STUDIES OF GASTRIC PEPSIN. II. SECRETION OF PEPSIN IN CASES OF DUODENAL ULCER AND PSEUDO-ULCER.
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Journal Article↗
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[Inhibition of the catalytic activity of pepsin(ogen) in human urine. II. Isolation of uropepsin inhibitors from human urine concentrates using affinity chromatography with pepsin sepharose 4B].
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Chromatography, Affinity↗
Molecular and crystal structures of monoclinic porcine pepsin refined at 1.8 A resolution.
The molecular structure of the archetypal aspartic proteinase, porcine pepsin (EC 3.4.23.1), has been refined using data collected from a single monoclinic crystal on a twin multiwire detector system to 1.8 A resolution. The current crystallographic R-factor (= sigma parallel to Fo/-/Fc parallel to/sigma/Fo/) is 0.174 for the 20,519 reflections with /Fo/ greater than or equal to 3 sigma (Fo) in the range 8.0 to 1.8 A (/Fo/ and /Fc/ are the observed and calculated structure factor amplitudes respectively). The refinement has shown conclusively that there are only 326 amino acid residues in porcine pepsin. Ile230 is not present in the molecule. The two catalytic residues Asp32 and Asp215 have dispositions in porcine pepsin very similar to the dispositions of the equivalent residues in the other aspartic proteinases of known structure. A bound solvent molecule is associated with both carboxyl groups at the active site. No bound ethanol molecule could be identified conclusively in the structure. The average thermal motion parameter of the residues that comprise the C-terminal domain of pepsin is approximately twice that of the residues in the N-terminal domain. Comparisons of the tertiary structure of pepsin with porcine pepsinogen, penicillopepsin, rhizopus pepsin and endothia pepsin reveal that the N-terminal domains are topographically more similar than the conformationally flexible C-terminal domains. The conformational differences may be modeled as rigid-body movements of "reduced" C-terminal domains (residues 193 to 212 and 223 to 298 in pepsin numbering). A similar movement of the C-terminal domain of endothia pepsin has been observed upon inhibitor binding. A phosphoryl group covalently attached to Ser68 O gamma has been identified in the electron density map of porcine pepsin. The low pKa1 value for this group, coupled with unusual microenvironments for several of the aspartyl carboxylate groups, ensures a net negative charge on porcine pepsin in a strongly acid medium. Thus, there is a structural explanation for the very early observations of "anodic migration" of porcine pepsin at pH 1. In the crystals, the molecules are packed tightly into a monoclinic unit cell. There are 190 direct contacts (less than or equal to 4.0 A) between a central pepsin molecule and the five unique symmetry-related molecules surrounding it in the crystalline lattice. The tight packing in this cell makes pepsin's active site and binding cleft relatively inaccessible to substrate analogs or inhibitors.
Amino Acid Sequence↗
Oxidation of methionine residues of porcine and bovine pepsins.
By treating porcine and bovine pepsins with H2O2 at pH 3.2, 3.5 of the 4 methionine residues of porcine pepsin and 1.6 of the 3 residues of bovine pepsin were oxidized to methionine sulfoxide. The effect of modification on activity varied with the substrate. There were no significant changes in catalytic constants in the hydrolysis of acetyl-L-phenylalanyl-L-tyrosine by both pepsins and in the hydrolysis of benzyloxycarbonyl-L-glutamyl-L-tyrosine by porcine pepsin. Hydrolysis of benzyloxycarbonyl-L-glutamyl-L-tyrosine by bovine pepsin was too slow to measure. With benzyloxycarbonyl-L-histidyl-L-phenylalanyl-L-tryptophan ethyl ester as substrate, the modification decreased the catalytic efficiency (kcat/Km) by two-thirds for porcine pepsin and by half for bovine pepsin. With hemoglobin substrate, digestion was significantly less than with native pepsin for modified porcine pepsin, and slightly less for modified bovine pepsin. The results are interpreted as indicating the presence of a methionine residue that participates in the binding of long substrates, but is not close enough to the active site to reach short substrates. Cleavage of the modified pepsins with cyanogen bromide identified the methionine nearest the carboxyl terminus of both pepsins as a resiude that remained partially unmodified.
