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/ . Biochem. 93, 743-754 (1983) Occurrence of Two Different Pathways in the Activation of Porcine Pepsinogen to Pepsin1 Takashi KAGEYAMA and Kenji TAKAHASHI Received for publication, September 9, 1982 Activation of porcine pepsinogen at pH 2.0 was found to proceed simultaneously by two different pathways. One pathway is the direct conversion process of pepsinogen to pepsin, releasing the intact activation segment. The isolation of the released 44-residue segment was direct evidence of this one-step process. At pH 5.5 the segment bound tightly to pepsin to form a 1 : 1 pepsin-activation segment complex, which was chromatographically indistinguishable from pepsinogen. The other is a stepwise-activating or sequential pathway, in which pepsinogen is activated to pepsin through intermediate forms, releasing activation peptides stepwisely. These intermediate forms were isolated and characterized. The major intermediate form was shown to be generated by removal of the amino-terminal 16 residues from pepsinogen. The released peptide mixture was composed of two major peptides comprising residues 1-16 and 17^t4, and hence the stepwise-activating process was deduced to be mainly a two-step process. Pepsinogen is converted to pepsin under acidic conditions. The reaction proceeds autocatalytically, releasing the so-called activation peptides from the amino(N)-terminal part of the pepsinogen molecule (7). In porcine pepsinogen, these activation peptides are derived from the N-terminal 44-residue segment (2-4). This activation follows predominantly an intramolecular mechanism below pH 3 (5-9). Essentially two reaction pathways are possible for the activation; i.e., the direct conversion pathway and the sequential conversion pathway. In porcine pepsinogen, evidence supporting the latter pathway has been presented by several investigators. Dykes and Kay reported that in the presence of pepstatin, a potent inhibitor of pepsin, the N-terminal 16-residue peptide (residues 1-16) was released first, suggesting the sequential release of the activation segment (10). They obtained similar results using bovine, chicken, and canine pepsinogens and bovine prochymosin (11). We also isolated an intermediate form2 (pseudopepsin) between pepsinogen and pepsin 1 2 This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Abbreviations: N,amino; C,carboxyl; SDS, sodium dodecyl sulfate; SP, sulfopropyl. Vol. 93, No. 3, 1983 The term 'intermediate form' used in the present paper and our previous papers (12, 14) differs from the conformational intermediates d, 6, and 4> of Marciniszyn et al. (9), and corresponds to the pseudopepsin presumed by Dykes and Kay (10) and Christensen et al. (13). 743 Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 18, 2016 Department of Biochemistry, Primate Research Institute, Kyoto University, Inuyama, Aichi 484 744 T. KAGEYAMA and K. TAKAHASHT upon activation of human pepsinogen in the presence of pepstatin {12). Further, Christensen ei al. reported that the initial cleavage of porcine pep16 17 MATERIALS AND METHODS Materials—Porcine pepsinogen (grade I, chromatographically prepared free from pepsin) was purchased from Sigma. A small amount of minor components present in the preparation was chromatographically removed prior to use. DEAEToyopearl was obtained from Toyo Soda Manufacturing Co., Tokyo. Sephadex G-50 and SP (sulfopropyl)-Sephadex were purchased from Pharmacia, fluorescamine from Japan Roche Co., Tokyo, and reagents for amino acid sequence determination from Wako Pure Chem. Ind., Tokyo. Carboxypeptidase Y was purchased from Oriental Yeast Co., Tokyo. Pepstatin was a Purification of Activation Peptides—The lyophilized activation mixture was dissolved in about 5 ml of 0.1 M sodium acetate buffer, pH 5.5, containing 8 M urea, and subjected to gel filtration on a column (1.6 x 150 cm) of Sephadex G-50 in the same buffer. The activation peptides were fractionated into a few peaks separated from the protein. Fractions containing a peptide mixture were further purified by chromatography on a column (1.6x40 cm) of SP-Sephadex in 0 . 