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PubMed1958

Comparative studies of three methods for measuring pepsin activity.

Comparison has been made of a simple method originated by Absolon and modified in our laboratories for assay of proteolytic activity using RISA (radioactive iodinated serum albumin-Abbott Laboratories), with the commonly used photometric methods of Anson and Kunitz. In this method, pepsin was incubated with an albumin substrate containing RISA, followed by precipitation of the undigested substrate with trichloroacetic acid and measurement of radioactive digestion products in the supernatant fluid. The I(131)-albumin bond was shown in the present studies to be altered only by the proteolytic activity, and not by the incubation procedures at various values of pH. Any free iodine present originally in the RISA was removed by a single passage through a resin column (amberlite IRA-400-C1). Pepsin was shown to be most stable in solution at a pH of 5.5. Activity of pepsin was shown to be maximal when it was incubated with albumin at a pH of 2.5. Pepsin activity was shown to be altered in the presence of various electrolytes. Pepsin activity measured by the RISA and Anson methods as a function of concentration or of time of incubation indicated that these two methods are in good agreement and are equally sensitive. Consistently smaller standard errors were obtained by the RISA method of pepsin assay than were obtained with either of the other methods.

Pepsin A↗

PubMed1984

Adsorption of pepsin by aluminum hydroxide I: Adsorption mechanism.

Adsorption of pepsin by gibbsite and boehmite, non-acid-reactive forms of aluminum hydroxide, was observed and related to the surface area of the adsorbent. Adsorption was pH dependent, with maximum adsorption occurring between pH 2.7-3.3 for gibbsite and pH 2.7-4.3 for boehmite. Electrostatic attraction was an important adsorption mechanism at the pH conditions encountered in the GI tract; the isoelectric point of pepsin was approximately 1, giving it a negative charge, and the point of zero charge for the adsorbents was greater than 9, giving them a positive charge. However, the pH-adsorption profile can not be fully explained by electrostatic considerations. Desorption studies indicate the importance of specific adsorption because pepsin was not desorbed by washing with acidified water, but was partly desorbed by exchange with phosphate. The IR spectrum of adsorbed pepsin also suggested that specific adsorption of pepsin occurred through anionic ligand exchange involving carboxylate groups of pepsin and surface aluminum ions.

Adsorption↗

PubMed1999

Different sensitivity of recombinant Aspergillus niger phytase (r-PhyA) and Escherichia coli pH 2.5 acid phosphatase (r-AppA) to trypsin and pepsin in vitro.

Proteolysis of two purified recombinant enzymes, namely, the Aspergillus niger phytase (r-PhyA) and the Escherichia coli pH 2.5 acid phosphatase (r-AppA), by pepsin and trypsin was investigated in this study. After r-PhyA and r-AppA were incubated with different concentrations of pepsin or trypsin, their residual phytase activities and amounts of inorganic phosphorus released from soybean meal were determined. Both enzymes retained more than 85% of their original activities at the trypsin/phytase ratios (w/w) 0.001 and 0. 005, while r-AppA and r-PhyA lost 60 and 20% of the original activity at the ratio of 0.01 or 0.025, respectively. In contrast, there was a 30% increase in phytase activity after r-AppA was incubated with pepsin at the ratios of 0.005 or 0.01. Meanwhile, r-PhyA lost 58 to 77% of its original activity under the same conditions. Trypsin and pepsin affected the hydrolysis of phytate phosphorus from soybean meal by r-AppA and r-PhyA in a similar way to their residual phytase activities. All of these in vitro proteolyses were confirmed by SDS-PAGE analysis. Our results demonstrate different sensitivities of r-AppA and r-PhyA to trypsin and pepsin, suggesting active trypsin resistant r-PhyA and pepsin resistant r-AppA polypeptides.

6-Phytase↗

PubMed1980

The effect of atropine and metiamide on pepsin secretion of the rat.

