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PubMed2006

Purification and characterization of chymosin and pepsin from kid.

The objective of this work was to study the characteristics of the gastric aspartic proteinases chymosin and pepsin which are constituents of the kid rennet. The two enzymes were extracted from abomasal tissue of one kid from a local indigenous breed, separated from each other by DEAE-cellulose chromatography and then were purified by gel filtration and anion-exchange chromatography. The molecular weights of the purified kid chymosin and pepsin as determined by gel filtration were 36 kDa and 40 kDa respectively. The isoelectric point of kid chymosin was as multiple forms of 3-6 zones at pH 4.6-5.1, while that of kid pepsin was at pH < or =3.0. Kid pepsin contained 0.37 molecules phosphorous per molecule and was totally inhibited by 5 muM pepstatin A, being more sensitive than kid chymosin. Both enzymes were almost equally as proteolytic as calf chymosin on total casein at pH 5.6. Kid pepsin activity was more pH and temperature dependent than kid chymosin activity. In comparison with the calf chymosin temperature sensitivity, the order of increased sensitivity was: calf chymosin <kid chymosin <kid pepsin.

Abomasum↗

PubMed1983

Isolation and characterization of pepsin-solubilized human basement membrane (type IV) collagen peptides.

Native type IV collagen was isolated from human placental tissue by pepsin digestion, fractional salt precipitation, reduction and alkylation, a second pepsin digestion, and chromatography on diethylaminoethyl- and carboxymethyl-cellulose. After denaturation, 10 distinct peptides were isolated from this material by molecular sieve, ion-exchange, and high-performance liquid chromatography. All of the peptides were found to have amino acid compositions characteristic of type IV collagen. Analysis of the eight major peptides by amino-terminal amino acid sequencing and by cyanogen bromide and tryptic peptide mapping has revealed the manner in which they are derived from type IV collagen. Pepsin liberates two large peptides by attacking non-triple-helical regions, one derived from the alpha 1 (IV) chain (F2, Mr 90 000) and one derived from the alpha 2 (IV) chain (F3, Mr 75 000). The alpha 1 (IV)-derived F2 peptide is also represented in the pepsin digest by amino-terminal and carboxy-terminal subfragments [F4c (Mr 41 000) and F4a (Mr 60 000)], as is the alpha 2 (IV)-derived F3 peptide [F5 (Mr 28 000) and F4b (Mr 50 000), respectively]. These findings indicate that the molecular regions from which the larger peptides are derived in themselves contain pepsin-sensitive (non-triple-helical) domains. In addition, several of the peptides examined were found to be present in two slightly different forms, suggesting that closely adjacent pepsin-sensitive sites often exist within the type IV collagen molecules. The methods outlined here provide a reliable means by which identifiable type IV collagen peptides can be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2003

Comparison of a salivary/sputum pepsin assay with 24-hour esophageal pH monitoring for detection of gastric reflux into the proximal esophagus, oropharynx, and lung.

The purpose of this study was to determine whether measurement of salivary/sputum pepsin could be used as a surrogate marker for detecting gastroesophageal reflux using 24-hr esophageal pH monitoring as the gold standard. Patients with gastroesophageal reflux symptoms underwent simultaneous 24-hr esophageal pH monitoring and collection of saliva and sputum samples for pepsin measurement using a recently developed assay. In all, 16 patients provided 19 positive (10.6%) and 161 negative pepsin assays. The mean pH values for the positive pepsin samples were lower then the negative samples at both the proximal [5.34 (95% CI, 4.94-5.75) vs 6.12 (95% CI, 6.03-6.20; P < 0.01)] and distal [4.97 (95% CI, 4.61-5.33) vs 6.03 (95% CI, 5.92-6.15; P < 0.01)] pH probes. Proximal esophageal reflux was not detected in patients who had a negative pepsin assay (N = 12); in contrast, proximal esophageal reflux was documented in three of four patients with a positive assay. In conclusion, detection of pepsin in the saliva and/or sputum may provide a noninvasive method to test for the proximal reflux of gastric contents.

Esophagoscopy↗

PubMed1975

The effect of acid proteinase inhibitors on chicken pepsin.