1 M sodium acetate buffer, pH 5.5, containing 8 M urea. Peptides were eluted with a linear gradient of NaCl from 0 to 0.75 M using two 300-ml chambers. To remove urea, the pooled fraction was diluted about 20-fold with 0.1 M sodium acetate /. Biochem. Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 18, 2016 sinogen occurred at the peptide bond Leu-Ile on activation without pepstatin, forming the intermediate form (pseudopepsin) (13). These results indicate that porcine pepsinogen is activated to pepsin through intermediate form(s) by sequential release of the activation peptides. However, quite recently, we isolated the intact activation segment upon activation of Japanese monkey pepsinogen, indicating that one-step activation occurred exclusively (14). Our results also showed that the intermediate species was formed only in the presence of pepstatin. These results for Japanese monkey pepsinogen strongly suggested the occurrence of direct conversion of pepsinogen to pepsin, which differed greatly from those obtained for porcine pepsinogen. We decided to clarify the activation pathways in porcine pepsinogen based on the structural evidence and compare them to those of Japanese monkey pepsinogen. In the present study, porcine pepsinogen was activated in solution in the absence of pepstatin, and both the released peptides and the intermediate protein species were isolated and characterized. The released peptides were shown to include the intact activation segment of residues 1-44 together with peptides of residues 1-16 and 17-44. These results demonstrate that one-step activation occurs in porcine pepsinogen along with the sequential process. A preliminary report on part of this study has appeared elsewhere (75). generous gift from Drs. H. Umezawa and T. Aoyagi. All other chemicals were of reagent grade. Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Disc Gel Electrophoresis—The procedure was similar to that described by Weber and Osborn (16). Fluorometric Determination of Peptides—The peptide concentration was determined by the fluorometric method using fluorescamine according to de Bernardo et al. (17). The fluorescence was measured with a Hitachi Model 203 spectrofluorometer with excitation at 390 nm and emission at 475 nm, using leucine as a standard. Activation of Pepsinogen—Pepsinogen (10-100 mg) was dissolved in 50 ml of 0.01 M sodium phosphate buffer, pH 7.0, and the solution was acidified to pH 2.0 by the addition of 12.5 ml of 0.1 N HC1. The reaction mixture was prepared and incubated with gentle stirring at 14°C. At desired intervals, aliquots were withdrawn to examine the extent of activation by assaying the remaining potential pepsin activity and by SDSdisc gel electrophoresis (for these methods, see the legend to Fig. 1). The activation was terminated by the addition of 1 M NH4OH to a final concentration of 0.2 M. The mixture was immediately frozen, lyophilized, and subjected to gel filtration. To isolate the resulting protein species, the activation reaction was stopped by raising the pH to near 5.5 by adding 2.5 ml of 5 M sodium acetate buffer, pH 5.5, containing an about 3-fold molar excess of pepstatin over the initial amount of pepsinogen used. This preparation was subjected to chromatography on DEAE-Toyopearl. ACTIVATION OF PORCINE PEPSINOGEN Vol. 93, No. 3, 1983 RESULTS Analysis of the Time Course of Pepsinogen Activation—Pepsinogen was activated at various concentrations at pH 2.0, and the activation process was analyzed by SDS-disc gel electrophoresis (Fig. 1). In all cases, pepsinogen disappeared rapidly after acidification. The resulting protein species were detected as two bands; one of them had the same molecular weight as the authentic pepsin and the other hand a molecular weight intermediate between those of pepsinogen