assay with the isolated rat containing uterine-contractile activity was dis uterus in the presence of 10-6 g/ml atropine solved in 0.2 N acetic acid and applied to a and 10-7 g/ml dibenamine) containing uterine 2.6x89 cm Sephadex G-25 column. The flow contractile activity was applied to a 1.3x14 cm rate was 14 ml/hr. Fractions of 10 ml each were SP-Sephadex C-25 column. The elution buffer collected, lyophilized, and a part of each fraction was 0.05 M Tris-HCI buffer (pH 8.0) containing was dissolved in 0.9% saline. The resultant 0.08 M NaCI. The flow rate was 6 ml/hr. solution was assayed with the isolated rat Fractions of 6 ml each were collected, and a part uterus in the presence of 10-6 g/ml atropine of each fraction was tested with the isolated rat and 10-7 g/ml dibenamine. uterus in the presence of 10-6 g/ml atropine and 10-7 g/ml dibenamine. Panel A shows the elution pattern of the preparation. Panel B shows the elution pattern of the standard materials 1: BK, 2: MLBK and 3: Kallidiri (60, 400 and 300 ug, respectively). observed by densitometry of the ninhydrin stained strip. The uterine-contractile activity was detected as a shoulder region (hatched area in Fig. 4) in the presence of atropine and dibenamine. HPLC: In Fig. 5, panel A shows the elution pattern of the standard materials detected at 220 nm, 0.32 absorbance units full scale (a.u.f.s.). Panels B and C show the elution Fig. 4. Analytical high voltage paper electro phoresis of the preparation obtained from pattern of the preparation (25 ug BK equi equilibrium chromatography. S shows the valent), obtained from gel chromatography, preparation (1 ug BK equivalent on assay with detected at 220 and 280 nm (2.56 a.u.f.s.), the isolated rat uterus in the presence of 10-6 g/ respectively. Each fraction was assayed with ml atropine and 10-7 g/ml dibenamine) con the isolated rat uterus. As shown in panel D, taining uterine-contractile activity. The densi tometry of the ninhydrin-stained spot of S on four regions containing the uterine-con the guide strip was carried out at 570 nm. The tractile activity were detected in the presence uterine-contractile activity was detected from of atropine and dibenamine. the hatched area of the preparative strip in the The uterine-contractile activity of the presence of 10-6 g/ml atropine and 10-7 g/ml dibenamine. Arg indicates arginine (10 leg) materials (a, b, c and d) contained in these used as the standard marker. Conditions: 30 min regions was 0.9, 0.9, 15 and 5.6 icg BK at 50 volts per cm, pH 1.9. equivalent, respectively; and the activity of In the detection at 220 nm (panel B of the materials (a, b, c and d) was abolished by Fig. 5), the peaks of materials c and d were chymotrypsin, but not by trypsin treatment. observed to be matched to peaks of the All the materials relaxed the isolated rat uterine-contractile activity; whereas materials duodenum in the presence of atropine, a and b did not coincide with contractile dibenamine, diphenhydramine and pro activity. In the detection at 280 nm (panel C pranolol. of Fig. 5), none of the peaks of materials a, As shown in Fig. 5, the retention times of b, c and d were found to match with the materials a, c and d were different from that uterine-contractile activity. of kallidin, MLBK, BK and neurotensin. The The content ratio of a: b: c: d was 4: 4: 67. retention time of material b was equal to 25 as calculated from panel D of Fig. 5. that of BK. Figure 6 shows the chromatographic

Fig. 5. High performance liquid chromatography of the preparation obtained from gel chromatography. The preparation (25 Pg BK equivalent as assayed with the rat uterus in the presence of 10-6 g/ml atropine and 10-7 g/ml dibenamine) containing uterine contractile activity and standard materials (8 ng each) were dissolved in distilled water, and they were applied to a Zorbax ODS reversed-phase column (two columns, each 25 cm long, joined in series). The mobile phase was 0.05 M potassium dihydrogen phosphate (pH 2.5)-acetonitrile (8:2). The flow rate was 0.1 ml/min. Ultraviolet detection was carried out at 220 and 280 nm. Fractions of 0.7 ml each were collected, adjusted to pH 7.0 with 3 N NaOH, lyophilized, and a part of each preparation was assayed with the isolated rat uterus in the presence of 10-6 g/ml atropine and 10-7 g/ml dibenamine. Panel A shows the elution pattern of the standard materials, 1: kallidin, 2: MLBK, 3: BK, 4: neurotensin, detected at 220 nm (0.32 a.u.f.s.). Panels B and C show the chromatographic profile of the preparation containing uterine-contractile activity obtained from gel chromatography, detected at 220 and 280 nm (2.56 a.u.f.s.), respectively. Panel D shows the distribution of uterine-contractile activity. The biologically active substances detected by assay with the rat uterus are indicated by a, b, c and d.