Amino Acids↗
Roles of Tyr13 and Phe219 in the unique substrate specificity of pepsin B.
Pepsin B is known to be distributed throughout mammalia, including carnivores. In this study, the proteolytic specificity of canine pepsin B was clarified with 2 protein substrates and 37 synthetic octapeptides and compared with that of human pepsin A. Pepsin B efficiently hydrolyzed gelatin but very poorly hydrolized hemoglobin. It was active against only a group of octapeptides with Gly at P2, such as KPAGF/LRL and KPEGF/LRL (arrows indicate cleavage sites). In contrast, pepsin A hydrolyzed hemoglobin but not gelatin and showed high activity against various types of octapeptides, such as KPAEF/FRL and KPAEF/LRL. The specificity of pepsin B is unique among pepsins, and thus, the enzyme provides a suitable model for analyzing the structure and function of pepsins and related aspartic proteinases. Because Tyr13 and Phe219 in/around the S2 subsites (Glu/Ala13 and Ser219 are common in most pepsins) appeared to be involved in the specificity of pepsin B, site-directed mutagenesis was undertaken to replace large aromatic residues with small residues and vice versa. The Tyr13Ala/Phe219Ser double mutant of pepsin B was found to demonstrate broad activity against hemoglobin and various octapeptides, whereas the reverse mutant of pepsin A had significantly decreased activity. According to molecular modeling of pepsin B, Tyr13 OH narrows the substrate-binding space and a peptide with Gly at P2 might be preferentially accommodated because of its high flexibility. The hydroxyl can also make a hydrogen bond with nitrogen of a P3 residue and fix the substrate main chain to the active site, thus restricting the flexibility of the main chain and strengthening preferential accommodation of Gly at P2. The phenyl moiety of Phe219 is bulky and narrows the S2 substrate space, which also leads to a preference for Gly at P2, while lowering the catalytic activity against other peptide types without making a hydrogen-bonding network in the active site.
Animals↗
Association of pepsin with type II collagen (CII) breaks control of CII autoimmunity and triggers development of arthritis in rats.
Lewis rats develop arthritis after immunization with heterologous but not homologous rat type II collagen (CII). We have observed that if the rat CII is prepared by pepsin digestion without subsequent extensive purification, it is arthritogenic in Lewis rats. To address whether pepsin in the CII preparations contributed to the development of arthritis and whether this was associated with the induction of an immune response to CII, Lewis rats were immunized with rat CII of various degrees of purity and with various pepsin contents. After immunization with a crude preparation of CII, containing relatively large amounts of pepsin, Lewis rats developed arthritis with an incidence of 80% together with a strong anti-CII autoantibody production. Further purification of the CII on DEAE-Sepharose, which removes pepsin, eliminated the arthritogenic properties and the capacity to activate CII-specific B cells. Likewise, lathyritic CII, prepared without pepsin, induced neither a CII-specific immune response nor arthritis. If, however, pepsin was added to non-arthritogenic batches of rat CII, arthritis appeared at an incidence of 40%. By using an ELISPOT technique to detect antigen-specific interferon-gamma-producing T cells and antibody-producing B cells, the immune response to CII and pepsin can be evaluated. Eleven days after immunization with lathyritic CII and pepsin, a B-cell response towards both CII and pepsin was seen. Pepsin-specific T cells were also seen at day 11, but CII-specific T cells did not appear until day 14 after immunization. In addition, a weak CII-specific proliferative response of the T cells could be demonstrated at day 14 but not at day 11 or 12. These data show that pepsin plays an important role in the triggering of a CII-specific immune response. We suggest a carrier-hapten mechanism where pepsin acts as a carrier and CII as a 'hapten' which will activate CII-specific B cells. Subsequently these CII-specific B cells will break the T-cell tolerance and evoke a T-cell-mediated immune response towards CII.
Animals↗
Pepsin 5 in gastric juice: determination and relationship to the alkali-stable peptic activity.