and pepsin. The intermediate form was relatively stable as compared with pepsinogen, but was gradually converted to pepsin during a long period of incubation. When analyzed by the proteolytic activity assay, the activation appeared to be complete within a few min (Fig. 2). The formation of the intermediate form became predominant when the initial pepsinogen concentration decreased. Released peptides were also detected as two bands. The amount of peptide in the high molecular weight peptide band appeared to be maximum at I or 2 min and to decrease rather rapidly, while that in the low molecular weight peptide band appeared to increase gradually with the progress of incubation time. However, when the initial pepsinogen concentration was 1.6 mg/ ml, both peptide bands were scarcely detectable after 2 min. This may be due to further cleavage of these peptides to smaller peptides, which were not retained in the gel. The activation experiments described in the following sections were carried out at an initial pepsinogen concentration of 0.16 mg/ml in the activation mixture. Isolation and Characterization of Activation Peptides—Peptides released after 2-min and 30-min activation were isolated and characterized. The lyophilized reaction products were fractionated by Sephadex G-50 gel filtration (Fig. 3). The protein mixture was eluted near the void volume, separated from peptides. Peptides in the 2-min activation mixture were separated into 3 peaks (Fractions 1, II, and III). Each fraction showed a single N-terminal amino acid, and Fractions IF and 11 f gave a single spot on thin layer electrophoresis at pH 3.5. These results indicated that each fraction was composed of a single peptide. Electro- Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 18, 2016 buffer, pH 5.5, and then applied to a column (1.2x2.5 cm) of SP-Sephadex equilibrated with the same buffer. After the column was washed with the buffer, the peptide was eluted with the same buffer containing 1 M NaCI. The peptide was freed from the salts by passage of the solution through a column (1.6x45 cm) of Sephadex G-15 equilibrated with 1 % acetic acid. Purity of each peptide fraction was examined by N-terminal amino acid analysis by dansylation (18), and further by electrophoresis on a thin-layer plate (Precoated TLC plate, Cellulose, Merck Co.) at pH 3.5 in pyridine-acetic acid-water ( 1 : 1 0 : 90, by volume) at 1,000 volts/20 cm for 40min. Isolation of Pepsinogen, Pepsin, and the Intermediate Form—The activation mixture adjusted to pH 5.5 was applied to a column (1.15x25 cm) of DEAE-Toyopearl previously equilibrated with 0.1 M sodium acetate buffer, pH 5.5, containing 7 //M pepstatin. The adsorbed protein was eluted with a linear gradient of NaCI from 0 to 0.5 M using two 300-ml chambers followed by 100 ml of 0.5 M NaCI in the same buffer. Amino Acid Analysis—Samples for analysis were hydrolyzed with 1 ml of 6 N HCI at 110°C for 24 and 72 h in evacuated sealed tubes. The amino acids were determined with a Hitachi Model 835 amino acid analyzer essentially according to Spackman et al. (19). Amino Acid Sequence Determination—The amino acid sequences of the N-terminal regions of pepsinogen, pepsin, and the intermediate forms were determined by a modification (20) of the manual Edman degradation method (21) using 0.5-1 mg of each protein. Phenylthiohydantoin derivatives of amino acids were identified by thin layer chromatography according to Kulbe (22), and/or high performance liquid chromatography using a Toyo Soda column LS410K. according to Omichi et al. (23). The carboxyl(C)-terminal amino acid sequence of the activation segment was analyzed with carboxypeptidase Y as follows. Two nmoles of each activation segment was incubated at 37°C with 20 //g of carboxypeptidase Y in 300 /i\ of 0.1 M sodium phosphate buffer, pH 6.5, containing 10% methanol. Aliquots were withdrawn at desired times and released amino acids were determined with the amino acid analyzer. 