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At least 19 recordsLinked to original sources
PubMed1986

Mucus degradation by pepsin: comparison of mucolytic activity of human pepsin 1 and pepsin 3: implications in peptic ulceration.

The ability to digest mucus, mucolytic activity of isolated pepsins and samples of human gastric juice has been assayed by measuring the fall in viscosity when incubated with purified pig gastric mucus glycoprotein. Pure human pepsin 1, the peptic ulcer associated pepsin, digested gastric mucus glycoprotein at a faster rate than did pure human pepsin 3 (the principal human pepsin), or the equivalent pig pepsin (pepsin A). At pH 2.0 pepsin 1 had twice the mucolytic activity of pepsin 3. Above pH 3.8 this difference became more marked and whereas pepsin 1 caused substantial mucolysis up to and including pH 5.1, pepsin 3 had minimal activity. At pH 4.0 pepsin 1 had six times the mucolytic activity of pepsin 3. Gastric juices from patients with duodenal ulcer each exhibited substantial mucolytic activity between pH 2 to 5, similar to that of pepsin 1. In contrast, gastric juice from non-symptomatic volunteers exhibited little mucolytic activity above pH 4. Analysis of the mucus glycoprotein by gel filtration showed that an increase in lower molecular weight, pepsin degraded, glycoprotein was associated with the fall in mucus viscosity for all enzyme preparations. These results showed that pepsin 1 can digest the mucus more effectively than pepsin 3 and at higher pH values. The raised concentrations of pepsin 1 in the juice of peptic ulcer patients may thus promote the ulcerative process by increased erosion of the mucus barrier under conditions likely to pertain in the duodenal bulb as well as the stomach.

Chromatography, Gel↗
PubMed1980

Monkey pepsinogens and pepsins. Monkey pepsinogens and pepsins. V. Purification, Characterization, and amino-terminal sequence determination of crab-eating monkey pepsinogens and pepsins.

Pepsinogens were purified from the gastric mucosa of the crab-eating monkey, Macaca fascicularis. Eight pepsinogens were shown to be present disc-electrophoretically and they were termed pepsinogens I-a, I-b, III-1-a, III-1-b, III-2-a, III-2-b, III-3, and C, based on the nomenclature used for Japanese monkey pepsinogens. The molecular weights were 43,000 for pepsinogens I-a and I-b, 40,000 for pepsinogens III-1-a, III-1-b, III-2-a, III-2-b, and III-3, and 38,000 for pepsinogen C, as determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Pepsinogens I-a and I-b contained carbohydrate amounting to about 4-5% by weight. Each was activated to pepsin by acidification at pH 2.0. Pepsinogen III-1 (a mixture of III-1-a and III-1-b) yielded a single pepsin, i.e. pepsin III-1, and pepsinogen III-2 (a mixture of III-2-a and III-2-b) also gave a single pepsin, i.e. pepsin III-2. The molecular weights were estimated to be 38,000 for pepsins I-a and I-b, 35,000 for pepsins III-1, III-2, and III-3, and 34,000 for pepsin C. Optimal pHs toward acid-denatured hemoglobin were 1.9, 2.3, 2.0, 2.0, and 2.3 for pepsins I-a, III-1, III-2, III-3, and C, respectively. Pepstatin, diazoacetyl-DL-norleucine methyl ester (DAN), 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), and p-bromophenacyl bromide inhibited each pepsin. Amino acid compositions of the pepsinogens and pepsins were determined. Pepsinogen C and pepsin C were distinct from the other pepsinogens and pepsins in their high ratios of glutamic acid to aspartic acid, and leucine to isoleucine. Amino acid sequences of the amino (N)-terminal 14 residues of pepsinogens were determined by the manual Edman procedure. One to three substitutions of amino acids were observed in the 14-residue segments among the pepsinogens except for pepsinogen C. There were 7 amino acid substitutions between pepsinogens C and III-3. These results suggest that the amino acid substitutions in the N-terminal region contribute considerably to the heterogeneity of pepsinogens.

Amino Acid Sequence↗
PubMed1967

Pepsin D. A minor component of commercial pepsin preparations.