1. The activity of chicken pepsin was partially inhibited by dimethyl-(2-hydroxy-5-nitrobenzyl)sulphonium bromide, but was unaffected by p-bromophenacyl bromide. 2. In the presence of Cu2+, diazoacetylnorleucine methyl ester completely inactivated chicken pepsin with the incorporation of 1 mol/mol. The mechanism of the reaction was similar to that with pig pepsin. 3. Chicken pepsin was completely inactivated by 2-diazo-4-bromoacetophenone in the presence of Cu2+. 4. Chicken pepsin was almost completely inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane at 25 degrees C, 3-4mol of inhibitor/mol being incorporated. The reaction at 10 degrees C was investigated briefly. 5. Calf chymosin was inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane at 10 degrees C, the incorporation of 1 mol/mol being required for complete inhibition. 6. The characteristics of the reactions of chicken pepsin with the above compounds were compared with those of other acid proteinases.

Acetophenones↗

PubMed1978

The preparation and purification of individual human pepsins by using diethylaminoethyl-cellulose.

A procedure was devised for isolating human pepsins 1, 2, 3 and 5 from gastric juice by repetitive column chromatography on DEAE-cellulose. The combined yields in four different experiments varied from 14% to 90% of the total peptic activity of the starting material. The isolated individual pepsins were shown to behave as single homogeneous proteins on agar-gel electrophoresis at pH 5.0 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. There is preliminary evidence, requiring further study, of two other pepsins, one migrating between pepsins 1 and 2 and the other a pepsin-3 component associating closely with pepsin 5 on chromatography.

Chromatography, DEAE-Cellulose↗

PubMed1998

Molecular cloning, expression and characterization of an Ascaris inhibitor for pepsin and cathepsin E.

Molecular cloning of a cDNA for a pepsin inhibitor in the round worm, Ascaris suum, was achieved. The ORF was found to encode a 20-residue potential signal peptide and a 149-residue inhibitor moiety. Northern analysis showed the mRNA for the inhibitor to be expressed in the body wall and not in the viscera. To obtain the active inhibitor, we constructed a yeast expression vector, pYES2API, containing the inducible galactosidase promoter and a DNA fragment encoding a fusion protein of the yeast alpha-factor leader and the Ascaris pepsin inhibitor. The active inhibitor was secreted in the culture medium, the yield being approximately 3 mg x l(-1) x day(-1), and purified by a two-step procedure that included HPLC. The inhibitor inactivated pepsin A and cathepsin E almost completely at amounts equimolar with the enzymes, but was 100-fold less effective against pepsin C and did not act on cathepsin D and renin. Ki values for the inhibition of pepsin A and cathepsin E were in the nanomolar range below pH 5. Since the inhibitor activity was lost by modification of specific Lys residues, including Lys110, an electrostatic interaction between these Lys residues and Asp/Glu residues of pepsin A or cathepsin E was thought to be essential for the inhibition.

Amino Acid Sequence↗

PubMed1996

Effects of aging and gastritis on gastric acid and pepsin secretion in humans: a prospective study.

BACKGROUND & AIMS: Recent studies suggesting that gastric secretion does not decrease with aging included few elderly individuals and measured only acid secretion. The aims of this study were to measure gastric acid and pepsin output in 206 health Americans (age range, 18-98 years) and to compare secretion rates with gastric histology. METHODS: Immediately after basal and pentagastrin-stimulated acid and pepsin outputs were measured, oxyntic biopsy samples were obtained. RESULTS: Gastric acid and pepsin output rates were similar in young (age range, 18-34 years) and middle-aged (age range, 35-64 years) groups. Stimulated acid output was reduced approximately 30% in the elderly (age range, 65-98 years). However, after adjustment for histology, Helicobacter pylori infection, and other variables, age had no independent effect on acid output. The decline in acid secretion in the elderly was primarily related to a higher prevalence of chronic atrophic gastritis and a lower prevalence of smoking. Pepsin output was reduced by approximately 40% in the elderly. After adjustment for other variables, age remained a robust predictor of reduced pepsin output. CONCLUSION: Although advancing age has no independent effect on gastric acid secretion, it is associated with reduced pepsin output independent of atrophic gastritis, H. pylori infection, and smoking.

Adolescent↗

PubMed1971

Bifunctional inhibitors of pepsin.

