The pepsins of normal human gastric juice

1. The frequency of occurrence, under defined conditions, of the different human pepsins in the gastric juices of 50 normal subjects was investigated by agar-gel electrophoresis. 2. From a total of eight proteolytic zones located in the zymograms, no significant differences of occurrence existed between the sexes, or between subjects with or without gastric symptoms. 3. Two zones, numbered 3 and 5, occurred in all normal gastric juices. Zone 3 always exhibited the greatest proteolytic activity, then zone 5. The remaining enzymic zones were less well-marked and occurred less frequently. 4. A minor zone, 3a, was demonstrated within zone 3. The corresponding pepsin, 3a, has a mobility towards the anode 6-7% greater than has pepsin 3. 5. Of the eight zones, 1,2,3,3a and 5, at least, represent unique pepsins.     

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.     

Studies on heterogeneity of human gastric zymogens

THE PEPTIC ZYMOGENS OF HUMAN GASTRIC MUCOSA HAVE BEEN STUDIED IN THE PAST BY THREE DIFFERENT TECHNIQUES: phosphate-gradient chromatography on a column of DEAE-cellulose, chloride-gradient chromatography on DEAE-cellulose and agar-gel electrophoresis. These techniques have given different results, each suggesting the presence of a different number of pepsin zymogens in human gastric mucosa. In the present experiments gastric mucosal homogenates were subjected to analysis by all three techniques. The two major zymogen peaks obtained from phosphate-gradient chromatography were found to be composed of the three fractions found on chloride-gradient chromatography; these fractions in turn were shown to be heterogeneous on agar-gel electrophoresis. The latter technique demonstrated the presence of seven separable enzymically active components in human gastric mucosal homogenates. The relationships of the components separated by the three techniques are discussed.     

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

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

Comparative Pepstatin Inhibition Studies on Individual Human Pepsins and Pepsinogens 1,3 and 5(gastricsin) and Pig Pepsin A

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

Re-formation of disulphide bonds in reduced antithrombin III.

The separation of pepsin isoenzymes 1, 2, 3 and 5 (gastricsin) in human gastric juice was effected by chromatography on Mono Q ion-exchanger, and slow-moving proteinase was purified to homogeneity by using a modified procedure incorporating a novel affinity-chromatography step. The pH-activity profiles of these enzymes with mucus glycoprotein and basement-membrane substrates were determined; the profiles for pepsin 2 were noticeably different, and, in general, the pH optima for the hydrolysis of basement membrane were more acidic. Pepsin 1 expressed larger specificity constants (kcat./Km) than pepsin 3 with a series of synthetic peptide substrates, reflecting greater binding (smaller Km) by pepsin 1. Inhibitor studies at pH 1.7 and 4.5 with a series of P2-substituted lactoyl-pepstatins implied that valine at position P2 was optimal for inhibiting pepsins 1, 2 and 3 but detrimental for pepsin 5, whereas lysine at position P2 was tolerated well by pepsin 5 but not by pepsins 1, 2 and 3. The potency of lactoyl-pepstatin with lysine at position P2 did not increase as a function of pH. P2-substituted lactoyl-pepstatins failed to show any inhibitory selectivity among pepsins 1, 2 and 3.     

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.     

Multiple forms of horse pepsin

Using ion-exchange and affinity chromatography and isoelectrofocusing, eight forms of pepsin with pI 1.6, 1.8, 2.1, 2.3, 2.6, 2.8, 3.2 and 3.6, were isolated from horse gastric juice. The molecular weights, amino acid composition, N-terminal sequence and functional activity of these multiple forms were determined. Partial primary structure of tryptic peptides of pepsin with pI 2.3 was investigated. The analyzed partial sequences of the forms with pI 1.8, 2.1, 2.3, and 2.6 have identical structures which differ from the amino acid sequence of pepsin with pI 3.2 by four substituents. In terms of their functional activity, horse pepsins differ only insignificantly. Presumably, the pepsins under study (at least the forms with pI 1.8, 2.1, 2.3, 2.6 and 3.2) arose comparatively recently as a result of duplication of the common precursor gene and exist at an early stage of structural and functional divergence. As far as their primary structure and functional properties are concerned, these pepsins are more related to pepsin A than to other isoenzymes of gastric aspartyl proteinases of mammalia, e. g., gastricsin or chymosin.     