745 ACTIVATION OF PORCINE PEPSINOGEN 747 200 — 100 — 0 5 TIME OF ACTIVATION (min) by SP-Sephadex chromatography. Amino acid analysis showed that it was identical with peptide 1-16 in Fraction III. Fraction VI contained one major peptide as shown by thin layer electrophoresis, and it was purified by preparative electrophoresis on Whatman 3 MM filter paper. It was composed of 8 residues corresponding to residues 17-24 of the activation segment. Fractions No. 110-120 (Fig. 3b) contained small peptides and/or free amino acids and they were not purified further. Isolation and Characterization of Pepsinogen, Pepsin, and Intermediate Forms—The activation mixtures were analyzed by chromatography on DEAE-Toyopearl in the presence of pepstatin. After activation for 2 min, several peaks (Fractions A through F) appeared (Fig. 5b). Fraction B was eluted at the same position as that of authentic pepsinogen. This was confirmed by cochromatography of authentic pepsinogen and the activation mixtre. Upon further incubation until 30 min, Fractions A and B disappeared and the relative contents of Fractions E and F increased as shown in Fig. 5c as Fractions J and K. The amino acid compositions of some of these fractions are shown in Table II. Fractions A and B had Vol. 93, No. 3, 1983 200 — Q. 0. 100 — 40 60 FRACTION 80 100 NUMBER Fig. 3. Gel filtration of the activation mixture. Activation' was carried out for 2 min (a) and 30 min (b), and stopped by the addition of NH4OH. The mixture was lyophilized, redissolved and subjected to gel filtration. A column (1.6 x 150 cm) of Sephadex G-50 was equilibrated and eluted with 0.1 M sodium acetate buffer, pH 5.5, containing 8 M urea. The fraction size was 3 ml. BD and«§alt indicate the positions of blue dextran and inorganic^alts, respectively. The fractions under the bars were pooled. - 100 120 FRACTION 140 NUMBER Fig. 4. SP-Sephadex chromatography of peptide fraction IV. The column (1.6x40 cm) was equilibrated with 0 . 1 M sodium acetate buffer, pH 5.5, containing 8 M urea. Peptides were eluted with a linear gradient'of NaCl in the same buffer. The fractions under the bars were pooled. The fraction size was 3 ml. Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 18, 2016 Fig. 2. Time course of activation of porcine pepsinogen analyzed by the proteolytic activity assay. Activation was carried out at pH 2.0 and 14°C. The initial concentration of pepsinogen in the activation mixture was 0.16mg/ml. Aliquots of the incubation mixture were withdrawn at desired intervals, diluted 10-fold with 0.01 M sodium acetate buffer, pH 5.5, and used partly for the assay of the total proteolytic activity at pH 2.0 and 37°C with bovine hemoglobin as a substrate according to Anson (27). For the assay of the residual pepsinogen, aliquots of the diluted reaction mixture were mixed with an equal volume of 0.1 M Tris, and incubated at 14°C for 30 min before the assay to inactivate pepsin formed. The extent of activation was calculated from the difference in the activities determined in these two assays. 748 T. KAGEYAMA and K. TAKAHASHI TABLE I. Amino acid compositions of purified peptide fractions.1 Number of residues per molecule of peptide t> Amino acid II in IV-2 IV-3 V VI Lys 9.3 (9) 5.9 (6) 2.9 (3) 5.6 (6) 5.9 (6) 2.7 (3) 2. 4 (3) His 1.8 (2) 1.8 (2) 1.8 (2) 2. 1 (2) Arg 2.2 (2) Asp 4.0 (4) 3.0 (3) 3.0 (3) 3.0 (3) 1.8 (2) 1.0 (1) 2. 0 (2) Thr 0.9 (1) Ser 0.9 (1) 1.8 (2) 2.0 (2) 0.9 (1) 0.9 (1) 0.9 (1) 1.0 (1) Glu 1.0 (1) 1.1 (1) 0.9 (1) 1.0 (1) 1. 1 (1) 1. 1 (1) Pro 2.5 (3) 1.6 (2) 0.7 (1) 1.4 (2) c 1.8 (2) Gly 1.5 (1)0 1.3 (1) Ala 3.5 (4) 3.9 (4) 1.3 (1) 3.5 (4) 1. 1 (1) 2. 1 (2) Val 3.1 (3) He Leu 1.1 (1) 7.4 (7) 0.9 (1) 3.1 (3) 0.8 (1) 3.3 (3) 1.0 (1) 2.3 (2) Tyr 0.2 ( l ) e Phe 1.8 (2) 0.8 (1) 1.9 (2) 0.1 (I) 8 1.6 (2) 0.2 (l) e 1.8 (2) Total N-Terminus f Yield «(%) 1.8 (2) 1.0 (1) 0. 9 (1) 3.1 (3) 2.5 (3) 3.9 (4) 0.9 (1) 1.1 (1) 0.7 (1) 3.9 (4) 0. 7 (1) 0. 