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PubMed1983

A comparison of pirenzepine and cimetidine inhibition of centrally-mediated gastric acid and pepsin secretion in the gastric-fistula cat.

The effects of pirenzepine, a muscarinic antagonist, and cimetidine, a histamine H2-receptor antagonist, on 2-deoxy-D-glucose-stimulated gastric secretion were investigated in the gastric-fistula cat. Both drugs inhibited gastric acid secretion in a dose-related manner; the oral ED50 of pirenzepine was 0.37 (0.22-0.59) mg/kg and cimetidine was 4.6 (3.3-5.8) mg/kg. The two drugs affected pepsin secretion in characteristically different ways. The inhibition of pepsin secretion by cimetidine was small and inconsistent when compared with the effect on acid. Throughout the dose-range cimetidine tended to reduce volume more than pepsin secretion. The resulting rise in concentration of pepsin suggested that cimetidine had an indirect effect on pepsin secretion. In contrast, pirenzepine exerted a direct effect on both acid and pepsin. Hence at doses producing a 50% inhibition of acid secretion pirenzepine and cimetidine reduced pepsin secretion by 78% and 25% respectively. These observations were consistent with the concept that muscarinic receptors and not histamine H2-receptors are involved in the regulation of pepsin secretion.

Animals↗

PubMed1983

Purification and characterization of pepsinogens and pepsins from Asiatic black bear, and amino acid sequence determination of the NH2-terminal 60 residues of the major pepsinogen.

Five pepsinogens were purified to homogeneity from the gastric mucosa of Asiatic black bear and termed pepsinogens I-1, I-2, II-1, II-2, and III. Pepsinogen II-1 was the major component and accounted for more than half of the total pepsinogens. Their molecular weights were estimated to be 40,000 for pepsinogens I-1 and I-2, 38,000 for pepsinogens II-1 and II-2, and 42,000 for pepsinogen III. They resembled each other in amino acid composition, except that pepsinogens I-1 and I-2 contained larger numbers of basic residues than the others. Pepsinogen III was a glycoprotein containing about 3.7% carbohydrate. Each was activated to the corresponding pepsin and their enzymatic characteristics were investigated. The optimal pH against hemoglobin was about 2.2 for pepsin I-1, and about 2.5 for pepsins II-1, II-2, and III. Each pepsin was inhibited by pepstatin as well as porcine pepsin and also by diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(p-nitrophenoxy)-propane, and p-bromophenacyl bromide. Each pepsin could hydrolyze N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine, but the specific activity was much lower than that of porcine pepsin. Activation peptides corresponding to residues 1-43, 1-25, and 26-43 were isolated from an activation mixture of pepsinogen II-1. The amino acid sequences of these peptides and of the NH2-terminal portions of pepsinogen II-1 and pepsin II-1 were determined, resulting in the complete NH2-terminal 60-residue sequence of pepsinogen II-1.

Amino Acid Sequence↗

PubMed1986

Generation of immunoreactive neurotensin(s) and enkephalin(s) by pepsin-treatment of plasma.

Treatment of mammalian plasmas with pepsin yielded extraordinary quantities of immunoreactive neurotensin (iNT) and methionine5-enkephalin (iENK). The concentrations measured after pepsin-treatment (iNT, 1-5 microM and iENK, 0.1-0.5 microM) were 1-100 thousand times the normal circulating levels of these peptides. The reactions were shown to be time, temperature and pH dependent and to involve the action of pepsin on albumin-like proteins (Mr, ca, 65,000). Pepsin-generated iNT from rat plasma differed from NT since it reacted only with C-terminal directed antisera and eluted earlier than NT during HPLC on mu-Bondapak C-18. Partially purified iNT was active in two bioassays for NT, one which senses changes in vascular permeability to protein after intradermal injection into rats and another which measures release of histamine from isolated rat mast cells. Other biologic activities generated by pepsin-treating plasma included effects on systemic blood pressure in rats and on the contractility of the isolated guinea pig ileum. Some of these, however, were attributable to the formation of angiotensin- and bradykinin-related peptides. Pepsin-generated iENK gave three major peaks during HPLC, one of which (ca, 25%) co-eluted with oxidized ENK and also registered in a radioreceptor assay for opiate-related substances. In addition, this material produced ENK-like effects on the isolated guinea pig ileum and on vascular permeability in rat skin. The precursor-like protein(s) for iENK were distinguished from adrenal proenkephalins since it did not liberate iENK upon digestion with trypsin and carboxypeptidase B. Since pepsin can mimic renin these results suggest the existence of systems in blood (analogous to the renin/angiotensin system) for the generation of biologically active NT- and ENK-related peptides and they also raise the question as to whether other neuropeptides might be found circulating in precursor form(s).