Methods are described for the isolation and purification of pepsin D, an enzyme which accounts for about 10% of the enzymic activity in commercial preparations of pepsin. Pepsin D is similar to pepsin in having a molecular weight of about 35000, the same C-terminal amino acid sequence, and an N-terminal isoleucine residue. It differs in having no phosphate residue. Pepsin D is similar to pepsin in its ability to digest haemoglobin, acetyl-l-phenylalanyl-l-di-iodotyrosine and gelatin but it is twice as active as pepsin in the clotting of milk. It has the same specificity as pepsin in its action on the B-chain of oxidized insulin. It is probable that the pepsin D in commercial preparations of pepsin arises from the activation of gastric pepsinogen D.

Amino Acid Sequence↗
PubMed1991

[Do ulcerogenic pepsins exist? Characterization of polymorphism of human pepsins].

Studies on the role of pepsin in the pathogenesis of peptic ulcer are hampered by lack of suitable methods for the separation, characterization and quantification of different forms of pepsin. As a contribution toward solving this problem, we have developed several electrophoretic systems which exhibit a high resolving power for pepsins and their precursors, pepsinogens. When human gastric juices were fractionated by discontinuous electrophoresis at pH 3.7 in polyacrylamide gel, pepsin was resolved into as many as 20 distinct forms: 3 major isoenzymes (C, A4, A3), 4 less abundant isoforms (C', A6, A4', A3' or A2), 8-10 minor isoforms of pepsin A1 and at least 3 dimeric isoforms of pepsin A (Ad). All the pepsin components were identified by means of bidimensional electrophoresis. Pepsinogens from gastric mucosa were separated by discontinuous electrophoresis at pH 5.5 in the first dimension, then converted to pepsins in the gel by acidification and resolved by electrophoresis at pH 3.7 in the second dimension. Based on discontinuous electrophoresis at low pH, a much needed method was elaborated for the quantification of different forms of pepsin(ogen) within the stomach. This technique will allow detailed clinical studies which may provide insight into the question whether or not there are specific forms of pepsin that are associated with peptic ulcer.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1984

Adsorption of pepsin by aluminum hydroxide II: Pepsin inactivation.

Pepsin adsorbed on gibbsite or boehmite, non-acid-reactive forms of aluminum hydroxide, had a significantly lower activity than pepsin in solution. IR and desorbed pepsin activity studies showed that the reduced activity of adsorbed pepsin was not due to denaturation of pepsin on adsorption. Steric occlusion of the active site, following pepsin adsorption, was responsible for the lower activity of pepsin adsorbed on gibbsite. The porous morphology of boehmite caused diffusional resistance and steric exclusion, contributing to the decreased activity of adsorbed pepsin. The specific inactivation of pepsin by adsorption on aluminum hydroxide may be important in ulcer therapy.

Adsorption↗
PubMed2003

Comparative pepstatin inhibition studies on individual human pepsins and pepsinogens 1,3 and 5(gastricsin) and pig pepsin A.

Human gastric juice contains 3 major proteolytic components (pepsins1,3 and 5 or gastricsin). Pepsin 1 is increased in peptic ulcer and it's properties are relatively poorly understood. Studies with pepstatin the highly specific aspartic-protease inhibitor have therefore been carried out on individual active and proenzymes to assess any enzymic similarities. Human pepsin 1 was inhibited with high affinity similar to pepsin 3, whereas pepsin 5(gastricsin) was at least 40 times less sensitive. Inhibition of human pepsinogens 1,3 and 5 and pig pepsinogen A showed similar trends to the active enzymes. Studies using Sephadex gel filtration showed that pepstatin does not bind to pepsinogens and inhibition arises from pepstatin binding the pepsins released upon activation. Pepstatin inhibition was shown to be relatively independent of pH between 1.5 and 3.8 although at higher pH inhibition was less effective. The evidence suggests that pepsin 1 is similar to pepsin 3 and pepstatin inhibits by a one to one molecular binding to the active site. The explanation for the reduced affinity of pepstatin to pepsin 5(gastricsin) needs further study by co-crystallisation X-ray analysis.

Animals↗
PubMed1976

Spectroscopic studies of pepsin and its complex with Streptomyces pepsin inhibitor.

Binding of Streptomyces pepsin (EC 3.4.23.1) inhibitor to the active site of pepsin causes a characteristic ultraviolet difference spectrum having a trough around 298 nm which suggests that tryptophan residue(s) are involved in a decreased refractive index or different charge density. The fluoreschat of the pepsin-inhibitor complex. Relatively large circular dichroism (CD) spectrum change at 280--310 nm was observed upon binding of the inhibitor. Solvent perturbation difference spectra of pepsin alone and the pepsin-inhibitor. Solvent perturbation difference spectra of pepsin alone and the pepsin-inhibitor complex obtained with 20% ethylene glycol as perturbant showed that the exposed 2.5 tryptophan residues were not buried upon binding of the inhibitor, whereas 1.5 tyrosine residues were buried. It is speculated that the microenvironmental change around tryptophan residue(s) which are not located at the inhibitor binding site is induced upon binding of the inhibitor.

