The pepsins from human gastric mucosal extracts

1. The pepsins and pepsinogens of the gastric mucosal extracts of two normal subjects, of seven patients with gastric adenocarcinoma and of two patients with duodenal ulcer have been investigated by agar-gel electrophoresis and by ion-exchange chromatography. 2. Of the eight zones of proteolytic activity that have previously been reported in normal human gastric juice, seven can be detected in activated fundic mucosal extracts. Of these seven, four can be attributed to discrete pepsins, numbered 1, 3a, 3 and 5. 3. Zone 7 results from the activity of one or more enzymes that are alkali-stable and are best referred to as gastric proteinases rather than as pepsins. Zone 7 is much more evident in mucosal extracts than in gastric juice. 4. Zones 4 and 6 may result respectively from the activity of a pepsin-inhibitor complex and of an unactivated zymogen. 5. It was not possible, by the chromatographic methods employed, to separate satisfactorily the individual pepsins from activated extracts or their precursors from unactivated extracts, so that the ascribing of a pepsin to a specific zymogen must be considered tentative. Even so, pepsin 3 appears to arise from at least two major precursors, if not from three, whereas pepsins 1 and 5 each arise from a single major precursor. 6. Pyloric mucosal extracts contain principally zone 5 but also zones 6 and 7. These zones in general behave similarly to the corresponding zones of fundic extracts, but pyloric pepsin 5 migrates slightly faster on agar-gel electrophoresis than does fundic pepsin 5 and is a different enzyme. Zones 1 to 4 are absent.     

The pepsins of normal human gastric juice

1. The frequency of occurrence, under defined conditions, of the different human pepsins in the gastric juices of 50 normal subjects was investigated by agar-gel electrophoresis. 2. From a total of eight proteolytic zones located in the zymograms, no significant differences of occurrence existed between the sexes, or between subjects with or without gastric symptoms. 3. Two zones, numbered 3 and 5, occurred in all normal gastric juices. Zone 3 always exhibited the greatest proteolytic activity, then zone 5. The remaining enzymic zones were less well-marked and occurred less frequently. 4. A minor zone, 3a, was demonstrated within zone 3. The corresponding pepsin, 3a, has a mobility towards the anode 6-7% greater than has pepsin 3. 5. Of the eight zones, 1,2,3,3a and 5, at least, represent unique pepsins.     

Re-formation of disulphide bonds in reduced antithrombin III.

The separation of pepsin isoenzymes 1, 2, 3 and 5 (gastricsin) in human gastric juice was effected by chromatography on Mono Q ion-exchanger, and slow-moving proteinase was purified to homogeneity by using a modified procedure incorporating a novel affinity-chromatography step. The pH-activity profiles of these enzymes with mucus glycoprotein and basement-membrane substrates were determined; the profiles for pepsin 2 were noticeably different, and, in general, the pH optima for the hydrolysis of basement membrane were more acidic. Pepsin 1 expressed larger specificity constants (kcat./Km) than pepsin 3 with a series of synthetic peptide substrates, reflecting greater binding (smaller Km) by pepsin 1. Inhibitor studies at pH 1.7 and 4.5 with a series of P2-substituted lactoyl-pepstatins implied that valine at position P2 was optimal for inhibiting pepsins 1, 2 and 3 but detrimental for pepsin 5, whereas lysine at position P2 was tolerated well by pepsin 5 but not by pepsins 1, 2 and 3. The potency of lactoyl-pepstatin with lysine at position P2 did not increase as a function of pH. P2-substituted lactoyl-pepstatins failed to show any inhibitory selectivity among pepsins 1, 2 and 3.     