9 (1) 44 28 16 28 25 16 8 Leu lie 24 Leu He He Leu nd 78 5 28 87 nd 10 a The composition of Fraction IV-1 is not shown, since this fraction was a mixture of a few peptides as clarified by N-terminal analysis. The composition of the high molecular weight peptide isolated from Fraction B after DEAE-Toyopearl chromatography was the same as that of Fraction I. i> The values were calculated by assuming the number of aspartic acids to be 4.0 in Fraction I, 3.0 in Fractions II, IV-2, and IV-3, 2.0 in Fraction VI, and 1.0 in Fractions III and V. <= Assumed as 2 residues. « Assumed as 1 residue. • Assumed value allowing for loss during acid hydrolysis. ' Determined by dansylation according to Gray and Hartley (75). s Yields of Fractions I, II, III, and V were calculated after Sephadex G-50 gel filtration, and those of Fractions IV-2 and IV-3 were calculated after SP-Sephadex chromatography. These values were based on the amounts of peptides determined by amino acid analysis, nd: Not determined. practically the same composition as pepsinogen, and Fractions E, F, J, and K the same composition as pepsin (data for Fractions J and K not shown). The compositions of Fractions C, D, G, H, and I were intermediate between those of pepsinogen and pepsin, and those of Fractions C, D , G, and I were nearly identical with one another (data for Fractions G and I not shown). The somewhat lower lysine value for Fraction D may be due to contamination of the pepsin fraction {i.e., Fraction E). The N-terminal sequences of these fractions were determined by manual Edman degradation (Table III). Analysis of the N-terminal 5-residue sequence of Fraction B indicated that this fraction contained the N-terminal sequences of both pepsinogen and pepsin. Moreover, two protein bands corresponding to pepsinogen and pepsin, and one peptide band corresponding to the high molecular weight peptide were detected in Fraction B by SDS-disc gel electrophoresis (Fig. 6). The peptide was isolated from Fraction B by adsorption with SP-Sephadex in the presence of 8 M urea. Amino acid analysis and N-terminal analysis showed that the peptide was identical with the 44-residue activation segment (see footnote of Table I). From these results Fraction B was judged to be a mixture of pepsinogen and a 1 : 1 complex of pepsin / . Biochem. Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 18, 2016 I ACTIVATION OF PORCINE PEPSINOGEN 749 TABLE II. Amino acid compositions of pepsinogens, pepsins, and the intermediate forms purified by DEAEToyopearl chromatography." Number of residues per molecule of protein b Amino acid B A C F E D H[ Pgc 11 P» Lys 11.4 (11) 9.5 (10) 6.7 ( 7) 5.4 ( 5) 1.3 ( 1) 0.9 ( 1) 4.7 ( 5 ) 10 7 1 His 2.8 ( 3) 2.2 ( 2) 2.5 ( 3) 2.0 ( 2 ) 0.9 ( 1) 0.8 ( 1) 1.8 ( 2 ) 3 3 1 Arg 3.6 ( 4 ) 3.7 ( 4) 4 2 2 46 45 42 46.0 (46) 46.0 (46) Thr 28. K (28) 28.8 f (29) 27.9 f (28) 27.9 f (28) Ser 43.5 f (44) 44. 3'(44) 44.7'(45) 45.5 f (46) 43.7 f (44) 45. If (45)' 45.7f(46) 46 44 44 Glu 27.0 (27) 28.2 (28) 27.7 (28) 28.9 (29) 27.5 (28) 27.3 (27) 27.5 (28) 28 27 26 Pro 17.2 (17) 18.2 (18) 17.7 (18) 15.7 (16) 18.3 (18) 18 17 15 26.6 f (27) 27.4f(27) 28. Of (28) 28 27 27 Gly 37.3 (37) 35.5 (36) 17.0 (17) 35.0 (35) 36.3 (36) 33.9 (34) 15.2 (15) 34.6 (35) 34.1 (34) 36 36 35 Ala 21.8 (22) 19.4 (19) 20.7 (21) 20.7 (21) 18.8 (19) 18.0 (18) 21.6 (22) 20 20 16 Val 24.3 (24) 25.0 (25) 23.6 (24) 24.3 (24) 23.7 (24) 23.9 (24) 23.9 (24) 25 22 22 2.3 ( 2) 2.6 ( 3) 2.5 ( 3) 2.6 ( 3) 2.7 ( 3) 2.2 ( 2) 4 4 4 lie 27.2 (27) 27.0 (27) 26.2 (26) 26.6 (27) 25.3 (25) 25.5 (26) 24.7 (25) 26 26 25 Leu 32. 1 (32) Met nd 32.6 (33) 29.4 (29) 29.7 (30) 27.3 (27) 26.7 (27) 28. 1 (28) 34 29 27 Tyr nd 15. 