Two bifunctional reagents designed to probe the active site of pepsin and other acid proteinases are described. One of these, the bisdiazoketone 1,1-bis(diazoacetyl)-2-phenylethane inactivates pepsin at pH 5.0 much more rapidly than the corresponding monodiazoketon 1-diazoacetyl-2-phenylethane, whereas the other, the bromodiazoketone dl-1-diazoacetyl-1-bromo-2-phenylethane is less effective in this regard. The inactivation is greatly accelerated by the presence of Cu(II), and the pH dependence of the process is consistent with the interaction of the enzyme with the metal complex of the carbene derived from the reagent. The bisdiazoketone appears to react stoichiometrically with pepsin in a 1:1 ratio to form a product whose apparent molecular size is the same as that of untreated pepsin. The inactivation of pepsin by the bromodiazoketone is accompanied by the release of stoichiometric amounts of bromide ions and the formation of a major product whose apparent size is similar to that of pepsin, and a minor component larger than the untreated enzyme.

Azo Compounds↗

PubMed1976

Hydrolysis and transpeptidation of peptide substrates by acetyl-pepsin.

Treatment of swine pepsin with acetylimidazole to acetylate approximately five of its 16 tyrosyl residues causes a significant enhancement of catalytic efficiency (kcat/Km) toward substrates such as dansyl-glycyl-glycyl-L-phenylalanyl-L-phenylalanine 3-(4-pyridyl)propyl ester and benzyloxy-carbonyl-(glycyl)n-p-nitroLphenylalnyl-Lphenylalanyl-L-tyrosine (where n = 0, 1,2). Stopped-flow kinetic studies, under conditions of enzyme excess, with the dansyl peptide have shown that, as with untreated pepsin, the rate-limiting step in the over-all catalytic process is associated with the decomposition of the first detectable enzyme-substrate complex, whose dissociation constant is approximately equal to the Km found in steady-state kinetic experiments. With substrates of the type benzoyl-(glycyl)n-nitro-L-phenylalanyl-L-tyrosine, an increase in the chain length of the peptide leads to an increase in the value of kcat/Km, supporting the view that secondary enzyme-substrate interactions may produce at the extended active site conformational changes that are reflected in higher catalytic efficiency. This effect is more marked with acetyl-pepsin than with untreated pepsin, and suggests that the conformational mobility of the active site is increased by partial acetylation. Acetyl-pepsin is less effective than untreated pepsin in catalyzing transpeptidation reactions in which acetyl-L-phenylalanyl-L-tyrosine and benzyloxycarbonyl-(glycyl)n-p-nitro-L-phenylalanine are the reactants; this finding is consistent with the more rapid hydrolysis of the product of transpeptidation.

Acetylation↗

PubMed2005

Protein profile of pepsin-digested carious and sound human dentine.

The purpose of this study was to describe the protein profile of pepsin-digested carious and sound dentine using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Carious and sound dentine powder was decalcified using 10% EDTA at pH 7.4 for 48 h. The decalcified pellet was digested using pepsin at pH 2 under sequenced conditions: at 4 degrees C for 24 h, a further 24 h at 23 degrees C, and finally for 24 h at 37 degrees C. After every step, the soluble fraction was separated by centrifugation and analyzed in 15% SDS-PAGE. Two bands at 56 and 62 kDa could be observed in carious dentine digests and were considered specific carious bands. Similar bands could be observed in sound dentine samples, but only after pepsin digestion at higher temperatures (23 degrees C and 37 degrees C). Pepsin digests non-helical collagen and the triple helix structure of collagen is lost when the temperature rises. The bands at 56 and 62 kDa in sound dentine specimens thus represent pepsin-cleaved collagen. There is a possibility that the specific carious bands in carious dentine represent collagen decomposed in a manner similar to the way pepsin digests native dentine collagen at 23 degrees C and 37 degrees C.

Collagen↗

PubMed2001

In vitro refolding of porcine pepsin immobilized on agarose beads.

Since in vitro refolding of pepsin has long been attempted without success, it has been suspected that pepsin has no intrinsic refolding ability. In the present study, in order to eliminate unfavorable intermolecular interactions bringing about aggregation and autoproteolysis, we immobilized pepsin onto agarose beads. This technique enabled us to search extensively for appropriate refolding conditions without limitation of the refolding period. Renaturation of immobilized pepsin was observed exclusively at pH 3-5. This process was extremely slow and reached equilibrium after 300 h. Sixty percent of the proteolytic activity was recovered at pH 5. Addition of salts raised the recovery to 80% but had no significant effect on the refolding rate, suggesting that the salts mainly stabilize the native state of pepsin. This is the first report on the successful in vitro refolding of pepsin.