Amino Acid Sequence↗

PubMed1981

Influence of metiamide and atropine on acid and pepsin secretion in the anaesthetized Heidenhain cat.

The inhibition of acid and pepsin secretion by metiamide and atropine was studied in the anaesthetized Heidenhain-pouch cat. Metiamide strongly inhibited acid secretion induced by histamine, pentagastrin or carbachol. Metiamide had not effect on pepsin secretion stimulated by pentagastrin and carbachol but was effective on histamine-stimulated pepsin secretion. Atropine strongly inhibited pepsin secretion but was not as effective as metiamide in the inhibition of acid secretion. Reperfusion of the pouch with acid + salicylic acid also increased pepsin secretion. This increased pepsin output was abolished by atropine, but not by metiamide. It is postulated that (1) the peptic cell probably has only cholinergic receptors which can be additionally triggered by HCl passing the peptic cell, and (2) H2-antagonists only inhibit pepsin secretion indirectly via reduction of acid output.

Anesthesia↗

PubMed1979

The formation and thermal stability of in vitro assembled fibrils from acid-soluble and pepsin-treated collagens.

The role of the non-helical regions of the collagen molecule in fibrillogenesis has been investigated by comparing the kinetics of fibril formation of pepsin-treated acid-soluble collagen, acid-soluble collagen and mixtures of the two and by comparison of the thermal stabilities of the fibrils formed. The acid-soluble collagen was found to aggregate more rapidly than the pepsin-treated collagen under physiological conditions of pH and ionic strength. Variations in ionic strength, at physiological pH, were found to have differing effects on the aggregation of these two forms of soluble collagen. Fibrils formed from the pepsinized-collagen had a lower thermal stability tha n those formed from the intact collagen. The behavior observed with mixtures of acid-soluble and pepsin-treated collagens was found to be quantitatively consistent with the pepsinized collagen being able to utilize the nuclei formed by the acid-soluble collagen for subsequent growth. However, the use of the acid-soluble nuclei by the pepsinized collagen for growth did not enhance its rate of precipitation during the growth phase, nor did it enhance the thermal stability of the fibrils formed from the pepsinized collagen.

Animals↗

PubMed1994

Aspartic proteinases--Fourier transform IR studies of the aspartic carboxylic groups in the active site of pepsin.

Fourier transform (FTIR) difference spectra of pepsin minus diazoacetylnorleucine methyl ester (DAN) or minus diazoacetyl-L-phenylalanine methyl ester (DAP) modified pepsin, respectively, demonstrated that Asp-215 is not deprotonated in pepsin. The FTIR difference spectrum of pepsin minus 1,2-epoxyparanitrophenoxypropane (EPNP) modified pepsin demonstrates that Asp-32 is present in pepsin as CO2- anion. The position of the v(C = O) vibration demonstrates that no (O...H...O)- hydrogen bond between Asp-215 and Asp-32 is formed. Furthermore, no H3O+ is present in the active center. Studies of the complex of pepsin with the inhibitor pepstatin prove that the inhibitor removes the water from the active site and Asp-32 becomes protonated.

Aspartic Acid↗

PubMed1984

Isolation and characterization of camel pepsins.