Pure human pepsins 1 and 3 are inactivated by incubation at pH 7.1-7.3 for 30 minutes, losing 90% or more of activity. Pepsin 5 is alkali-stable, retaining 100% of activity. Mixtures of pure pepsins 1 and/or 3 with pepsin 5 were found to have greater alkali-stable activity than predicted. Two published methods for determining the alkali-stable fraction of the peptic activity of gastric juice gave, respectively, in our hands values of 45.4-80.0% and 27.5-43.9% of the total activity. These values seemed too high to be attributable only to pepsin 5 in gastric juice, as agar gel electrophoresis shows pepsin 3 to have the principal activity. Electrophoretograms of alkaline incubated gastric juice revealed that large amounts of pepsin 3 retained activity as well as pepsin 5, and a proteolytic zone "4" appeared between them. Alkali inactivation thus does not allow the estimation of pepsin 5 individually in gastric juice. Pepstatin, at a final concentration of 100 to 170 pmol/ml, may be used to estimate pepsin 5 in gastric juice and gave values of 18.0 to 27.6% of the total peptic activity. Pepsin 5, in gastric juice and in mixtures of pepsins, appears to protect pepsin 3 from alkaline-inactivation, and to a lesser extent from pepstatin inhibition.
Alkalies↗
Partial purification of pepsins from adult and juvenile salmon fish Oncorhynchus keta. Effect of NaCl on proteolytic activities.
1. Two pepsins, designated Pepsin I and Pepsin II, were isolated and partially characterized from the stomach of the adult stage salmon Oncorhynchus keta. This stage is developed in a marine environment. 2. One pepsin, designated Pepsin II, was isolated from the stomach of the juvenile stage salmon Oncorhynchus keta. This stage is developed in an estuarine environment. 3. The enzymes were partially purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration. 4. Pepsins I and II from adults and Pepsin II from juvenile showed proteolytic activity on acid-denatured hemoglobin with a pH optimum of 3. 5. The mol. wt determined by gel filtration on Sephadex G-100 of Pepsin I from juvenile species was found to be 32,000 whereas a value of 27,000 was determined for Pepsin II from juvenile and adult fish. 6. In contrast with Pepsin II, Pepsin I was activated by NaCl. It is suggested that the appearance of NaCl-activated pepsin would represent and adaptive response of the organism to the change from a low to a high salinity environment.
Aging↗
Variation in the proportions of individual pepsins secreted by the cat in response to vagal stimulation and hypoglycaemia.
1. Gastric juice was collected at regular intervals during electrical stimulation of the vagus in anaesthetized cats and during insulin hypoglycaemia in both anaesthetized and conscious cats. The total amounts of acid and pepsin secreted were similar in the three groups. 2. Pepsins were examined by agar-gel electrophoresis. Resting juice contained two pepsins, and up to nine pepsins could be detected after stimulation. Three patterns of pepsin secretion were found. 3. The most noticeable feature was the variation in the proportion of total pepsin attributable to the pepsin which migrated most rapidly during electrophoresis (pepsin 1). In response to insulin hypoglycaemia, anaesthetized cats secreted only a small proportion of total pepsin 1 and conscious cats secreted a large proportion as pepsin 1. During direct electrical stimulation of the vagus, the proportion of pepsin 1 rose. 4. The possibility of a dependence of pepsin 1 secretion on vagal stimulation is discussed and the relevance of this to peptic ulcer and to vagotomy is considered.
Animals↗
Production of acute gastric erosions in the cat by individual human pepsins.
1. Differing amounts of human pepsins 1, 3 (pepsin A) and 5 (pepsin C or gastriscin) in aqueous HCl/NaCl at pH 1.3 were placed in Perspex rings on the exposed luminal surface of the perfused cat stomach to test for mucolytic and erosive activity in vivo, with the acid medium in control rings. 2. After incubation at 37 degrees C for 60 or 120 min, the test and control solutions were replaced by the same volume of each test or control solution containing 18 mg of aspirin and incubated for a further 120 min. 3. The number of bleeding points or erosions was counted at 15 min intervals. None was observed with human pepsins 1, 3 and 5 or with pig pepsin A or in the control rings. 4. With aspirin, erosions developed in all ringed areas. Their rate of development with time for pepsin 1 and pig pepsin did not differ from controls. Significantly more erosions developed with human pepsins 3 (5 mg per ring, P < 0.001) and 5 (0.32 mg per ring, P < 0.05). 5. Human pepsins 3 and 5, applied at pH 1.3 to the mucosal surface of the perfused cat stomach, therefore cause erosions when administered with aspirin, but do not produce erosions when given alone. In man, pepsins secreted in increased amount or concentration may therefore be unlikely to cause significant mucosal erosion unless human gastric mucosa is less resistant to pepsins than cat mucosa. Pepsins may perhaps facilitate the action of a second factor, such as aspirin, or infection with Helicobacter pylori.