The inhibition of pepsin secretion by metiamide and atropine was studied by perfusing the stomach lumen of anaesthetized rats. Atropine was more effective than metiamide in the inhibition of pepsin secretion induced by pentagastrin or carbachol. Perfusion of the stomach with a weak acid solution stimulated pepsin secretion, which was only abolished by atropine. Metiamide was ineffective. It is postulated that (1) hydrogen ions probably stimulate pepsin secretion, (2) a cholinergic-like reflex may be involved in this stimulation, (3) anticholinergics are direct and strong inhibitors of pepsin secretion, (4) histamine H2-receptor antagonists probably inhibit pepsin secretion indirectly via reduction of acid output.

Animals↗

PubMed1994

Pepsin concentration in gastroduodenal biopsy homogenates in chronic ulcer disease.

A modification of Berstad's spectrophotometric method was tested and proved capable of detecting pepsin concentrations in mucosal perendoscopic biopsy homogenates. The relationship between this parameter and pepsin in gastric juice and pepsinogen group I in serum and in biopsy homogenates was analyzed. From the biochemical point of view, the assay was found sufficiently accurate. Mucosal pepsinogen group I, but not mucosal pepsin, concentration was found higher in gastric and duodenal ulcer patients than in controls. Patients with corpus-fundic gastric ulcer showed significantly lower mucosal pepsin and mucosal pepsinogen group (PG) I. Aging and smoking did not influence either parameter but male duodenal ulcer subjects presented higher mucosal pepsinogen group I concentration. The lack of any relationship between serum and mucosal PG I and between pepsin in gastric juice and in mucosa raises a question, at least in methodological terms, about the validity of using serum pepsinogen group I and pepsin as indicators of peptic output.

Adult↗

PubMed1983

A new substrate for porcine pepsin possessing cryptic fluorescence properties.

A fluorescent substrate for porcine pepsin, 50-dimethylaminonaphthalene-1-sulfonyl (Dns)-Ala-Ala-Phe-Phe-3-[4-(N-CH3)-pyridyl]propyl-1-oxy ester has been synthesized. It is stable, soluble from pH 1 to 7, and is readily hydrolyzed by pepsin with values of 288 (+/- 40) s-1 for kcat, 0.039 mM (+/- 0.005) for Km, and 7510 s-1 mM-1 (+/- 500) for kcat/Km in sodium formate, pH 3.1. Kinetic studies were carried out by following the increased fluorescence (300-nm excitation, 525-nm emission) as hydrolysis occurred. The products of hydrolysis were identified and established that the peptide bond between the phenylalanine residues is cleaved by pepsin. The inhibition of pepsin catalysis by pepsinogen (1-12) activation peptide was studied in order to compare the inhibition of the reaction of pepsin with Dns-Ala-Ala-Phe-Phe-OP4P-CH3+ with that obtained by the standard milk-clotting assay. The inhibition results were comparable. Dns-Ala-Ala-Phe-Phe-OP4P-CH3+ should be a valuable tool for studies of pepsin because of its solubility over an extended pH range, its excellent turnover rate, and the ease with which the hydrolysis can be followed.

Animals↗

PubMed1981

Cleavage of human serum immunoglobulin G by an immobilized pepsin preparation.

In order to obtain an efficacious and safe immunoglobulin G (IgG) preparation for intravenous use, the digestion of IgG with an immobilized pepsin (EC 3.4.23.1) preparation was studied. Thus, pepsin was immobilized onto glutaraldehyde-activated AH-Sepharose 4B under acidic conditions. THe enzymatic properties, such as proteolytic activity, pH-activity profile and heat stability, of the immobilized pepsin preparation were examined. The immobilized pepsin retained more than 40% of its proteolytic activity toward N-acetyl-L-phenylalanyl-L-3,5-diiodo-tyrosine and more than 30% toward IgG, and also remarkable stability as compared with free pepsin. The immobilized pepsin thus prepared was efficiently used for the limited cleavage of IgG and the gel-filtration effect of the column made it easily possible to yield the F(ab')2-rich fraction for intravenous use.

Chromatography, High Pressure Liquid↗

PubMed1978

The differential enzyme susceptibility of bovine immunoglobulin G1 and immunoglobulin G2 to pepsin and papain.