Fig. 6. Rechromatography of the preparation containing material c obtained from high performance liquid chromatography (HPLC) on the Zorbax ODS reversed-phase column. HPLC was repeated with the desalted preparation (5 !ig BK equivalent on assay with the isolated rat uterus in the presence of 10-6 g/ml atropine and 10-7 g/ml dibenamine) containing uterine-contractile activity obtained previously by HPLC. The sample size was 20 II. Ultraviolet detection was carried out at 220 nm (0.04 a.u.f.s.). profile, detected at 220 nm, on HPLC repeated with the region containing the major mateial c indicated in panel B of Fig. 5. The retention time of the peak containing the uterine-contractile activity was equal to that of material c. Estimation of molecular weight: Figure 7 shows the elution positions of material c and standard materials of known molecular weight. The apparent molecular weight of material c obtained from this plot was 1650. Fig. 7. Molecular weight determination of material c obtained from rechromatography on the Biological studies: In Fig. 8, panels A, B Zorbax ODS reversed-phase column. The pre and C show the effects of the preparation paration (10 eg BK equivalent on assay with containing material c on the isolated rat the isolated rat uterus in the presence of 10-6 g/ uterus, isolated rat duodenum, and rabbit ml atropine and 10-7 g/ml dibenamine) con taining material c was dissolved in 0.2 N acetic blood pressure, respectively. The preparation acid and applied to the Sephadex G-25 column. containing material c contracted the uterus The column size was 0.9x97 cm (the void (1.2x10-10 g/ml) in the presence of atropine volume determined with blue dextran 2000 was and dibenamine, relaxed the duodenum 33 ml), and the flow rate was 7.0 ml/hr. Fractions of 2.0 ml each were collected and (2x10-10 g/ml) in the additional presence of tested with the isolated rat uterus in the presence diphenhydramine and propranolol, and pro of 10-6 g/ml atropine and 10-7 g/ml dibenamine. duced a fall in the blood pressure (4.4x10-8 Each point indicates the elution positions of the following substances of known molecular g/kg) after administration of atropine, dibe weight (M.W.): (1) neurotensin (M.W.: 1672), namine and propranolol. (2) kallidin (M.W.: 1188), (3) leu5-enkephalin (M.W.: 556), respectively. DISCUSSION We reported the existence of KFE which intestine, and the KFE had characteristics has a pH optimum of 4.8 in the rat stomach similar to cathepsin D which was distinct (1). The KFE content in the rat stomach was from gastrointestinal and pepsin (2). much higher than that in any region of the Cathepsin D occurs in a variety of tissues: al. (11) reported that two pharmacologically active , leukokinin M and PMN, were isolated from incubates of human kininogen and enzyme fractions of macro phages and polymorphonuclear leukocytes. Greenbaum (12) showed that leukokinin M and PMN were biologically BK-like sub stances, but they do not contain BK in the crucial part of the amino acid sequence. Greenbaum et al. (13) demonstrated the release of leukokinin H from leukokininogen

Fig. 8. Effects of the preparation containing by cathepsin D in human ascite fluid of a material c obtained from rechromatography on patient with ovarian carcinoma. Greenbaum the Zorbax ODS reversed-phase column on the (14) reported that in neoplastic disease, isolated rat uterus and duodenum and on the pepstatin, an inhibitor of acid kininogenases rabbit blood pressure. The preparation containing such as cathepsin D, showed a therapeutic material c was tested with the isolated tissues suspended in De Jalon's solution containing the effect by inhibition of leukokinin release. following antagonists: A: uterus, atropine (10-6 The KFE had characteristics similar to g/ml) and dibenamine (10-7 g/ml); B: cathepsin D, so it was reasonable that the duodenum, additional diphenhydramine (10-8 g/ ml) and propranolol (10-8 g/ml) to A. Panel C KFE as well as cathepsin D catalyzes the shows the effect on the rabbit blood pressure. formation of kinin (leukokinin)-like sub The blood pressure was recorded from the stance. carotid artery of urethanized (1 .5-2.0 g/kg, s.c.) The product from rat plasma produced by rabbits. The test samples were administered into the cannule tied to the vena marginalis after KFE was classified as a biologically BK-like intravenous administration of atropine (2 mg/ substance (1). The results of the present kg, 1 min pretreated), dibenamine (5 mg/kg, 40 investigations showed that four uterine min pretreated) and propranolol (0.3 mg/kg, 1 min pretreated). BK indicates bradykinin as the contractile substances (a, b, c and d) existed standard material. in the reaction mixture of KFE with rat plasma (Fig. 5), therefore the pharmacological effects of the product from rat plasma by ox spleen (4), rabbit liver (5) and chicken KFE in the previous paper (1) can be ascribed muscles (6). Greenbaum and Yamafuji (7) to the sum total of the activities of the four