Biochemical and immunological characterizations of rat pepsinogens and pepsins

Biochemical and immunological properties of two kinds of pepsinogens isolated from the gastric mucosal extracts of adult Wistar rats were studied. Their activated enzymes were prepared from the zymogens using a DEAE-Sepharose CL-6B column. The isoelectric points of pepsinogens I and II were estimated to be 3.90 and 3.75, respectively, by isoelectric focusing, and those of pepsins I and II to be 3.60 and 3.45, respectively. Amino acid compositions of the two pepsinogens or pepsins were strikingly similar to each other and neither pepsinogen I nor II contained organic phosphate. The biochemical properties of rat preparations compared with porcine pepsinogens A and C and pepsins A [EC 3.4.23.1] and C [EC 3.4.23.3] showed that rat pepsinogens and pepsins resembled porcine pepsinogen C and pepsin C, respectively. Pepsinogens I and II were demonstrated to share a similar immunogenic molecular structure by double diffusion analysis and Laurell immunoelectrophoresis. Rabbit antipepsinogen I serum cross-reacted with the mouse preparation but did not with the rabbit and porcine preparations. The possibility of the genetically controlled occurrence of pepsinogens I and II in the rat is discussed.     

A pepsinogen from rainbow trout

1. A pepsinogen, Ia on the basis of its electrophoretic mobility, from rainbow trout stomach, has an optimum pH near 2 for activation. 2. The cognate pepsin is denatured at pH values above 7, in contrast to the zymogen, which is slightly more alkali-stable. It has an optimum pH of 3 for proteolysis of denatured hemoglobin. 3. The intrinsic reactivity of the zymogen and pepsin (rates of activation and of proteolysis, respectively) are quite high, but as they operate at the environmental temperature of the fish, are remarkably similar to rates of activation and proteolysis by mammalian pepsinogens and pepsins.     

Studies on human gastric mucosal zymogen granules and their zymogens

Intact human gastric mucosal zymogen granules (ZG) were detected in specimens from surgical resections of one patient with gastric adenocarcinoma and two with benign gastric ulcer. Both large ZG with unilaminar membranes and smaller ZG with trilaminar membranes were identified by electron microscopy. The zymogens in the ZG and in mucosal extracts were separated by gel electrophoresis. Slow-moving Protease (SMP) was seen in the whole mucosal extracts but was absent from ZG. One specimen of pyloric mucosa showed a striking absence of ZG. Despite the absence of ZG, pyloric mucosa showed Pepsinogens 2-5 (constituents of PG I) as well as Pepsinogens 6 and 7 (constituents of PG II) and SMP.     

Gastric secretion in dogs following three day fasting and resumed feeding

The investigation into the influence of a three-day starvation on the gastric secretion in dogs with Pavlov pouches stimulated by meat, histamine and pentagastrin, was carried out. A 72-hour starvation did not change the summary volume of the gastric juice, debit of the gastric acid, and quantity of pepsin. At the same time the starvation decreased the average rate of gastric juice secretion, gastric acid and pepsin secretion in response to histamine and decreased the pepsin secretion in response to pentagastrin. In this way re-feeding enhanced the average rate of gastric juice secretion and gastric acid secretion on 3-day and pepsin on 5-day in response to meat. The average rate of gastric juice secretion increased on the 5-day after refeeding in response to histamine and the average rate of gastric juice, gastric acid and pepsin secretion in response to pentagastrin.     

Partial characterization of pepsins and gastricsins and their zymogens from human and toad gastric mucosae.

1. Three zymogens have been isolated from human gastric mucosae and two from the stomachs of the toad Caudiverbera caudiverbera. 2. Human zymogens I and III were immunologically related and cross-reacted with antisera prepared against porcine pepsinogen. The third, (II), showed no cross-reactivity. 3. Human zymogens I and III and toad zymogen ZII gave rise to two human pepsins and to a pepsin-like enzyme, respectively. 4. Human zymogen II (gastricsinogen) and toad zymogen ZI gave rise to human gastricsin and to a gastricsin-like enzyme respectively. 5. The toad enzymes showed much greater stability at neutral and alkaline pH values than the human enzymes.     

