Monkey pepsinogens and pepsins. III. Carbohydrate moiety of Japanese monkey pepsinogens and the amino acid sequence around the site of its attachment to protein.
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Purified Japanese monkey pepsinogens I and II contain carbohydrate as a part of the enzyme molecule. By gel filtration on Sephadex G-100, chromatography on DE-32 cellulose, and polyacrylamide disc gel electrophoresis, the carbohydrate moiety could not be separated from the enzyme protein, and the content did not decrease on repeated chromatography. Glycopeptides were obtained by successive digestion of pepsinogens with thermolysin and aminopeptidases and isolated by chromatography on Sephadex G-25 and G-50. Identification and determination of carbohydrate components was performed by paper and gas-liquid chromatographies. The presence of 4 glucosamines, 6 galactoses, 6--8 mannoses, and 8--11 fucoses per molecule of the glycopeptide of both pepsinogens was observed, of which the high content of fucose is especially unique. The molecular weight of the carbohydrate chains should be around 4,000--5,000. The amino acid sequence of a major glycopeptide was deduced to be Ile-Gly-Ile-Gly-Thr-Pro-Gln-Ala-Asn, in which the asparagine residue is the site of attachment of the carbohydrate chain. (+info)
Binding of pepsinogen to the 78 kDa gastrin binding protein.
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An endogenous ligand of the 78 kDa gastrin-binding protein (GBP) has been purified from detergent extracts of porcine gastric mucosal membranes by ion exchange chromatography and preparative gel electrophoresis. The ligand bound to the GBP with high affinity (mean IC50 value of 0.31+/-0.09 microgram/ml, or 8 nM), as assessed by inhibition of cross-linking of iodinated gastrin2,17 to the GBP. Both the N- and C-terminal halves of the GBP, which had been expressed individually as glutathione-S-transferase fusion proteins in Escherichia coli, and purified on glutathione-agarose beads, bound the ligand. Two peptides derived from the ligand were purified by reversed-phase high-performance liquid chromatography (HPLC), and characterised by mass spectrometry and Edman sequencing. The peptides were 97% and 100% identical, respectively, to amino acids 119-157 and 199-219 of porcine pepsinogen A. Commercial samples of pepsinogen also bound to the GBP, with a mean IC50 value of 3.9+/-1. 2 micrograms/ml (100 nM). We conclude that the ligand is closely related, but not identical, to pepsinogen A. (+info)
Significance of serum pepsinogens and their relationship to Helicobacter pylori infection and histological gastritis in dialysis patients.
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BACKGROUND: Previous investigations reported that patients undergoing dialysis therapy had significantly higher serum pepsinogen (PG) levels than patients with normal renal function. However, in dialysis patients, the relationship between serum PG levels and Helicobacter pylori infection remains unknown. METHODS: Sixty three maintenance dialysis patients (54 haemodialysis and nine continuous ambulatory peritoneal dialysis) who required endoscopic examination were enrolled in the study. Sixty four age- and sex-matched patients with normal renal function served as controls. We performed endoscopic examination and obtained both the gastric antral and corpus mucosa for histopathological evaluation and H. pylori identification. Twenty three patients on dialysis underwent H. pylori eradication therapy. RESULTS: In dialysis patients, H. pylori-positives had significantly higher serum PG II levels than H. pylori-negatives (26.6+/-21.5 vs 14.1+/-7.1 ng/ml, P<0.05), but no significant difference was found in serum PG I between H. pylori-positives and H. pylori-negatives (228.8+/-158.5 vs 179. 4+/-113.5 ng/ml). There was no significant difference in serum PG II between dialysis patients and controls (19.9+/-16.5 vs 18.6+/-14.9 ng/ml), while serum PG I levels were significantly higher in dialysis patients than in controls (201.7+/-136.8 vs 77.6+/-85.8 ng/ml, P<0.05). Serum PG II levels, but not those of PG I, significantly correlated with the inflammation and activity scores of antrum in dialysis patients, and these scores were highly influenced by H. pylori infection. Dialysis patients in whom H. pylori was eradicated successfully showed significant reductions of serum PG II levels but not of PG I. CONCLUSIONS: In dialysis patients, high serum levels of PG II, but not PG I, are significantly related to H. pylori infection and mucosal inflammation. A significant decrease in serum PG II levels could be used as a predictor of the eradication of H. pylori infection in dialysis patients. (+info)
Mechanism of intramolecular activation of pepsinogen. Evidence for an intermediate delta and the involvement of the active site of pepsin in the intramolecular activation of pepsinogen.