Acute Disease↗
Pepsin as a marker for pulmonary aspiration.
BACKGROUND: Although assessment for aspiration of small volumes of gastric contents in tube-fed patients receiving mechanical ventilation is important, available methods for this purpose are not wholly satisfactory. A potential method is immunoassay of tracheal secretions for the gastric enzyme pepsin. OBJECTIVES: To determine the frequency with which pepsin in suctioned tracheal secretions from acutely ill, tube-fed patients receiving mechanical ventilation could be detected via an immunoassay. METHODS: A convenience sample of 136 specimens of suctioned tracheal secretions was collected from 30 acutely ill, tube-fed adults receiving mechanical ventilation. Multiple samples were obtained from 26 of the 30 patients (range, 2-11 per subject). An immunoassay with rooster polyclonal antibodies to purified human pepsin was used to detect pepsin in the secretions. RESULTS: Fourteen specimens tested positive for pepsin. Secretions from 5 patients accounted for the 14 pepsin-positive results. A significant relationship was found between the position of the head of the bed and the presence of pepsin in tracheal secretions (P<.001). Of the 14 pepsin-positive specimens, 13 (92.9%) were obtained from subjects in a flat position. CONCLUSIONS: A pepsin immunoassay can be used to detect pepsin in human tracheal secretions. If pepsin in tracheal secretions is considered an indicator of aspiration of gastric contents, aspiration occurred in 5 of the 30 subjects. A flat position is strongly associated with the presence of pepsin in tracheal secretions.
Enteral Nutrition↗
Lipase and pepsin activity in the gastric mucosa of infants, children, and adults.
The range of activity and the location of lipase and pepsin were determined in the stomach and duodenum of infants, children, and adults. The range of lipase activity in biopsy specimens from the gastric body, in 29 subjects aged from 3 months to 26 years, was 1.8-5.3 U/mg protein (1 U is 1 mumol [3H]oleic acid released from tri-[3H]olein per minute). There were no significant differences among age groups (5-19 months, 2-4 years, 6-10 years, 11-13 years, and 15-26 years). Lipase activity was low or undetectable in the gastric antrum of all subjects. Pepsin activity in specimens from the gastric body ranged from 180 to 780 pepsin units/mg protein (using hemoglobin as substrate). The antrum had significantly lower pepsin activity (P less than 0.001) than the gastric body. As with lipase activity, there were no statistically significant differences in pepsin activity among age groups. Lipase and pepsin activity was also quantified in pinch biopsy specimens from the duodenum and duodenal bulb in 13 subjects. Contrary to lipase activity, which was almost completely absent from the duodenum or duodenal bulb, these sites contained low pepsin activity (9-78 pepsin units/mg protein). The data show that in infants and children, as previously reported in adults, gastric lipase is localized primarily in the gastric body. Tissue pepsin levels and localization, reported here for the first time, are similar to those of lipase, although, contrary to lipase, the gastric antrum has considerable pepsin activity. The identical levels of lipase and pepsin activities in infants, children, and adults indicate that the gastric phase of nutrient digestion is well developed at birth.
Adolescent↗
[Effect of the degree of phosphorylation of bovine pepsin A on its enzymatic activity].