Animals↗

PubMed1995

Aspirin renders the oesophageal mucosa more permeable to acid and pepsin.

OBJECTIVE: To examine the effects of aspirin on the oesophageal mucosa and on acid- and pepsin-induced oesophagitis. DESIGN AND METHODS: The effects both of intraluminal (18 mg/ml) and of parenteral (100 mg/kg per h) aspirin on an in-vivo rabbit model of oesophagitis induced by acidified pepsin (pH 2) were studied. Oesophageal injury was assessed by macroscopic and microscopic scoring including the cell proliferation immunohistochemical parameter mib1. The mucosal barrier function was determined by hydrogen, potassium and haemoglobin flux rates. RESULTS: Acidified saline alone caused no damage, but the addition of aspirin induced mucosal barrier damage (P < 0.05). The exposure of the oesophageal mucosa to acidified aspirin and then acidified pepsin significantly increased mucosal injury and mucosal barrier dysfunction compared with control experiments (exposure to acidified saline and acidified pepsin). This damage was significantly (P < 0.05) reduced (> 40%) by prostaglandin cotherapy (prostaglandin E2) administered before acidified aspirin exposure. Mucosal damage was less severe (P < 0.05) when the oesophageal mucosa was exposed to a pH 6 aspirin solution. Parenterally administered aspirin also increased the oesophageal damage induced by acidified pepsin compared with control experiments, but the damage was 23% lower than that obtained with intraluminal aspirin. Cell proliferation studies showed a significant increase in the number of positive cells in those experiments with a higher degree of damage and in those treated with prostaglandins. CONCLUSION: Aspirin renders the oesophageal mucosa more permeable to acid and pepsin. These effects are in part pH-dependent and might be partially reversed by prostaglandin E2 cotherapy.

Acids↗

PubMed2003

Structural dissection of alkaline-denatured pepsin.

It has been established in a number of studies that the alkaline-denatured state of pepsin (the I(P) state) is composed of a compact C-terminal lobe and a largely unstructured N-terminal lobe. In the present study, we have investigated the residual structure in the I(P) state in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C-terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N-terminal lobe contributes a substantial amount of additional residual structure to the I(P) state of pepsin. CD spectra indicate in addition that significant nonnative alpha-helical structure is present in the C-terminal lobe of the structure when the N-terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the I(P) state is significantly more complex than that of a fully folded C-terminal lobe connected to an unstructured N-terminal lobe.

Amino Acid Sequence↗

PubMed1992

Pepsin as a catalyst of peptide synthesis. Enzyme co-precipitation with emerging peptide products.

Pepsin successfully catalyzed the synthesis of several peptide derivatives from N-protected di- or tripeptides and amino acid or peptide esters or p-nitroanilides in dimethylformamide-water solutions at pH 4.6. An optimal substrates:pepsin ratio depended on the structure of starting peptides, especially their fit to the substrate binding sites of the enzyme. For hexapeptide Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 formation, an equilibrium yield was attained at 1:3.10(5) enzyme-substrates ratio that indicated high efficiency of pepsin in synthesis reactions. In the course of the equilibrium peptide synthesis, pepsin gradually disappeared from the liquid phase due to its entrapment within a gel, formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin, so far as inert proteins, lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin, an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.

Amino Acid Sequence↗

PubMed1994

The gastroduodenal mucus barrier and its role in protection against luminal pepsins: the effect of 16,16 dimethyl prostaglandin E2, carbopol-polyacrylate, sucralfate and bismuth subsalicylate.

Mucus and bicarbonate secretions have been widely implicated as an important pre-epithelial protective barrier against autodigestion of the gastric mucosa by acid and pepsin. Evidence from several independent studies shows there is a continuous layer of resilient viscoelastic mucus gel adherent to the surface of the gastroduodenal mucosa. The median thickness of the adherent gastric mucus layer in humans is 180 microns, range 50-450 microns. The epithelial bicarbonate secretion permeates the unstirred matrix of mucus gel neutralizing luminal acid and establishing a pH gradient within the gel. In the duodenum, evidence supports the mucus bicarbonate barrier as a major protective mechanism against acid aggression. The adherent mucus gel, by acting as an effective 'permeability' barrier to pepsin, protects the underlying sensitive mucosa from digestion. However, pepsin slowly digests mucus gel at its luminal surface to produce soluble degraded mucin. In a rat gastric damage model in vivo, pepsin in excess digests the gastric mucus barrier sufficiently rapidly to outweigh new mucus secretion and lead to breaching of the mucus barrier with the formation of small punctate ulcers in the epithelium accompanied by mucosal haemorrhage. The mucus secretagogue 16,16 dimethyl prostaglandin E2 and the muco-adhesive carbopol-polyacrylate both fully protected the mucosa against pepsin damage by enhancing the protective properties of the mucus barrier. Sucralfate and bismuth subsalicylate were partially effective in protection against pepsin damage but this protection was mainly mediated at the level of the mucosa. In peptic ulcer disease, there is increased mucolytic (mucus degrading) activity in gastric juice and this is associated with an impaired mucin polymeric structure and a weaker mucus barrier.(ABSTRACT TRUNCATED AT 250 WORDS)