Purified bovine immunoglobulins IgG1 and IgG2 were subjected to enzymatic degradation with pepsin and papain. Results were monitored using density gradient ultracentrifugation, acrylamide electrophosesis and immunodiffusion employing subclass- and light chain-specific antisera. The results indicated a marked enzymatic susceptibility of IgG1 to digestion with pepsin. This differential susceptibility can also be demonstrated in unfractionated bovine gamma-globulin. No striking differences between the two subclasses were observed during treatment with papain in the presence of cysteine and after 24 h, most IgG1 and IgG2 was degraded to Fc and Fab fragments. The pepsin Fc fragment generated from IgG2 was larger than that generated from IgG1 although the F(ab')2 fragments were simialr in size. These results are consistent with the hypothesis that the Fc region of IgG1 contains multiple cleavage sites for pepsin whereas IgG2 has few. Rabbits immunized with the first elution peak from a 30 h pepsin digest of bovine gamma-globulin fractionated on Sephadex G-150, responded primarily to common gamma-chain and IgG2-specific determinants. Thus, the differential susceptibility of bovine subclasses to pepsin provides a method for stimulating IgG2-specific antibodies in rabbits.

Animals↗

PubMed1990

Pepsin, an aspartic protease, converts porcine big endothelin to 21-residue endothelin.

Porcine big endothelin (big ET-39) at 1 nM, a concentration with no influence on contractile activity in isolated rat aorta, induced a slow-onset and sustained contraction by the pre-incubation with pepsin. When the incubation mixture of big ET-39 with pepsin was analyzed by high-performance liquid chromatography on an octadecyl silica column, two major products of pepsin hydrolysis were obtained; their amino acid sequences were identical with those of 21-residue endothelin (ET-21) and a C-terminal peptide of big ET-39, big ET (22-39), respectively. On the other hand, no degradation of ET-21 was observed by pepsin treatment. These results indicate that pepsin specifically cleaves a Trp21-Val22 bond in the big ET-39 molecule, producing ET-21 and big ET (22-39). Thus, the possibility that pepsin-like aspartic protease may participate in the conversion of big ET-39 to ET-21 in vivo warrants further attention.

Animals↗

PubMed1991

A critical analysis, with appropriate controls, of gastric acid and pepsin secretion in clinical esophagitis.

Because esophagitis is a presumed "acid-peptic" disease, fasting gastric contents (volume and acid and pepsin concentrations) and basal and pentagastrin-stimulated acid and pepsin outputs were studied in 155 patients with endoscopically defined (and graded 1-4) esophagitis and 508 control patients without esophagitis. Basal pepsin and maximal acid and pepsin outputs were lower in the patients with esophagitis than in those without esophagitis. In further analysis, the patients were subdivided into three categories, duodenal ulcer, nonulcer with no disease other than esophagitis, and postgastric surgery, because these categories affect gastric secretion independently of esophageal disease and in the rank order given. Each category was subdivided by sex, because men secreted more than women. Within each category there was no systematic difference in fasting, basal, or maximal gastric acid or pepsin secretion between patients with and patients without esophagitis. Severity of esophagitis was not related to any secretion parameters. Hiatal hernia was present in 50% of patients with esophagitis vs. 15% of controls without the condition (P less than 0.01); however, this did not independently influence the gastric secretion findings. Lower esophageal sphincter pressure was also measured in 62 of the patients, 31 with and 31 without esophagitis. Below 10 mm Hg (incompetent sphincter), 9 of 10 patients had esophagitis but accounted for only less than 30% of the patients with esophagitis, whereas none of 11 patients with basal acid output of less than 0.1 mEq/h and lower esophageal sphincter pressure of greater than 10 mm Hg had esophagitis. Because neither the composition of gastric juice nor basal or stimulated gastric acid or pepsin output could be correlated to the presence or severity of esophagitis, factors other than amount or composition of gastric juice per se must be responsible for susceptibility to esophagitis.

Adult↗

PubMed1983

Pepsin and autoimmune disease mouse.