Binding Sites↗
PubMed1975

Fluorescence studies on the active sites of porcine pepsin and Rhizopus-pepsin.

Fluorescence studies on the interaction, with porcine pepsin, of oligopeptides bearing a mansyl (Mns, 6-(N-methylanilino)-2-naphthalenesulfonyl) or dansyl (Dns, 5-dimethylaminonaphthalene-1-sulfonyl) group at the NH2 or COOH terminus have provided further evidence showing that the probe group is drawn into the extended active site largely as a consequence of the specific binding of the peptide portion of the substrate. The active site does not appear to have appreciable intrinsic affinity for the mansyl or dansyl group, and the principal contribution to the specific peptide-protein interaction is provided by the sensitive L-phenylalanyl-L-phenylalanyl (Phe-Phe) unit of the substrates tested. The pepsin inhibitor pepstatin can displace substrates such as Mns-(Gly)n-Phe-Phe-OR or Gly-Gly-Phe-Phe-NHNH-Mns from the active site of porcine pepsin; in these circumstances the mansyl group is bound weakly at a separate, nonspecific locus, distinct from the active site, which can accept the mansyl group of Mns-Gly-Gly-OR or mansylamide. In the interaction with substrates such as Mns-(Gly)n-Phe-Phe-OR or Dns-(Gly)n-Phe-Phe-OR, the above conclusions for porcine pepsin also apply to Rhizopus-pepsin. With substrates such as Gly-Gly-Phe-Phe-NHNH-Mns, however, the active site of Rhizopus-pepsin shows less affinity for the fluorescent probe group than does that of porcine pepsin, suggesting structural differences between the two acid proteinases in the region of their extended active sites which bind the COOH-terminal portion of small oligopeptide substrates.

Anilino Naphthalenesulfonates↗
PubMed2000

Structural basis for the inhibition of porcine pepsin by Ascaris pepsin inhibitor-3.

The three-dimensional structures of pepsin inhibitor-3 (PI-3) from Ascaris suum and of the complex between PI-3 and porcine pepsin at 1. 75 A and 2.45 A resolution, respectively, have revealed the mechanism of aspartic protease inhibition by this unique inhibitor. PI-3 has a new fold consisting of two domains, each comprising an antiparallel beta-sheet flanked by an alpha-helix. In the enzyme-inhibitor complex, the N-terminal beta-strand of PI-3 pairs with one strand of the 'active site flap' (residues 70-82) of pepsin, thus forming an eight-stranded beta-sheet that spans the two proteins. PI-3 has a novel mode of inhibition, using its N-terminal residues to occupy and therefore block the first three binding pockets in pepsin for substrate residues C-terminal to the scissile bond (S1'-S3'). The molecular structure of the pepsin-PI-3 complex suggests new avenues for the rational design of proteinaceous aspartic proteinase inhibitors.

Amino Acid Sequence↗
PubMed1977

Anhydride intermediates in catalysis by pepsin: is pepsin an enzyme with two active sites?

By the use of sulfite ester substrates together with hydroxylamine as a highly reactive trapping agent, we have been able to obtain strong evidence for the intermediacy of enzyme-bound anhydride species in the pepsin-catalyzed hydrolysis of these substrates. From our observations that in the trapping experiments hydroxamate functions are introduced at the beta-carboxylates of Asp-32, Asp-215 and at least one additional Asp residue, it appears that several reactive carboxylate species can function as nucleophiles against sulfite esters, leading to the formation of anhydride species. Because the location of the hydroxamate incorporated into pepsin other than at the Asp-32 and Asp-215 residues is unknown, it remains conceivable that, at least for the action of pepsin on sulfite substrates, there are two distinct active site regions. If the possibility is considered that peptides possessing common amino-terminal residues but different acyl residues may bind productively in different fashions so that in some cases the beta-carboxylate of Asp-32 acts as the attacking nucleophile while in others the beta-carboxylate of Asp-215 acts in this way (as has been observed for sulfites), much of the confusion in the literature concerning the reactions of pepsin with peptidase may be explained.