Camel (Camelus dromedarius) pepsins were precipitated from the extract of the fundic gastric mucosa by ammonium sulfate between 35 and 80% saturation. DEAE--cellulose chromatography of this fraction produced two isoenzymes, I and II, which were further purified to homogeneity on sephadex G-100. Their Km with N-acetyl-L-phenylalanyl-diiodotyrosine were 0.10 and 0.90 mM, and their molecular weights which were determined on sodium dodecylsulfate gel electrophoresis exhibited 35,500 and 34,700, respectively. The two pepsins were essentially free of carbohydrates, but contained 0.3 and 1.0 mol of organic phosphate per 1 mol of protein, respectively. The apparent mobilities of the phospho- and dephosphoforms of each pepsin were indifferent in polyacrylamide gel electrophoresis at pH 8.9. The N-terminal residues of pepsins I and II were found to be alanine and leucine, respectively. Pepsin I was inactivated at a faster rate than that of pepsin II at pH 8 and 0 degrees C, and at pH 7.5 and 37 degrees C; but both were denatured under these conditions. The properties of these enzymes are compared with those of other mammalian and avian pepsins.

Animals↗

PubMed2005

Response properties of the brainstem neurons of the cat following intra-esophageal acid-pepsin infusion.

Studies in humans have documented that acute acid infusion into the esophagus leads to decrease in threshold for sensations to mechanical distension of the esophagus. It is not known whether acid infusion leads to sensitization of brainstem neurons receiving synaptic input from vagal afferent fibers innervating the esophagus. The aim of this study was to investigate the correlation of responses of vagal afferents and brainstem neurons after acute infusion of acid (0.1 N HCl)+pepsin (1 mg/ml) into the esophagus of cats. The vagal afferent fibers (n=20) exhibited pressure-dependent increase in firing to graded esophageal distension (5-80 mm Hg). Infusion of acid+pepsin into the esophagus produced a significant increase in ongoing resting firing of five of 16 fibers (31%) tested. However, their responses to graded esophageal distension did not change when tested 30 min after infusion. Pepsin infusion did not change the resting firing and response to esophageal distension (n=4). Twenty-one brainstem neurons were recorded that responded in an intensity-dependent manner to graded esophageal distension. Responses of 12 excited neurons were tested after intra-esophageal acid+pepsin infusion. Neurons exhibited a decrease in threshold for response to esophageal distension and increase in firing after acid+pepsin infusion. The sensitization of response after intra-esophageal acid remained unaffected after cervical (C1-C2) spinal transection (n=3). Results indicate that the esophageal distension-sensitive neurons in the brainstem exhibit sensitization of response to esophageal distension after acute acid+pepsin exposure. The sensitization of brainstem neurons is possibly initiated by increased spontaneous firing of the vagal afferent fibers to acid+pepsin, but not to sensitized response of vagal distension-sensitive afferent fibers to esophageal distension. Results also indicate that spinal pathway does not contribute to sensitization of brainstem neurons.

Animals↗

PubMed2003

The active site of pepsin is formed in the intermediate conformation dominant at mildly acidic pH.

Pepsin is an aspartic protease that acts in food digestion in the mammal stomach. An optimal pH of around 2 allows pepsin to operate in its natural acidic environment, while at neutral pH the protein is denatured. Although the pH dependence of pepsin activity has been widely investigated since the 40s, a renewed interest in this protein has been fueled by its homology to the HIV and other aspartic proteases. Recently, an inactive pepsin conformation has been identified that accumulates at mildly acidic pH, whose structure and properties are largely unknown. In this paper, we analyse the conformation of pepsin at different pHs by a combination of spectroscopic techniques, and obtain a detailed characterisation of the intermediate. Our analysis indicates that it is the dominant conformation from pH 4 to 6.5. Interestingly, its near UV circular dichroism spectrum is identical to that of the native conformation that appears at lower pH values. In addition, we show that the intermediate binds the active site inhibitor pepstatin with a strength similar to that of the native conformation. Pepsin thus adopts, in the 6.5-4.0 pH interval, a native-like although catalytically inactive conformation. The possible role of this intermediate during pepsin transportation to the stomach lumen is discussed.

Binding Sites↗

PubMed1982

Study of the heterogeneity of bovine pepsin A by pseudo isoelectric focusing.