Pepsin-mediated processing of the cytoplasmic histone H2A to strong antimicrobial peptide buforin I

The intestinal epithelium forms a first line of innate host defense by secretion of proteins with antimicrobial activity against microbial infection. Despite the extensive studies on the antimicrobial host defense in many gastrointestinal tracts, little is known about the antimicrobial defense system of the stomach. The potent antimicrobial peptide buforin I, consisting of 39 aa, was isolated recently from the stomach tissue of an Asian toad, Bufo bufo gargarizans. In this study we examined the mechanism of buforin I production in toad stomach tissue. Buforin I is produced by the action of pepsin isozymes, named pepsin Ca and Cb, cleaving the Tyr39-Ala40 bond of histone H2A. Immunohistochemical analysis revealed that buforin I is present extracellularly on the mucosal surface, and unacetylated histone H2A, a precursor of buforin I, is localized in the cytoplasm of gastric gland cells. Furthermore, Western blot analysis showed that buforin I is also present in the gastric fluids, and immunoelectron microscopy detected localization of the unacetylated histone H2A in the cytoplasmic granules of gastric gland cells. The distinct subcellular distribution of the unacetylated histone H2A and the detection of the unacetylated buforin I both on the mucosal surface and in the lumen suggest that buforin I is produced from the cytoplasmic unacetylated histone H2A secreted into the gastric lumen and subsequently processed by pepsins. Our results indicate that buforin I along with pepsins in the vertebrate stomach may contribute to the innate host defense of the stomach against invading microorganisms.     

Gastric antibacterial efficiency is different for pepsin A and C

The gastric lumen represents a bactericidal barrier, whose major components are an acidic pH and a family of isoenzymes of the gastric aspartate protease, pepsin. To evaluate whether specific pepsins are specialized in antibacterial protection, we tested their effects on the gastric pathogen Helicobacter pylori. In a recent study we found pepsin to affect the motility of the bacteria, one of its most important virulence factors. We were able to show that the antibacterial effect of pepsin occurs in two phases: rapid loss of motility and subsequent destruction. In the present study we used the rapid pepsin-induced bacterial immobilization as a marker of antibacterial efficiency. The proteolytic activity of different pepsins was normalized to values between 2 and 200 U/ml in the hemoglobin degradation test of Anson, performed at pH 2 and 5. We found that pepsin C completely inactivates H. pylori at proteolytic activities of 2 (pH 5) and 20 (pH 2) U/ml. In contrast, the activities of pepsin A and chymosin required to affect Helicobacter motility were ten times higher.     

Pepsin and the esophagus

Esophagitis results from excessive exposure of the esophagus to gastric juice through an ineffective or dysfunctional lower esophageal sphincter mechanism. A possible role of pepsin in damaging the esophageal mucosa with consequent esophagitis may be examined directly by testing pepsin under various conditions in experimental models of esophagitis. Since gastric juice contains both acid and pepsin, all experiments examine separately effects of perfusion of the esophagus by acid without and with pepsin in various combinations. Acid perfusion alone at concentrations represented by pH 1.3 or above does not produce esophagitis. The addition of pepsin to acid between pH 1 and 3.5 causes considerable acute esophageal damage. Outside the proteolytic range, i.e., higher than pH 3.5, pepsin does not damage the esophagus. The damage caused by acidified pepsin may be made much worse by the further addition of aspirin or other NSAIDs, presumably by further breaking down mucosal barriers.     

电刺激室旁核对大鼠应激性胃粘膜损伤的影响

电刺激室旁核(PVN)有加重大鼠应激性胃粘膜损伤的作用;PVN内微量注射神经元胞体兴奋剂L-谷氨酸钠和电刺激PVN的效应相同;电解损毁双侧PVN或对其电刺激后,使应激性胃粘膜损伤明显减轻,切断膈下迷走神经或皮下注射阿托品后,可显著减轻电刺激PVN加重大鼠应激性胃粘膜损伤的效应;电刺激PVN使胃粘膜血流量减少,但对胃液量、胃酸排出量、胃蛋白酶活性及胃壁结合粘液量均无显著影响。从而表明,PVN是影响应激性胃粘膜损伤的特异性中枢部位之一,当其兴奋时,可加重应激性胃粘膜损伤,并可能是通过迷走神经胆碱能纤维起作用的,且与胃粘膜血流量的减少有关;电刺激PVN加重胃粘膜损伤似不是由胃液量、胃酸、胃蛋白酶活性及胃壁结合粘液量等因素的改变引起的。     