Pepsinogens and pepsins from Japanese seabass (Lateolabrax japonicus)

Three pepsinogens (PG1, PG2, PG3) were highly purified from the stomach of Japanese seabass (Lateolabrax japonicus) by ammonium sulfate fractionation, DEAE-Sephacel anion exchange column chromatography and Sephacryl S-200 gel-filtration. Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis revealed that the molecular masses of the three PGs were 35, 37, and 34kDa, and their isoelectric points were 5.3, 5.1, and 4.7, respectively. Zymography analysis showed that the three pepsinogens had different mobilities and enzymatic activities under native conditions. Pepsinogens converted into their active form pepsins under pH 2.0 by one-step pathway or stepwise pathway. All three pepsins were completely inhibited by pepstatin A, a typical aspartic proteinase inhibitor. The N-terminal amino acid sequences of the three pepsinogens were determined to the 30th, 30th and 28th amino acid residue and those of their corresponding active form pepsins were also determined to the 19th, 18th and 20th amino acid residue, respectively. All amino acid sequences of Japanese seabass PGs revealed high identities to reported fish and mammalian pepsinogens. The effective digestion of fish and shrimp muscular proteins by pepsins indicated their physiological function in the degradation of food proteins.     

Isolation and characterization of sheep pepsin.

Sheep pepsin was isolated (approx. 120-fold purification) from aqueous abomasal homogenates by (1) pH fractionation, (2) chromatography on Sepharose 4B-poly-L-lysine columns and (3) gel filtration on Sephadex G-100. The enzyme had mol.wt. approx. 34000, N-terminal valine and C-terminal alanine. The amino acid composition of sheep pepsin was generally similar to that of pig and ox pepsins, with a very low content of basic residues and a high content of acidic and hydroxy-amino acids. The pH optimum for NN-dimethyl-casein and NN-dimethyl-haemoglobin as substrates was approx. 1.8. The Km and kcat. for NN-dimethyl-haemoglobin were 46micronM and 1100min-1 respectively, and for NN-dimethyl-casein the corresponding parameters were 50micronM and 420min-1. These values were generally similar to those for pig and ox pepsins. At the pH optimum of 4.6, the sheep pepsin was about 50% as active on benzyloxycarbonyl-L-histidyl-L-phenyl-alanyl-L-tryptophan ethyl ester as was pig pepsin. The pH optimum for the hydrolysis of N-acetyl-L-phenylalanyl-L-di-iodotyrosine by sheep, ox and pig pepsins was approx. 1.85.     

A comparative study on the NH2-terminal amino acid sequences and some other properties of six isozymic forms of human pepsinogens and pepsins

Six pepsinogen isozymogens, including five forms of pepsinogen A (PGA) and an apparently single form of pepsinogen C (PGC), were isolated simultaneously from the purified total pepsinogen fraction of human gastric mucosa by fast protein liquid chromatography on a Mono Q column, and their NH2-terminal amino acid sequences and some other properties were compared. Upon activation at pH 2.0, all the isozymogens were converted to the corresponding pepsins in a stepwise manner through intermediate forms. The activation rates and the cleavage sites in the activation peptide segment to generate intermediate forms were significantly different among the isozymogens. The NH2-terminal 85-residue amino acid sequences of these isozymogens were determined, including the sequences of the activation peptide segments and the NH2-terminal regions of the corresponding pepsins. Differences in amino acid sequence were found at positions 43 and 77 among the pepsinogen A isozymogens; the residue at position 43 was Lys in PGA-5, PGA-4, and PGA-3a, and Glu in PGA-3 and PGA-2, and the residue at position 77 was Leu in PGA-5 and PGA-4 and Val in PGA-3 and PGA-2. Phosphate was not found in any of the isozymogens. The corresponding pepsins also showed significant variations in properties such as specific activities toward synthetic and protein substrates, pH dependence of activity, susceptibility to various inhibitors, and thermal and alkaline stabilities.     

Purification and characterization of two pepsins from the stomach of pectoral rattail (Coryphaenoides pectoralis)