Pseudo isoelectric focusing (PIEF), i.e. focusing stopped before the equilibrium is reached, has been successfully applied to analyse bovine pepsins A obtained from abomasum mucosa extracts and from 3 individual abomasal juices. Whole bovine pepsin A from mucosa extracts, was resolved in PIEF, using an acidic pH gradient(2-4), into five major and active components. Its fractionation on hydroxyapatite only gave four components which were not homogeneous in PIEF, suggesting that interactions between the different components occur during the chromatographic procedure. After treatment with potato acid phosphatase, whole bovine pepsin A showed only one band in PIEF, displaying enzymic activity and with a mobility identical to that of the less anodic band in untreated pepsin. These results, together with organic phosphate determinations, obviously confirm that the heterogeneity of bovine pepsin A is due to the phosphate content which appears to range between 0 and 3 mol./mol. for the 5 components. Two of them, the phosphate content of which appears to be 1 mol./mol., seem to differ in the location of this phosphate group in the molecule. Whole bovine pepsin A preparations from abomasal juices shared the same pattern in PIEF, identical to that observed with bovine pepsin A from mucosa extracts, thus excluding that dephosphorylation might be involved in the secretory process, as well as disproving the theory that dephosphopepsinogen might be an intermediate in the synthesis of pepsinogen, as it was previously suggested.

Abomasum↗

PubMed1999

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.

Abomasum↗

PubMed2006

Extensive deuterium back-exchange in certain immobilized pepsin columns used for H/D exchange mass spectrometry.

Pepsin digestion prior to mass analysis increases the spatial resolution of hydrogen exchange mass spectrometry experiments. Online digestion with immobilized pepsin is advantageous for several reasons including better digestion efficiency. We have found that certain immobilized pepsin columns cause substantial deuterium back-exchange, rendering the data unusable. When pepsin immobilized on a POROS support was used for online digestion, back-exchange was within the expected range and was similar to the back-exchange of deuterated peptides produced by in-solution pepsin digestion. However, when pepsin immobilized onto selected polystyrene-divinylbenzene supports was used for online digestion with the same system, deuterium loss was extremely high. The effect seems linked to the properties of the solid support used to conjugate the pepsin.

Deuterium Exchange Measurement↗

PubMed2006

Comparison of solution structures and stabilities of native, partially unfolded and partially refolded pepsin.

A zymogen-derived protein, pepsin, appears to be incapable of folding to the native state without the presence of the prosegment. To better understand the nature of the irreversible denaturation of pepsin, the present study reports on the characterization of the stability and low-resolution tertiary and secondary structures of native, alkaline unfolded and acid refolded porcine pepsin. Through a combination of small-angle neutron scattering (SANS), CD, and DSC, acid refolded pepsin (Rp) was shown to have secondary and tertiary structures intermediate between the alkaline denatured and native forms but was found to be thermodynamically stable relative to the native state. It was also observed that the acid refolded state of pepsin was dependent on the protein concentration during refolding because CD and SANS data revealed that both the secondary and tertiary structures of concentrated-refolded pepsin (>10 mg/mL) (CRp) were native-like, in contrast to the intermediate nature of Rp, refolded under dilute concentration (<10 mg/mL). Despite a native-like conformation, CRp was more stable and had substantially reduced activity compared to that of the native state, suggesting that the protein was misfolded. It is proposed that the stable but misfolded, acid-refolded states are evidence that pepsin in its native conformation was metastable. Furthermore, the disruption of the active site cleft in the denatured states could be discerned by modeling of the SANS data.

Animals↗

PubMed1977

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.

Amino Acids↗

PubMed1985

The development of acid and pepsin (EC 3.4.23.1) secretory capacity in the pig; the effects of age and weaning. 1. Studies in anaesthetized pigs.