1 (15) 15.5 (16) 16.2 (16) 15.5 (16) 14.4 (14) 16 16 15 Phe nd 17.1 (17) 16.3 (16) 17.6 (18) 15.8 (16) 15.1 (15) 16.3 (16) 16.3 (16) 16 16 14 Yield g(%) 1.7 10.3 13.5 Relative yield (%) •»,»— 23 8.4 1 13.3 5.7 -v*'— •v^-— 41 36 6.6 a A through H indicate the fractions purified by DEAE-Toyopearl chromatography. The compositions of Fractions G, 1, J, and K are not shown, since they are essentially the same as those of Fractions C, D, E, and F, respectively. b The values were calculated by assuming the number of aspartic acids to be 46.0 in Fractions A and B, 45.0 in Fractions C and D, 43.0 in Fraction H, and 42.0 in Fractions E and F. Except for serine and threonine, each value is an average of values obtained for 24- and 72-h hydrolysis. The values in parentheses are nearest integers. Half-cystine and tryptophan were not determined. The compositions of the intermediate species and pepsin may include one alanine and two valine residues per molecule derived from the bound pepstatin. c Composition of pepsinogen from the sequence of the activation segment (2-4) and pepsin (26). i Composition of the intermediate form expected from residues 17-370 of pepsinogen. « Composition of pepsin from the known amino acid sequence (26). f Values extrapolated to zero time of hydrolysis, e Yield after DEAE-Toyopearl chromatography. The yields of Fractions G, I, J, and K were 10.4, 10.4, 24.4, and 8.5 percent, respectively, nd: Not determined. and the activation segment. This was also supported by the fact that Fraction B had the same elution position and amino acid composition as authentic pepsinogen as previously mentioned. On quantitative determination of the N-terminal residues in Fraction B, the complex was estimated to occupy about 5 0 % of Fraction B. These results indicate that the activation segment formed Vol. 93, No. 3, 1983 a tight complex with pepsin at pH 5.5 and that the complex cochromatographed with pepsinogen at the same pH. When the activation was terminated by raising the pH to 5.5 and the activation mixture was chromatographed in the absence of pepstatin, almost all the intermediate forms (Fractions C, D, G, and 1) were converted to corresponding pepsins during chromatography, whereas Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 18, 2016 Asp 2.0 ( 2) 2.1 ( 2 ) 1.9 ( 2) 1.9 ( 2) 2.0 ( 2) 45.0 (45) 45.0 (45) 42.0 (42) 42.0 (42) 43.0 (43) ACTIVATION OF PORCINE PEPSINOGEN 751 10 20 30 40 LVKVPLVRKKSLRQNLIKNGKLKDFLKTHKHNPASKYFPEAAALIGDEP pepsinogen (Pg) : -intermediate (C,D,G,I ) \ -intermediate'(H) ' -activation segment ( I ) - -m,v- IV3 contained at least 2 isozymes which were chromatographically separable from each other after the activation segment was released completely or partially. The molar ratio of these two isozymes was estimated to be about 7 : 3. Although Fraction H was contaminated by Fractions G and I, the major component was shown to have the N-terminal sequence :Phe-Leu-Lys-. Fraction H is therefore thought to have been generated by removal of the N-terminal 24-residues from pepsinogen. The sequence of Fraction A could not be determined because of the small amount. Assignment of Peptides and Proteins, and the Ratio of Two Activation Pathways—Figure 7 shows the assignment of the released peptides and the protein species isolated and identified. Isolation and characterization of the 44-residue activation segment together with shorter activation peptides as well as the intermediate protein species from the activation mixture of pepsinogen clearly showed that porcine pepsinogen is largely activated simultaneously by two different pathways, i.e., one-step activation with initial cleavage at the 44 45 Leu-Ile bond and a stepwise-activating process 16 17 with first cleavage at the Leu-Ile bond, followed 44 45 by cleavage at the Leu-Ile bond. The yields of released peptides and resulting protein species are shown in Tables I and II, respectively. The yield of the 44-residue activation segment was about 10% after 2-min activation. This value appears to be lower than expected from the proportion of the one-step activation pathway, since the 44-residue segment was cleaved to smaller Vol. 93, No. 3, 1983 peptides rather rapidly (Fig. 1). Assuming that in the early period of activation the 28-residue peptide was formed exclusively by the cleavage of the 44-residue segment