Two pepsins (A and B) were purified from the stomach of pectoral rattail (Coryphaenoides pectoralis) by acidification, ammonium sulfate precipitation, gel filtration chromatography and anion exchange chromatography to obtain a single band on native-PAGE and SDS-PAGE. The purities of pepsin A and B were increased to 7.1- and 13.0-fold with approximately 5.7% and 2.2% yield, respectively. Pepsin A and B had the apparent molecular weights of 35 and 31 kDa, respectively, when analyzed using SDS-PAGE and Sephacryl S-200 gel filtration. Pepsin A and B showed maximal activity at pH 3.0 and 3.5, respectively, and had the same optimal temperature at 45 °C using hemoglobin as a substrate. Both pepsin A and B were stable in the pH range of 2.0–6.0 but were unstable at the temperatures greater than 40 °C. Activity of both pepsins was inhibited by pepstatin A and was activated by divalent cations, indicating pepsin characteristics. Activities of both pepsins continuously decreased as NaCl concentration increased (0–30%). The enzymes had high affinity and activity toward hemoglobin with K_m and K_cat values of 98–152 μM and 32–50 S^− 1, respectively. Purified pepsins generally showed the similar characteristics to other fish pepsins.     

The pathway of pepsin-catalysed transpeptidation. Evidence for the reactive species being the anion of the acceptor molecule

Several minor pepsinogens, present in extracts of bovine fundic mucosa obtained from the fourth stomach or abomasum, were separated from the main pepsinogen by chromatography on hydroxyapatite at pH7.3. The major pepsinogen and two of these minor pepsinogens were studied in detail. All three zymogens have N-terminal Ser-Val-, C-terminal -Val-Ala and not more than 1mol of glucose/mol of protein; no significant differences in amino acid composition were found. The pepsinogens differ in their organic phosphate content, which accounts for their chromatographic separation. By activation at 0 degrees C and pH2, a corresponding series of pepsins is formed. These enzymes were separated by hydroxyapatite chromatography at pH5.7. All the pepsins have N-terminal valine, C-terminal alanine and are free from carbohydrate. Again the only difference detected among them is in their organic phosphate content. The pepsins of high phosphate content are converted by an acid phosphatase in vitro into pepsins of low phosphate content.     

Purification and characterization of rat pepsinogens whose contents increase with developmental progress.

Two pepsinogens, the contents of which increase with developmental progress, were purified from the gastric mucosa of the adult rat by ammonium sulfate fractionation and chromatography on DEAE-cellulose and DEAE-Sepharose CL-6B columns. The purified zymogens, designated as pepsinogens I and II, were each shown to be homogeneous by polyacrylamide gel disc electrophoresis. Pepsinogen II had a greater electrophoretic mobility toward the anode at pH 8.0 than pepsinogen I. The molecular weights of both zymogens were estimated to be 38,000 by SDS-polyacrylamide gel electrophoresis. The activated enzymes, pepsins I and II, each had the same molecular weight of 32,000. The pH optima for both enzymes were found to be 2.0. The enzymes showed high stabilities at pH 8.0, while they lost their activities within 60 min at pH 10.0. The enzymes were inhibited by pepstatin and diazoacetyl-DL-norleucine methyl ester (DAN). The activities of the enzymes in hydrolyzing N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine (APDT) were about 1/8 of that of porcine pepsin. These results suggest that pepsins I and II are very similar.     

Tuna pepsinogens and pepsins. Purification, characterization and amino-terminal sequences

Three pepsinogens (pepsinogens 1, 2, and 3) were purified from the gastric mucosa of the North Pacific bluefin tuna (Thunnus thynuus orientalis). Their molecular masses were determined to be 40.4 kDa, 37.8 kDa, and 40.1 kDa, respectively, by SDS/polyacrylamide gel electrophoresis. They contained relatively large numbers of basic residues when compared with mammalian pepsinogens. Upon activation at pH 2.0, pepsinogens 1 and 2 were converted to the corresponding pepsins, in a stepwise manner through intermediate forms, whereas pepsinogen 3 was converted to pepsin 3 directly. The optimal pH of each pepsin for hemoglobin digestion was around 2.5. N-acetyl-L-phenylalanyl-L-diiodotyrosine was scarcely hydrolyzed be each pepsin. Pepstatin, diazoacetyl-DL-norleucine methyl ester in the presence of Cu2+, 1,2-epoxy-3-(p-nitrophenoxy)propane and p-bromophenacyl bromide inhibited each pepsin, although the extent of inhibition by each reagent differed significantly among the three pepsins. The amino acid sequences of the activation segments of these pepsinogens were determined together with the sequences of the NH2-terminal regions of pepsins. Similarities in the activation segment region among the three tuna pepsinogens were rather low, ranging over 28-56%. A phylogenetic tree for 16 aspartic proteinase zymogens including the three tuna pepsinogens was constructed based on the amino acid sequences of their activation segments. The tree indicates that each tuna pepsinogen diverged from a common ancestor of pepsinogens A and C and prochymosin in the early period of pepsinogen evolution.     