The development of gastric secretory capacity of hydrochloric acid and pepsin (EC 3.4.23.1) was studied in thirty-eight Large White X Landrace pigs from the litters of six sows (three pairs of two), 9-38 d of age. The pigs of each pair of sows were born within 24 h of each other. The pigs of a litter were paired according to sex and size and cross-fostered, i.e. one pig from each pair was allocated to each sow. One litter from each pair was reared entirely by the sow (milk-fed, MF) whereas the other litter was reared by the sow for 21 d, but was allowed access to solid food (210 g crude protein (nitrogen X 6.25)/kg) at 12 d and was entirely dependent on solid food after 21 d (creep-fed, CF). Following a 14-18 h fast, pigs were anaesthetized (Halothane-sodium pentobarbitone) and their stomachs perfused at a constant rate with Ringer solution. Gastric secretion was stimulated by intravenous infusion of betazole hydrochloride (Histalog) at 3 mg/kg per h for 2 h. Hydrochloric acid and pepsin were measured in the perfusate which was collected at 15-min intervals. There were significant positive correlations between stomach weight and body-weight for both MF and CF pigs. The slope of the regression line for CF pigs was significantly greater than that for MF pigs (P less than 0.01). There were significant positive correlations between maximal acid output and stomach weight for both MF and CF pigs. There were significant positive correlations between maximal pepsin output and stomach weight for both MF and CF pigs. The slope of the regression line for CF pigs was significantly different from that for MF pigs (P less than 0.01). There were also significant positive correlations between maximal pepsin output per unit stomach weight and stomach weight for both MF and CF pigs. The pattern of development of pepsin secretory capacity in both CF and MF pigs was different from that for acid secretion. Maximal outputs of acid per unit stomach weight for MF and CF pigs remained relatively constant. Maximal outputs of pepsin per unit stomach weight and per unit body-weight increased with age for both MF and CF pigs. The results indicate that pigs given access to solid food before weaning and weaned on to solid food have heavier stomachs and greater acid and pepsin secretory capacity than pigs fed entirely on sows' milk.

Aging↗

PubMed1976

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.

Amino Acids↗

PubMed1998

Gastric lipase and pepsin activities in the developing ferret: nonparallel development of the two gastric digestive enzymes.

BACKGROUND: Gastric lipase has an important compensatory function in neonatal fat digestion. The activity level of pepsin and its role in protein digestion is less well understood. We have, therefore, studied the ontogeny of lipase and pepsin in the ferret, a species with a neonatal fat digestion pattern similar to that of humans. METHODS: Gastric lipase and pepsin activities were quantified from the late fetal period throughout lactation, and were compared with those of the adult. RESULTS: The data show earlier ontogeny and much more rapid rise of lipase activity than of pepsin. Lipase activity was present during the last week of fetal development, whereas pepsin was detected only postnatally. Lipase activity was 72.8% +/- 14.2% and 153% +/- 9.95% and pepsin activity was 11.6% +/- 1.3% and 30.1% +/- 1.3% of the adult level at 2 and 4 wk of age, respectively. CONCLUSIONS: We conclude that lipase activity develops early and exceeds adult activity during the suckling period, when fat intake is very high. The low pepsin activity and high postprandial pH probably limit gastric proteolysis, thereby contributing to the structural and functional stability of milk proteins, many with protective or bioactive function in the gastrointestinal tract of the newborn.

Animals↗

PubMed2002

Assay of tracheal pepsin as a marker of reflux aspiration.

OBJECTIVES: Aspiration of gastric contents is a relatively common cause of acute and chronic pulmonary disease. However, a reliable method of diagnosing recurrent aspiration is currently lacking. The aim of this study was to determine whether the presence of gastric pepsin in tracheal aspirates of infants and children might be used as a reliable marker of the microaspiration of refluxed gastric contents. METHODS: Ninety-eight children undergoing general anesthesia and tracheal intubation participated in the study. Sixty-four of 98 children underwent endoscopy for clinically significant gastroesophageal reflux. Thirty-four children from routine operative lists were nonreflux controls. These two groups were further subdivided based on the presence or absence of associated respiratory symptoms. After endotracheal intubation, tracheal aspirates were obtained and subsequently assayed for gastric pepsin using a fluoroscein isothiocyanate casein. RESULTS: Pepsin was detected in 7 of 27 children with reflux symptoms alone and in 7 of 8 of those with chronic respiratory symptoms. In addition, pepsin was present in 31 of 37 children with a history of both reflux and chronic respiratory symptoms. Tracheal pepsin was not detected in any of the 26 children without gastroesophageal reflux or respiratory symptoms. Tracheal pepsin was found significantly more frequently in children with reflux symptoms than in those without, particularly in children with both reflux and respiratory problems. CONCLUSION: Tracheal pepsin assay as a reliable marker of gastroesophageal reflux aspiration.

Biomarkers↗