16,16-Dimethylprostaglandin E2↗

PubMed1992

Bile acids do not modify the effects of pepsin on the fine structure of human oesophageal epithelium.

Oesophageal mucosal specimens (n = 250) were taken from 25 normal subjects (14 females, 11 males; median age 52 years; range 19-63 years) and incubated in physiological saline, pepsin and bile acid solutions to determine whether conjugated bile acids modify the epithelial cytotoxic action of pepsin. Short (5 min) and long (22 min) incubations were carried out and the results were assessed by transmission electron microscopy. Six different parameters of epithelial damage were scored (0-4) by a single 'blinded' pathologist for each of four epithelial layers. The scores after incubation in saline (pH 7 and titrated to pH 2 with HCl) were not different from those of the controls (P = 0.35). Both pepsin and bile acids (pH 2) caused more damage than saline at pH 2 (P less than 0.001) which was similar for the two substances (P = 0.136). Conjugated bile acids in combination with pepsin (pH 2) did not alter the overall extent or pattern of damage caused by pepsin alone (pH 2); P = 0.142). Conjugated bile acids, in concentrations commonly encountered during gastro-oesophageal reflux, did not appear to modify the cytopathic effects of pepsin on oesophageal mucosal cells in vitro. Conjugated bile acids may not be important in the pathogenesis of oesophagitis in patients with acid/peptic gastro-oesophageal reflux.

Adult↗

PubMed1971

Pepsin secretion, gastric motility and mucosal blood flow in the anaesthetized cat.

1. There is a weak relationship between pepsin output and gastric mucosal blood flow (MBF) during vagal stimulation. When compared with the strong relationship between H(+) output and MBF, this suggests little dependence of pepsin secretion on MBF.2. Gastric MBF per unit H(+) output was significantly greater during vagal stimulation, when H(+) secretion was accompanied by pepsin secretion, than during pentapeptide stimulation when no pepsin was secreted.3. Pepsin output was not reduced during splanchnic stimulation whereas MBF and acid output were, when blood pressure was kept constant.4. Splanchnic stimulation did not produce any pepsin secretion.5. Gastric motor responses to vagal stimulation and to I.A. injections of acetylcholine were equally reduced by splanchnic stimulation.

Acetylcholine↗

PubMed1977

The effects of somatostatin and metiamide on tachyphylaxis of pentagastrin stimulated gastric acid and pepsin secretion in the conscious cat.

1. Pentagastrin stimulated gastric acid and pepsin secretions show parallel rates of tachyphylaxis in the conscious cat. The responses to histamine show only slight tachyphylaxis. 2. Somatostatin 10 microng.kg(-1).hr(-1) inhibits pentagastrin but not histamine stimulated acid secretion and inhibits pentagastrin stimulated pepsin secretion. 3. The inhibition of pentagastrin stimulated acid and pepsin secretion by Somatostatin delays the tachyphylaxis of these responses, but the rates of tachyphylaxis when they do subsequently occur are identical. 4. Metiamide 10 mg-kg(-1)-hr(-1) equally inhibits histamine and pentagastrin stimulated acid secretion but does not inhibit pentagastrin stimulated pepsin secretion. 5. Inhibiton of acid secretion during metiamide infusion neither prevents nor delays acid nor pepsin tachyphylaxis. 6. It is suggested that tachyphylaxis of acid and pepsin secretion is a gastrin receptor phenomenon and that Somatostatin occupies or modifies the behaviour of these receptors, preventing tachyphylaxis. Metiamide, however, exerts its action only on the histmine H2-receptor and not the gastrin receptor mechanism, and this apparently does not prevent or delay acid tachyphylaxis.

Animals↗