Purification, characterization, and amino acid sequences of pepsinogens and pepsins from the esophageal mucosa of bullfrog (Rana catesbeiana)

Two pepsinogens (pepsinogens 1 and 2) were purified from the esophageal mucosa of the bullfrog (Rana catesbeiana), and their molecular weights were determined to be 40,100 and 39,200, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal 70-residue sequences of both pepsinogens are the same, including the 36-residue activation segment. Furthermore, a cDNA clone encoding frog pepsinogen was obtained and sequenced, which permitted deduction of the complete amino acid sequence (368 residues) of one of the pepsinogen isozymogens. The calculated molecular weight of the protein (40,034) coincided well with the values obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results are incompatible with the previous report (Shugerman R. P., Hirschowitz, B. I., Bhown, A. S., Schrohenloher, R. E., and Spenney, J. G. (1982) J. Biol. Chem. 257, 795-798) that the major pepsinogen isolated from the bullfrog esophageal gland is a unique "mini" pepsinogen with a molecular weight of approximately 32,000-34,000. The two pepsinogens were immunologically indistinguishable from each other and related to human pepsinogen C. The deduced amino acid sequence was also more homologous with those of pepsinogens C than those of pepsinogens A and prochymosin. These results indicate that the frog pepsinogens belong to the pepsinogen C group. They were both glycoproteins, and therefore, this is the first finding of carbohydrate-containing pepsinogens C. Both pepsinogens were activated to pepsins in the same manner by an apparent one-step mechanism. The resulting pepsins were enzymatically indistinguishable from each other, and their properties resembled those of tuna pepsins.     

Catalytic properties and chemical composition of pepsins from Atlantic cod (Gadus morhua)

1. Three pepsins were purified from the gastric mucosa of Atlantic cod (Gadus morhua). 2. The enzymes, called Pepsin I and Pepsin IIa and b, had isoelectric points 6.9, 4.0 and 4.1, respectively, and digested hemoglobin at a maximal rate at a pH of approximately 3. 3. They resembled bovine cathepsin D in being unable to digest the mammalian pepsin substrate N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine. 4. Specificity constants (kcat/Km) for the cod pepsins were lower than for porcine pepsin, and they expressed higher substrate affinity and physiological efficiency at pH 3.5 than at pH 2. 5. The cod pepsins are glycoproteins, and their amino acid composition resembles that of porcine cathepsin D more than that of porcine pepsin. 6. The N-terminal sequence of Atlantic cod pepsins is substantially different from that of porcine pepsin. This indicates a significant evolutionary gap between fish and mammalian pepsins.     

Aspartic proteinases in fishes and aquatic invertebrates

1. The literature on molecular properties and physiological role of aspartic proteinases in fishes and aquatic invertebrates has been reviewed. 2. Pepsins have not been detected in invertebrates, and apparently cathepsin D, as well as other cathepsins, act both as digestive and lysosomal enzymes in many of these animals. The molecular properties of invertebrate cathepsin D correspond with cathepsin D in fishes and mammalians. 3. Fishes with a true stomach have pepsinogen secretion. Fish pepsins have higher pH optimum and are less stable in strong acid conditions than mammalian pepsins. They are very efficient at low temperatures, but less thermostable than mammalian pepsins. 4. Many fishes have two significantly different pepsins: Pepsin I and Pepsin II, which digest haemoglobin at a maximal rate in the pH ranges 3-4 and 2-3 respectively. Usually the pI of Pepsin I is in the range 6.5-7, whereas pI of Pepsin II is about 4. 5. Fish Pepsin I and cathepsin D have very similar molecular properties, and a hypothesis proposing that cathepsin D is the ancestor enzyme of aspartic proteinases in higher animals is presented.     