Proteolytic and clotting activities of bovine pepsin A with respect to its degree of phosphorylation were studied on various substrates. The occurrence of phosphate group(s) on bovine pepsin A more or less strongly affects its enzymic properties according to the substrate and its environment. This is particularly obvious as far as kappa-casein is concerned. The specific flocculating activity of unphosphorylated (fA0) as well as dephosphorylated (treated with potato acid phosphatase) bovine pepsin A, determined on a 0.2% kappa-casein solution, is significantly higher than that observed with phosphorylated pepsins, especially after kappa-casein was treated with alpha-D.N-acetyl galactosaminyl oligosaccharidase, while specific milk clotting activity remains unchanged regardless to the level of phosphorylation of bovine pepsin A is. Using haemoglobin as substrate, unphosphorylated pepsin A exhibits the highest specific proteolytic activity and the less acidic pH optimum. Conversely, the amount of phosphate groups does not seem to have any effect on the peptidase activity assayed towards the synthetic chromophoric hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe. By treating whole bovine pepsin A with potato acid phosphatase during 24 h at 37 degrees C and pH 5.6, using a 1/100 E/S ratio, almost complete dephosphorylation can be reached. The stability of different bovine pepsin A preparations, more or less phosphorylated, treated or not with phosphatase was also investigated. At pH 2.2, phosphorylated bovine pepsin A is twice more stable at 37 degrees C than the dephosphorylated enzymes while dephosphorylated pepsin does not exhibit any degradation at pH 5.6, judging by isoelectric focusing patterns, or loss of activity. Such a result suggests that post-translational phosphorylation might play an essential physiological function by improving the stability and integrity of pepsin in the bovine abomasum, the pH of which is very acidic (between 1.0 and 2.0).
Animals↗
Catalytic properties and chemical composition of pepsins from Atlantic cod (Gadus morhua).
1. Three pepsins were purified from the gastric mucosa of Atlantic cod (Gadus morhua). 2. The enzymes, called Pepsin I and Pepsin IIa and b, had isoelectric points 6.9, 4.0 and 4.1, respectively, and digested hemoglobin at a maximal rate at a pH of approximately 3. 3. They resembled bovine cathepsin D in being unable to digest the mammalian pepsin substrate N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine. 4. Specificity constants (kcat/Km) for the cod pepsins were lower than for porcine pepsin, and they expressed higher substrate affinity and physiological efficiency at pH 3.5 than at pH 2. 5. The cod pepsins are glycoproteins, and their amino acid composition resembles that of porcine cathepsin D more than that of porcine pepsin. 6. The N-terminal sequence of Atlantic cod pepsins is substantially different from that of porcine pepsin. This indicates a significant evolutionary gap between fish and mammalian pepsins.
Amino Acid Sequence↗
Cooperativity between pepsin and crystallization of calcium carbonate in distilled water.
Cooperativity between pepsin and crystallization of calcium carbonate in distilled water was studied. The results show that vaterite was formed under the influence of pepsin and the crystalline product was a composite of vaterite and pepsin. The component of this material was similar to that of nacre. At the same time, the crystallization of calcium carbonate had also an important effect on the secondary structure of the pepsin. The secondary structure of the pepsin was characterized through FT-IR technology. The result indicated that the pure pepsin is composed of 24.38% alpha-helices, 29.91% beta-sheets, 39.32% beta-turns and 6.49% random structures and the pepsin in the CaCO(3)-pepsin solution is composed of 2.09% alpha-helices, 93.304% beta-sheets, 4.592% beta-turns and 0.006% random structure. During the crystallization of the calcium carbonate from the pepsin solution, the secondary structure of the pepsin transformed. These results showed that there was cooperativity between the crystallization of vaterite and the pepsin. The cooperative mechanism is discussed.
Calcification, Physiologic↗
The diurnal profile of gastric pepsin activity is reduced with Helicobacter pylori infection.
Both Helicobacter pylori and pepsin are proven mucosal damaging agents and implicated in the aetiology of peptic ulcer disease. Historically studies of pepsin over time have proved methodologically difficult, and as a result little work has been done on the effect of H. pylori on luminal pepsin secretion. Our objectives were to determine pepsin activity over 24 hr in normal human subjects and to examine luminal pepsin activity in relation to H. pylori infection. Twenty-seven healthy volunteers had gastric juice samples aspirated every 2 hr for 24 hr. All subjects had H. pylori status determined by C13 urea breath test and serology. Meals were standardized throughout the study period. Gastric juice samples were measured for pH, diluted, and frozen in acetate buffer pH 4.1 for up to 1 month, conditions shown to cause no loss of activity. Individual samples were measured for pepsin activity by assaying for new N-terminal peptide formation. Mean pepsin activity (microg enzyme/ml) in 21 normal H. pylori-negative subjects ranged from 114 to 1030 microg/ml, with a characteristic diurnal profile of increasing activity to maximum after the evening meal. Mean pepsin activity in subjects with H. pylori was consistently below that for age-matched H. pylori-negative subjects at each time point. Overall mean pepsin activity was significantly lower in those with H. pylori compared to those without (P < 0.001). There is significant pepsin activity in the stomach throughout the 24-hr period, with a trend for the highest activity through the night. Subjects with H. pylori infection have lower luminal pepsin activity.