Amino Acids↗
PubMed1989

Induction of anti-pepsin antibodies after immunization with pepsin-extracted collagen.

Immunization with pepsin-extracted human type II collagen purified by different precipitation steps, although not showing any contamination with the enzyme on SDS-polyacrylamide gel electrophoresis, resulted in the generation of antibodies to the enzyme in addition to an immune response to collagen. These antibodies could be removed by immune absorption on a pepsin affinity column, leaving reactivity to type II collagen unaltered. High performance liquid chromatography on hydroxylapatite columns indicated that pepsin remained associated with the collagen molecules even after repeated precipitation and coeluted with a fraction of the collagen preparation. These results demonstrate that pepsin-extracted collagens may contain minimal amounts of the enzyme. On immunization, these impurities may induce the formation of unwanted antibodies, which might simulate a false specificity of the antibody preparation.

Antibodies↗
PubMed1982

Monkey pepsinogens and pepsins. VI. One-step activation of Japanese monkey pepsinogen to pepsin.

When Japanese monkey pepsinogen was activated at pH 2.0 in the absence of pepstatin, the activation segment of the amino(N)-terminal 47 residues was released as a single intact polypeptide. This clearly shows that the pepsinogen was activated to pepsin directly. This direct activation was called a 'one-step' process. On the other hand, when pepsinogen was activated at pH 2.0 in the presence of pepstatin, an appreciable amount of pepsinogen was converted to an intermediate form between pepsinogen and pepsin, although a part of pepsinogen was activated directly to pepsin. The intermediate form was generated by releasing the N-terminal 25 residues of pepsinogen. This activation through the intermediate form is thought to be a 'two-step' or 'stepwise-activating' process involving a bimolecular reaction between pepstatin-bound pepsinogen and free pepsin.

Animals↗
PubMed1976

Synthetic peptides for chymosin and pepsin assays: pH effect and pepsin independent-determination in mixtures.

Peptide I [H-Phe-Gly-His-Phe(NO2)-Phe-Ala-Phe-OMe] hydrolyzed by chymosin with kcat=.3+/-.3 s-1 and KM=7+/-3 mM (pH 4.7) inhibited competitively peptide II [H-Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe] hydrolysis by chymosin with KI=.23 +/- .12 mM at pH 4.7. In reference conditions (.4 mM peptide, .01 M acetate buffer pH 4.7), the specific activities of porcine pepsin and chymosin on peptide I were 470 +/- 70 nM S-1 and .8 nM S-1 per mg of enzyme. This difference in specific activity for peptide I allowed development of a chymosin-independent pepsin assay for mixtures of these enzymes. In addition, peptide II with a specific activity of 2400 +/- 300 nM S-1 and 154 +/- 20 nM S-1 per mg of porcine pepsin and chymosin provides an alternative to measurement of milk clotting for measurement of chymosin- and pepsin-like activities in commercial rennets. Hydrolysis products of peptide II by chymosin exhibited one ionized group of apparent pK of 3.5 +/- .2 and a molar absorption coefficient change of 1000 +/- 100 at pH 4.7 and at 310 nm. From measurements of the kinetic constants, kcat and KM, from pH 2.5 to 7 with peptide II, chymosin activity depends on the protonation of one group of apparent pK 5.3 +/- .2 in the free enzyme. Rennet powder proved to be fairly stable after a 17-month storage at 4 C. Within the same period, a crystalline chymosin solution kept at --18 C lost 30 to 50% of its activity.

Animals↗
PubMed1999

Pepsin as a catalyst for peptide synthesis: formation of peptide bonds not typical for pepsin substrate specificity.

Porcine pepsin in water solutions containing 15-28% of dimethylformamide at pH 5 and 20-37 degrees C catalysed the formation of peptide bonds between Z-Ala-Ala-Phe-OH and various amino acid or peptide derivatives. Substrate binding subsite S1' of pepsin demonstrated broad specificity in these reactions but revealed a certain preference for hydrophobic amino acid residues, including non-proteinous homophenylalanine, p-nitrophenylalanine, S-methylcysteine, as well as for those that contained, in addition to the hydrophobic elements, a group capable of donating a hydrogen bond, e.g. o-nitrotyrosine. This observation increases the range of peptides that might be prepared by pepsin-catalysed synthesis.

Amino Acid Sequence↗