reported kinin formation by spleen cathepsins. materials (a, b, c and d). All the materials Greenbaum and Kim (8) showed that poly contracted the rat uterus and relaxed the rat morphonuclear leukocytes collected from duodenum. The uterine-contractile activity peritoneal exudates of rabbits contain enzyme of these materials was abolished by chymo activity that can catalyze the formation of trypsin, but not by trypsin treatment. The kinin-like material from human and bovine major material (c, 67%) produced a fall in bradykininogen. Back and Steger (9) demon rabbit blood pressure. The retention times of strated that the acid proteinase kinin-forming materials a, c and d on HPLC were different enzyme in the lymphosarcoma most closely from that of kallidine, MLBK, BK and neuro resembles a cathepsin. Greenbaum et al. (10) tensin; but the retention time of material b reported that the macrophages and leukemic was equal to that of BK. From these results, cells contain enzymes that form kinins from material c was classified as a biologically kininogen substrates at acidic pH. Chang et BK-like peptide which is distinct from kallidin, MLBK, BK and neurotensin. The content have marked actions on blood ratios of materials a and b both were only 4%, vessels and smooth muscles in vivo through so materials a and b may be impurities from the release of pharmacologically active pep the KGN or KFE preparation itself derived tides, i.e., kallidin and BK (17). It was shown from rat plasma, and material b seems to be that kallikreins exist in the gastrointestinal BK according to its retention time on HPLC. tract (18-22). The dog colon kallikrein Material d is a kinin-like peptide which is releases kallidin and BK (20). Zeitlin (23) distinct from kallidin, MLBK, BK and neuro reported that the kinin release by kallikrein tensin from the results of HPLC. Materials c could well be one of factors controlling the and d seem to be peptides with low polarity normal vasomotor tone, vascular permeability from their mobility on HVPE and HPLC. It and motility of the intestine. Habermann (24, is unknown if materials c and d are products 25), Hochstrasser and Werle (26) and from the same substrate by the same KFE Guimaraes et al. (27) demonstrated that because the substrate KGN and the KFE pepsin releases MLBK and its analogs from preparation used in the present study were a various KGN. Pepsin may be also one of the complex mixture of many components. factors controlling the function of the Materials c and d separated in the present stomach. The content of KFE and pepsin in investigation as well as leukokinins were the rat stomach is much higher than that in generated from the KGN preparation by any region of the intestine. The kallikrein catheptic enzyme. Materials c and d may have content in the rat stomach was lower than the common parts in the amino acid se that of any other region of the alimentary quences with the leukokinins. The leukokinins tract (22, 23). The KFE and pepsin both may do not contain BK in their amino acid se function as possible substitutes in the quences. It suggests the possibility that stomach for the kallikrein, the optimum pH materials c and d are also peptides which do being 8.5. not contain BK as part of the molecule. The We propose that materials c and d released ultraviolet detection on HPLC indicates the by KFE as well as the kinins (MLBK and its absence of tryptophan and tyrosine in the analogs) released by pepsin in the rat molecules of materials c and d since peaks of stomach function as possible substitutes for materials c and d could not be detected at the kinins (kallidin and BK) released by 280 nm. It was reported that the leukokinins kallikreins. have tyrosine in their amino acid com Erspamer and Melchiorri (28) presented positions (12). The absence of tyrosine in the concept of a skin-gut-brain peptide materials c and d distinguish materials c and triangle, i.e., the various peptide families of d from the leukokinins. The result of the the amphibian skin exist as their counter rechromatography on HPLC and the bioactive parts in mammalian gut and brain (29). potency of the preparation containing material Materials c and d, along with the kinins c suggested that material c was highly generated by kallikrein and pepsin, may be purified. We reported the existence of a BK possible candidates as biologically BK-like like substance (PI) in the rat stomach (15); peptides in the gastro-intestinal tract. and PI is a biologically BK-like peptide which REFERENCES is distinct from kallidin, MLBK and BK (3, 16). Whether or not materials a, b, c and d 1) Kobayashi,M., Shikimi, T., Miyata,S. and Ohata, K.: Studies on kinin-formingenzyme in rat are equivalent to PI is the subject of ongoing stomach. Japan. J. Pharmacol.29, 947-950 investigations. (1979) 2) Kobayashi, M. and Ohata, K.: Property of kinin atropine on kinin-forming and destroying forming enzyme in rat stomach. Japan. 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