The effects of pepsin on autoimmune glomerulonephritis of MRL/1 mice were investigated. Intravenous administration of pepsin significantly improved survival rate and suppressed progressive increase in urinary excretion of protein and various histopathological changes in kidney. Increased serum levels of immune complex and anti-DNA antibody in MRL/1 mice were decreased by pepsin. Pepsin ameliorated abnormalities in lymphocyte subsets and lymphocyte functions. The fact that pepsin ameliorated abnormalities in immune function may contribute to the preventive effects of pepsin against pathogenesis and progress of immune complex nephritis.

Animals↗

PubMed1984

The effects of pepsin on the natural progression of autoimmunity in glomerulonephritis in the NZB/W F1 mouse.

The effects of pepsin on autoimmune glomerulonephritis of New Zealand Black and White F1 hybrid (NZB/W F1) mice were investigated. Intravenous pepsin significantly improved survival rate and suppressed progressive increase in urinary protein and histopathological changes in kidney. Increased serum levels of immune complexes, anti-DNA antibody and natural thymocytotoxic autoantibody were decreased and abnormalities in lymphocyte subsets were ameliorated by pepsin. Pepsin suppressed autoantibody production and enhanced antibody production against xenogeneic substance in these mice. The fact that pepsin ameliorates abnormalities in immune function may contribute to the preventive effects of pepsin against pathogenesis and progression of immune complex nephritis.

Animals↗

PubMed1984

Hydrolysis of the synthetic chromophoric hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe catalyzed by bovine pepsin A. Dependence on pH and effect of enzyme phosphorylation level.

Steady-state kinetic parameters for the hydrolysis of the chromophoric hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe catalyzed by bovine gastricsin and pepsin A were determined. It was shown that the phosphate content of bovine pepsin A is without any significant effect on that parameters. At pH 4.7, the specificity constant (kcat/Km) was 2455 and 2150 mM-1 X s-1 for the most phosphorylated bovine pepsin A (2.58 phosphate groups per molecule), before and after treatment by potato acid phosphatase, respectively. The kcat/Km ratio found for bovine gastricsin (1314 mM-1 X s-1) was closer to that of bovine pepsin A than that previously reported for chymosin (25 mM-1 X s-1). The spectral properties of the chromophoric tripeptide Leu-Ser-Phe(NO2) in the pH range 1-3.6 were investigated. We have shown that the hexapeptide hydrolysis could be followed by difference spectrophotometry at 295 nm (delta epsilon = -235 M-1 X cm-1 at pH 1.0) thus allowing to study the effect of pH on bovine pepsin A activity in a pH range which could not be explored earlier. The pH-dependence of kcat/Km ratio of unphosphorylated bovine pepsin A indicated that enzyme activity was dependent upon the ionization of two groups of the enzyme whose pK are 1.2 and 5.0. These pK values strongly suggest the involvement of two carboxyl groups probably corresponding to the two reactive aspartyl residues (Asp32 and Asp215) identified through active site-directed reagents for all the aspartic proteinases so far tested.

Animals↗

PubMedDate not supplied

Does motilin control interdigestive pepsin secretion in the dog?

Pepsin output in the Heidenhain pouch, plasma motilin concentration, and contractile activity in the pouch and the main stomach were investigated in five dogs. During the interdigestive state, the pepsin output was significantly increased with a cyclic increase in contractile activity in both the pouch and main stomach at approximately 100-min intervals. The plasma immunoreactive motilin (IRM) concentration fluctuated during the interdigestive state, and, peaks of IRM concentration coincided with the maximum pepsin secretory activity. Exogenous administration of motilin (0.5 micrograms/kg-hr) increased contractile activity in the main stomach and pouch quite similar to the natural interdigestive migrating contractions (IMC), and increased pepsin output significantly. Atropine pre-treatment suppressed the naturally-occurring and motilin-induced pepsin output and contractions in the pouch. It is concluded that pepsin output and contractions in the Heidenhain pouch increase in close association with the IMC in the main stomach during the interdigestive state and these cyclic motor and secretory events in the vagally denervated fundic pouch are most likely regulated by motilin through the intramural cholinergic pathway.