Adolescent↗
A history of pepsin and related enzymes.
Studies on gastric digestion during 1820-1840 led to the discovery of pepsin as the agent which, in the presence of stomach acid, causes the dissolution of nutrients such as meat or coagulated egg white. Soon afterward it was shown that these protein nutrients were cleaved by pepsin to diffusible products named peptones. Efforts to isolate and purify pepsin were spurred by its widespread adoption for the treatment of digestive disorders, and highly active preparations were available by the end of the nineteenth century. There was uncertainty, however, as to the chemical nature of pepsin, for some preparations exhibited the properties of proteins while other preparations failed to do so. The question was not settled until after 1930, when Northrop crystallized swine pepsin and provided convincing evidence for its identity as a protein. The availability of this purified pepsin during the 1930s also led to the discovery of the first synthetic peptide substrates for pepsin, thus providing needed evidence for the peptide structure of native proteins, a matter of debate at that time. After 1945, with the introduction of new separation methods, notably chromatography and electrophoresis, and the availability of specific proteinases, the amino acid sequences of many proteins, including pepsin and its precursor pepsinogen, were determined. Moreover, treatment of pepsin with chemical reagents indicated the participation in the catalytic mechanism of two aspartyl units widely separated in the linear sequence. Studies on the kinetics of pepsin action on long chain synthetic peptides suggested that the catalytic site was an extended structure. Similar properties were found for other "aspartyl proteinases," such as chymosin (used in cheese making), some intracellular proteinases (cathepsins), and plant proteinases. After 1975, the three-dimensional structures of pepsin and many of its relatives were determined by means of x-ray diffraction techniques, greatly extending our insight into the mechanism of the catalytic action of these enzymes. That knowledge has led to the design of new inhibitors of aspartyl proteinases, which are participants in the maturation of human immunodeficiency virus and in the generation of Alzheimer's disease.
Animals↗
Pepsin secretion in the anaesthetized cat and the effect of sympathetic nerve stimulation.
1. Experiments were performed on chloralose anaesthetized cats and gastric mucosal blood flow, acid and pepsin secretions were measured. Gastric mucosal pepsin and protein contents were measured at the end of the experiments which were done in three groups: gastrin (A), vagal (B), vagal and sympathetic nerve (C) stimulations. 2. Vagal stimulation significantly reduced (76%) the pepsin content of gastric mucosa compared with gastrin stimulated animals, none of which secreted pepsin. 3. The sum of the secreted and extracted pepsins for all the three groups was not significantly different. There was no significant difference in the extracted protein from any of the groups. 4. Gastric mucosal blood flow, acid and pepsin outputs all had significant correlations with time during the first 70 min of vagal stimulation. During the period 80-160 min of vagal stimulation acid secretion and mucosal blood flow were not correlated with time but pepsin output declined significantly. From 170 to 220 min of vagal stimulation, acid and pepsin outputs and mucosal blood flow were not correlated with time. 5. The assumed pepsin store during each period was calculated and after 40 min of vagal stimulation there is a constant percentage pepsin output from this assumed store. 6. There is some data to suggest that pepsinogen synthesis was occurring during the period 170-220 min of vagal stimulation. 7. Sympathetic nerve stimulation which started at 160 min after the beginning of sustained vagal stimulation, significantly inhibited gastric acid secretion and mucosal blood flow, and in addition it significantly inhibited pepsin secretion. This is consistent with the hypothesis that sympathetic nerve inhibition of gastric mucosal function is mediated by a vasoconstrictor mechanism.
Animals↗