Purification and characterization of pepsins from the arctic fish capelin (Mallotus villosus)

Two pepsins from the stomach of the arctic fish capelin (Mallotus villosus) have been isolated and characterized. The purification was achieved by ammonium sulphate precipitation, ion exchange chromatography and gel filtration. Both pepsins resemble mammalian pepsins regarding pepstatin sensitivity, amino acid composition, stability and specific activity. The major capelin pepsin has optimum activity at significantly higher pH than is common for mammalian pepsins, and the optimum pH is different with different substrates. Both pepsins have relatively high activity at low temperatures. The pepsins have mol. wt of about 25,000 which is significantly lower than that of mammalian pepsins.     

Purification and characterization of pepsinogens from the gastric mucosa of African coelacanth, Latimeria chalumnae, and properties of the major pepsins

Two major pepsinogens, PG1 and PG2, and one minor pepsinogen, PG3, were purified from the gastric mucosa of African coelacanth, Latimeria chalumnae (Actinistia). PG1 and PG2 were much less acidic than PG3. Their molecular masses were estimated by SDS-PAGE to be 37.0, 37.0 and 39.3 kD, respectively. When incubated at pH 2.0, PG1 and PG2 were converted autocatalytically to the mature pepsins through an intermediate form, whereas PG3 was converted to an intermediate form, but not to the mature pepsin autocatalytically. The N-terminal sequencing indicated that the 42 residue sequences of the propeptides of PG1 and PG2 were essentially identical with each other, but different from that of PG3. A phylogenetic tree based on the N-terminal propeptide sequences indicates that PG1 and PG2 belong to the pepsinogen A group, and PG3 to the pepsinogen C group. From the phylogenetic comparison, coelacanth PG1 and PG2 appear to be evolutionally closer to tetrapod pepsinogens A than ray-finned fish pepsinogens A, consistent with the traditional systematics. Pepsins 1 and 2 were essentially identical with each other and rather similar to mammalian pepsins A in the pH optimum toward hemoglobin (pH 2-2.5), the cleavage specificity toward oxidized insulin B chain and strong inhibition by pepstatin, except that they possessed a significant level of activity in the higher pH range unlike mammalian pepsins A.     

The isolation and properties of a non-pepsin proteinase from human gastric mucosa.

1. A non-pepsin proteinase, proteinase 2, was successfully isolated free from pepsinogen (by repetitive chromatography on DEAE- and CM-celluloses) from the gastric mucosa of a patient with a duodenal ulcer and the uninvaded mucosa of a patient with a gastric adenocarcinoma. 2. Proteinases 1a and 1b, found in gastric adenocarcinoma, were not found in the gastic mucosa of these patients. 3. Proteinase 2 was shown to have an asymmetrical broad pH-activity curve with a maximum over the pH range 3.0-3.7. 4. Proteolytic activity of proteinase 2 was inhibited by pepstatin; the concentration of pepstatin giving 50% inhibition is of the order of 3nm. 5. Inhibition of proteolytic activity by carbenoxolone and related triterpenoids indicated that at pH 4.0 proteinase 2 possesses structural characteristics relating it to the pepsins and at pH 7.4 to the pepsinogens. 6. The sites of cleavage of the B-chain of oxidized insulin for proteinase 2 at pH 1.7 and pH 3.5 were shown to be similar to those previously established for human pepsin 3 and for the cathepsin E of rabbit bone marrow. 7. The non-pepsin proteinase 2 (cathepsin) of human gastric mucosa has properties more similar to cathepsin E than to the cathepsins D.     