Animals↗

PubMedDate not supplied

Pepsin-mediated processing of synthetic precursor-like sequence yields neurotensin-like peptide.

A 15 amino acid synthetic peptide, which spanned the dibasic cleavage site C-terminal to neurotensin (NT), in its 170-residue canine precursor, was synthesized by solid-phase methods. Using this substrate in combination with a radioimmunoassay specific for the C-terminal region of NT, a simple assay was developed to monitor protease-mediated cleavage of the Leu8-Lys9 bond in the substrate. Hog pepsin and the related enzymes, rhizopus pepsin, bovine cathepsin D, and mouse renin, were found to be effective in this assay, pepsin cleaving only this bond to liberate the NT-like sequence. The pH dependence of the reaction indicated that pepsin, cathepsin D, and renin exhibited significant activity at pH's characteristic for secretory vesicles (pH 5.5-6.5). In addition, pepsin and cathepsin D were shown to process the native precursor at pH's as high as 5.5. These results, although not proof, are consistent with the idea that endoproteases with pepsin-like specificity may be involved in the processing of the NT precursor in neural/endocrine cells.

Amino Acid Sequence↗

PubMed2005

Bioadhesive oesophageal bandages: protection against acid and pepsin injury.

The rate of acid and pepsin diffusion through solutions of sodium alginate was measured using in vitro techniques. Previous work has demonstrated that solutions of alginate may adhere to the oesophagus for up to 60 min; this work measured their ability to protect the oesophageal epithelial surface from damage caused by refluxed acid and pepsin. Franz diffusion cells were used to measure the rate of acid and pepsin diffusion through an alginate layer. The effect of the type of alginate, alginate concentration and depth of alginate applied were investigated. The rate of both acid and pepsin diffusion was significantly reduced (ANOVA analysis; P<0.05) in the presence of an alginate solution compared to the control. A 2% (w/v) alginate solution with a high guluronic acid component, in a layer of 0.44 mm depth, demonstrated the greatest reduction in acid diffusion with a permeation coefficient 14% than that of a control value. All three alginates demonstrated significant reductions in acid diffusion with both increasing depth and increasing concentration, as expected. Pepsin diffusion was also significantly reduced as the depth and concentration of applied alginate increased. This study demonstrates that an adhesive layer of alginate present within the oesophagus will limit the contact of refluxed acid and pepsin with the epithelial surface.

Alginates↗

PubMed2006

Pepsin in bronchoalveolar lavage fluid: a specific and sensitive method of diagnosing gastro-oesophageal reflux-related pulmonary aspiration.

OBJECTIVES: Gastro-oesophageal reflux (GOR)-related aspiration is associated with respiratory disease, but the current "gold standard" investigation, the lipid-laden macrophage index (LLMI), is flawed. A specific marker of GOR-related aspiration should originate in the stomach, but not the lung. An assay to detect gastric pepsin in the bronchoalveolar lavage (BAL) of children was developed and validated. METHODS: Gastro-oesophageal reflux was diagnosed in 33 children using intra-oesophageal pH monitoring. Thirteen asymptomatic negative controls requiring endotracheal intubation for elective surgery and 5 positive control patients with observed aspiration were recruited. All subjects received a BAL; the fluid obtained was analysed for the pepsin content and the LLMI. RESULTS: All subjects in the negative control group were negative for pepsin. The positive control group had a significantly greater median pepsin level (P < .01) compared with negative controls. Patients with proximal oesophageal GOR and chronic cough also had significantly elevated pepsin levels (P = .04). The LLMI was not significantly elevated by the presence of cough or GOR. CONCLUSIONS: This study suggests that GOR-related aspiration plays a role in chronic cough in children with known GOR. Detecting pepsin in BAL fluid may therefore become an important adjunct in patient selection for antireflux surgery.

Bronchoalveolar Lavage Fluid↗

PubMed2004

A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins.

Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10U of pepsin activity/microg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60min. Protein digestibility was assessed from stained gels following SDS-PAGE of digestion samples and controls. Results. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%). Conclusions. These data demonstrate that this common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories.

Clinical Laboratory Techniques↗