Gastric proteases of the Greenland cod Gadus ogac. II. Structural properties

Three gastric proteases were isolated from the stomach mucosa of the Greenland cod (Gadus ogac). The cod proteases were all less stable to heating and protease 1 retained less activity at 5 degrees C when the pH was greater than 5 in comparison with porcine pepsin. The activities of cod proteases 1 and 2, with hemoglobin as the substrate, were doubled in the presence of 25 mM NaCl, while cod protease 3 and porcine pepsin were not stimulated by the salt. The cod proteases did not cross-react with antibodies raised against porcine pepsin. However, some cross-reactivity was noted with antibodies raised against proteases from psychotrophic pseudomonads. The molecular weights of all the cod proteases were in the range of 36,000-38,000. The amino acid compositions of the cod proteases as compared by the Metzger difference index differed from the mammalian gastric proteases by about the same extent that pepsin, gastricsin, and chymosin differ from each other. Of the cod enzymes, protease 1 differed from mammalian gastric proteases, while cod proteases 3 was more like chymosin with respect to amino acid composition. Cod protease 1 had the lowest hydrophobicity index and chymosin had the highest. The hydrophobicity indices of cod proteases 2 and 3 were intermediate between that of porcine pepsin and bovine chymosin. It is suggested that the Greenland cod proteases represent less differentiated forms of gastric proteases than the mammalian pepsins, gastricsins, and chymosins.     

Tuna pepsinogens and pepsins. Purification, characterization and amino-terminal sequences

Three pepsinogens (pepsinogens 1, 2, and 3) were purified from the gastric mucosa of the North Pacific bluefin tuna (Thunnus thynuus orientalis). Their molecular masses were determined to be 40.4 kDa, 37.8 kDa, and 40.1 kDa, respectively, by SDS/polyacrylamide gel electrophoresis. They contained relatively large numbers of basic residues when compared with mammalian pepsinogens. Upon activation at pH 2.0, pepsinogens 1 and 2 were converted to the corresponding pepsins, in a stepwise manner through intermediate forms, whereas pepsinogen 3 was converted to pepsin 3 directly. The optimal pH of each pepsin for hemoglobin digestion was around 2.5. N-acetyl-L-phenylalanyl-L-diiodotyrosine was scarcely hydrolyzed be each pepsin. Pepstatin, diazoacetyl-DL-norleucine methyl ester in the presence of Cu2+, 1,2-epoxy-3-(p-nitrophenoxy)propane and p-bromophenacyl bromide inhibited each pepsin, although the extent of inhibition by each reagent differed significantly among the three pepsins. The amino acid sequences of the activation segments of these pepsinogens were determined together with the sequences of the NH2-terminal regions of pepsins. Similarities in the activation segment region among the three tuna pepsinogens were rather low, ranging over 28-56%. A phylogenetic tree for 16 aspartic proteinase zymogens including the three tuna pepsinogens was constructed based on the amino acid sequences of their activation segments. The tree indicates that each tuna pepsinogen diverged from a common ancestor of pepsinogens A and C and prochymosin in the early period of pepsinogen evolution.     

Regional variation of Puccinia coronata avenae in Australia and its implications for oat breeding

A virulence survey of Puccinia coronata avenae was conducted in Australia from 1977 to 1980 with a chosen series of resistant oat lines as a tester set, viz., Ascencao, Pc-38, Pc-39, Pc-45, Pc-48, Pc-50, Pc-55, Pc-56 and TAM 0–312. Isolates of the pathogen were classified by using these nine tester lines in combination with the International Differential Set. The survey area was divided into six zones (1) -Queensland; (2) - northern New South Wales; (3) - southern New South Wales; (4) - Victoria; (5) - South Australia and (6) - Western Australia. Virulence for Pc-38 was widespread throughout Australia and virulences or partial virulences for lines Pc-39, Pc-45, Pc-48 and Pc-55, although not as frequent, were common in Zones 1, 2 and 3. No isolates were found with virulences for oat lines Pc-56 and TAM 0–312, while virulences for Ascencao or Pc-50 were rare. The combination of virulences or partial virulences for lines Pc-39, Pc-45 and Pc-48 was relatively common only among samples from Zones 1, 2 and 3. The diversity of the pathogen population varied between zones. Isolates from Zones 1 to 6 comprised 16, 21, 9, 8, 5 and 4 different standard races and mean virulence values were 5-0, 3–8, 3-0, 2–7, 2–6, 3-1 and 3-0, respectively. The proportion of complex strains (virulence value 5 or higher) decreased from 48-5% (Zone 1) and 17-0% (Zone 2) to 1–7% (Zone 3), 6-3% (Zone 4), 0% (Zone 5) and 13-9% (Zone 6). Greater diversity of the pathogen population in Zones 1 and 2was indicated by a higher incidence of strains with virulence for one or more of the nine resistant oat tester lines.