Purification and characterization of goat pepsinogens and pepsins

Three type-A and two type-C pepsinogens, namely, pepsinogens A-1, A-2, A-3, C-1, and C-2, were purified from adult goat abomasum. Their relative levels in abomasal mucosa were 27, 19, 14, 25, and 15%, respectively. Amino acid compositions were quite similar between isozymogens of respective types, but different between the two types especially in the Glx/Asx and Leu/Ile ratios. NH₂-terminal amino acid sequences of pepsinogens A-3 and C-2 were SFFKIPLVKKKSLRQNLIEN- and LVKIPLKKFKSIRETM-, respectively. Pepsins A and C showed maximal hemoglobin-digestive activity at around pH 2 and 3, respectively, and specific activities of pepsins C were higher than those of pepsins A. Two subtypes of pepsin A were obvious, namely pepsin A-2/3 which maintains its activity in the weakly acidic pH region over pH 3 and pepsin A-1, which does not. Hydrolysis of oxidized insulin B chain by goat pepsins A occurred primarily at Ala14-Leu15 and Leu15-Tyr16 bonds.     

Pepsinogen C and pepsin C from gastric mucosa of Japanese monkey. Purification and characterization.

A new pepsinogen component, pepsinogen C, was purified from the gastric mucosa of Japanese monkey. The chromatographic behavior of this component on DE-32 cellulose was coincident with that of pepsinogen III-2 previously reported (1), and final purification was performed by large-scale polyacrylamide disc gel electrophoresis. The molecular weight was 35,000 as determined by gel filtration. The ratios of glutamic acid to aspartic acid and of leucine to isoleucine were higher than those of other Japanese monkey pepsinogens. The activated form, pepsin C, had a molecular weight of 27,000 and contained a large number of glutamic acid residues. The optimal pH for hemoglobin digestion was 3.0. Pepsin C could scarcely hydrolyze the synthetic substrate, N-acetyl-L-phenylalanyl-3, 5-diiodo-L-tyrosine (APDT). 1, 2-Epoxy-3-(p-nitrophenoxy)propane (EPNP), p-bromophenacyl bromide, and diazoacetyl-DL-norleucine methyl ester (DAN) inhibited pepsin C [EC 3.4.23.3] in the same way as pepsin III-3 of Japanese monkey. The susceptibility to pepstatin of pepsin C was lower than that of pepsin III-3, and 500 times more pepstatin was required for the same inhibitory effect. The classification and nomenclature of Japanese monkey pepsinogens and pepsins are discussed.     

Protein-bound phosphorylserine in acid hydrolysates of brain tissue. The determination of [32P]phosphorylserine by ion-exchange chromatography and electrophoresis

1. Partial acid hydrolysates of proteins derived from cortical slices of guinea-pig brain were divided into two parts and fractionated by ion-exchange chromatography and high-voltage electrophoresis. 2. The apparent yield of protein-bound phosphorylserine by the ion-exchange method was about three times that obtained by electrophoresis. 3. The specific radioactivity of phosphorylserine isolated from (32)P-labelled slices by electrophoresis was twice that isolated by chromatography. 4. The discrepancies were found to be due to the presence of unlabelled phosphates of unknown composition in the ;phosphorylserine' fraction obtained by the ion-exchange method. 5. Electrical stimulation of slices respiring in the presence of [(32)P]phosphate increased the specific radioactivity of the total phosphate in the chromatographic ;phosphorylserine' fraction by 53+/-11%, as compared with only 19+/-5% for the phosphorylserine isolated by electrophoresis.     

Partial purification of pepsins from adult and juvenile salmon fish Oncorhynchus keta. Effect of NaCl on proteolytic activities

1. Two pepsins, designated Pepsin I and Pepsin II, were isolated and partially characterized from the stomach of the adult stage salmon Oncorhynchus keta. This stage is developed in a marine environment. 2. One pepsin, designated Pepsin II, was isolated from the stomach of the juvenile stage salmon Oncorhynchus keta. This stage is developed in an estuarine environment. 3. The enzymes were partially purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration. 4. Pepsins I and II from adults and Pepsin II from juvenile showed proteolytic activity on acid-denatured hemoglobin with a pH optimum of 3. 5. The mol. wt determined by gel filtration on Sephadex G-100 of Pepsin I from juvenile species was found to be 32,000 whereas a value of 27,000 was determined for Pepsin II from juvenile and adult fish. 6. In contrast with Pepsin II, Pepsin I was activated by NaCl. It is suggested that the appearance of NaCl-activated pepsin would represent and adaptive response of the organism to the change from a low to a high salinity environment.