released, the proportion of the one-step pathway could be estimated to be maximally about 40%. This assumption was based on the results in Fig. 1, which showed the intermediate form to be rather stable when formed and its further conversion to pepsin by release of the 28-residue peptide proceeded gradually. On the other hand, this relatively stable character of the intermediate form enabled us to estimate directly the proportion of the sequential pathway from the relative yields of resulting protein species (i.e., pepsin, intermediate forms and the complex). At 2-min activation, the value was about 46%. These results indicate that under the present activation conditions the two activation pathways operated nearly equally. DISCUSSION As described in the preceding section, porcine pepsinogen was shown to be activated to pepsin at pH 2.0 simultaneously through two different pathways. These are schematically illustrated in Fig. 8. The activation reaction at this pH should proceed predominantly intramolecularly as shown by several investigators (5-7). The occurrence of the onestep process, in which the intact activation segment of 44 residues is released directly from porcine pepsinogen, had not been reported previously. Therefore the isolation, in this study, of this intact activation segment from the activation mixture of pepsinogen is the first direct evidence of Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 18, 2016 Fig. 7. Assignment of protein species and various peptides obtained on activation of pepsinogen for 2min and 30min. The symbols are the same as those in Figs. 3, 4, and 5. Fraction I corresponds to the high molecular weight peptide band, and Fractions II to VI correspond to the low molecular weight peptide band in Fig. 1. T. KAGEYAMA and K. TAKAHASHI 752 \pH5.5 \ X pepsin-activation • segment complex N —t this conversion in the activation of porcine pepsinogen. As the initial pepsinogen concentration in the activation mixture was increased, the proportion of the one-step pathway seemed to increase. This appears to indicate that the one-step pathway may be partly due to the intermolecular action of the pepsin formed, although the intramolecular mechanism is thought to be predominant at pH 2. At pH 2 the activation segment was further cleaved into smaller peptides rather rapidly. At pH 5.5, however, it formed a tight complex with pepsin and the complex behaved chromatographically like pepsinogen. The activation segment could not be removed from the complex even by adsorption to a cation exchange resin such as SP-Sephadex but it could finally be released from the complex by denaturation of the complex with 8 M urea after alkali treatment. These results seem to indicate that the 44-residue segment has a much higher affinity to pepsin than other shorter activation peptides including the Nterminal 16-residue peptide which was reported to have a Kx value of 5.7 x lO"8 M (24) or 2.5 x 10~8 M (25). Previously, Dykes and Kay could obtain only the N-terminal 16-residue peptide from an activa- tion mixture upon activation of porcine pepsinogen at pH 2.5 in the presence of pepstatin, suggesting that the occurrence of a one-step process was unlikely (10). It may be possible, however, that the presence of pepstatin somewhat altered the course of the activation reaction. The failure in obtaining the intact activation segment may also be due to the rather short life span of the activation segment around pH 2 and the high affinity of the segment to pepsin around pH 5.5. The other type of activation pathway is a sequential or stepwise-activating process, in which activation proceeds through intermediate forms. In the present study, the intermediate forms (Fractions C, D, G, and I) were generated by removal of the N-terminal 16-residue peptide from pepsinogen. These intermediate forms are thought to be the same protein presumed by Dykes and Kay (10) and Christensen et al. (13) to be pseudopepsin. A part of these intermediate forms was converted to another intermediate (Fraction H), which was formed by releasing further an 8-residue peptide from the N-terminal region. The major part of the intermediate forms (Fractions C, D, G, and I), however, is though to be converted to pepsin directly, since only a small amount of / . Biochem. Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 18, 2016 intermediate Fig. 8. The proposed activation process of porcine pepsinogen at pH 2.0. A pepsinogen molecule at neutral pH is expressed as a square form with an unreleased activation segment and an undeveloped active site. A pepsinogen molecule after a conformational change at acidic pH, molecules of the intermediate forms, and a pepsin molecule are expressed as circular forms with a developed active site. The dashed double-lined arrow indicates the intermolecular attack of pepsin formed against pepsinogen. ACTIVATION OF PORCINE PEPSINOGEN 44 45 segment to pepsin: i.e., Leu-Ile (porcine pepsino47 48 gen) and Leu-Ile (Japanese monkey pepsinogen), and the bond in the middle region of the activation 16 17 segment: i.e., Leu-Ile (porcine pepsinogen) and 25 26 Asp-Phe (Japanese monkey pepsinogen). This suggests that these regions may be located structurally close to the active site exposed by a conformational change at acidic pH. The following mechanism may therefore be assumed. In porcine pepsinogen, both cleavage sites may come close to the active site and are similarly susceptible to the proteolysis and therefore either bond is cleaved first to form pepsin or the intermediate form. In monkey pep47 48 sinogen, the Leu-Ile bond may come closer to the 25 26 active site than the Asp-Phe bond and/or the former may be more susceptible than the latter. Thus the former is cleaved first, leading exclusively to one-step activation. This hypothesis implies that in pepsinogens two activation pathways are always probable depending on the structure of the activation segment and its location relative to the active site. Porcine pepsinogen has the same 2-residue sequences as Japanese monkey pepsinogens, i.e., 24 25 Asp-Phe, in the middle region of the activation Vol. 93, No. 3, 1983 segment, but this bond was not cleaved in the early period of activation. This may be due to a slight difference in the length of the activation segment. The intact activation segment of porcine pepsinogen is composed of 44 residues and this is 3 residues shorter than those of human and monkey pepsinogens. The lack of 2 residues was especially observed in the C-terminal region 16 17 of the porcine segment. Thus the Leu-Ile bond 24 25 rather than the Asp-Phe bond in the porcine segment may come close to the exposed active site and be cleaved off. However, another possibility cannot be excluded that the difference is partly due to the difference in the susceptibility of the peptide bonds themselves to the proteolysis. In 16 17 porcine pepsinogen the Leu-Ile bond may be more 24 25 susceptible than the Asp-Phe bond. On the other 25 26 hand, in monkey pepsinogen the Asp-Phe bond 17 18 may be more susceptible than the Leu-Ser bond 16 17 which corresponds to the Leu-Ile bond in porcine pepsinogen. We thank Drs. H. Umezawa and T. Aoyagi at the Institute of Microbial Chemistry, Tokyo, for the generous supply of pepstatin. REFERENCES 1. Herriott, R.M. (1962) J. Gen. Physiol. 45, 57-76 2. Ong, E.B. & Perlmann, G.E. (1968) / . Biol. Chem. 243, 6104-6109 3. Pedersen, V.B. & Foltmann, B. (1973) FEBS Lett. 35, 255-256 4. Stepanov, V.M., Baratova, L.A., Pugacheva, L.B., Belyanova, L.P., Revina, L.P., & Timokhina, E.A. (1973) Biochem. Biophys. Res. Commun. 54, 11641170 5. Bustin, M. & Conway-Jacobs, A. (1971) /. Biol. Chem. 246, 615-620 6. Al-Janabi, J., Hartsuck, J.A., & Tang, J. (1973) / . Biol. Chem. 247, 4628-4632 7. McPhie, P. (1974) Biochem. Biophys. Res. Commun. 56, 789-792 8. Sanny, C.G., Hartsuck, J.A., & Tang, J. (1975) / . Biol. Chem. 250, 2635-2639 9. 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