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Intramolecular pepsinogen activation is inhibited either by pepstatin, a potent pepsin inhibitor, or by purified globin from hemoglobin, a good pepsin substrate. Also, pepsinogen at pH 2 can be bound to a pepstatin-Sepharose column and recovered as native zymogen upon elution in pH 8 buffer. Kinetic studies of the globin inhibition of pepsinogen activation show that globin binds to a pepsinogen intermediate. This interaction gives rise to competitive inhibition of intramolecular pepsinogen activation. The evidence presented in this paper suggests that pepsinogen is converted rapidly upon acidification to the pepsinogen intermediate delta. In the absence of an inhibitor, the intermediate undergoes conformational change to bind the activation peptide portion of this same pepsinogen molecule in the active center to form an intramolecular enzyme-substrate complex (intermediate theta). This is followed by the intramolecular hydrolysis of the peptide bond between residues 44 and 45 of the pepsinogen molecule and the dissociation of the activation peptide from the pepsin. Intermediate delta apparently does not activate another pepsinogen molecule via an intermolecular process. Neither does intermediate delta hydrolyze globin substrate. (+info)
Probable genetic linkage between a locus for human urinary pepsinogen and the HL-A loci.
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The genetic basis of familial variation in the relative intensities of human urinary pepsinogen isozymes is not completely clear from family studies. An investigation of the linkage relationships of pepsinogen isozyme 5, considering only segregation for the presence or absence of Pg 5, yields a peak lod score of 4.1 at theta = .1 for linkage with HL-A1 or HL-A2. Added to data from segregation interpreted according to a scheme proposed for the inheritance of intensity differences in Pg 5, the peak lod score becomes 3.0 at theta = .2. Data derived from the segregation of pepsinogen isozyme 4, possibly determined by an allele to that controlling the presence or absence of Pg 5, further reduces the total lod score at theta = .2 to 2.9. The results indicate probable linkage between a locus for urinary pepsinogen and the HL-A loci, but are insufficient to permit any conclusion concerning possible heterogeneity in the linkage relationships of Pg 4 and Pg 5 to HL-A. (+info)
Pepsinogen C and pepsin C from gastric mucosa of Japanese monkey. Purification and characterization.
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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. (+info)
Helicobacter pylori eradication therapy improves atrophic gastritis and intestinal metaplasia: a 5-year prospective study of patients with atrophic gastritis.
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AIM: : To investigate the effect of the eradication of Helicobacter pylori on histological gastritis. METHODS: : Twenty-six patients with moderate to severe atrophy received successful eradication therapy of H.pylori. Four patients dropped out and 22 were followed up prospectively for 5 years. The grades of gastritis were estimated from gastric biopsy specimens. The grade of intestinal metaplasia was also evaluated by dye-endoscopy using methylene blue (methylthioninium chloride). The serum levels of pepsinogen, gastrin and anti-parietal cell antibody were also determined. RESULTS: : The grades of atrophy decreased in patients with successful eradication therapy in the gastric corpus (before vs. 5 years after eradication, 2.09 +/- 0.15 vs. 0.91 +/- 0.17; P < 0.01) and in the antrum (2.14 +/- 0.17 vs. 1.36 +/- 0.17; P < 0.01). The levels of intestinal metaplasia were also decreased in the corpus (0.91 +/- 0.24 vs. 0.50 +/- 0.16; P < 0.05) and in the antrum (1.41 +/- 0.20 vs. 1.00 +/- 0.16; P < 0.05), which was also demonstrated by the methylene blue (methylthioninium chloride) staining method (33.4 +/- 8.2% vs. 23.0 +/- 6.5%; P < 0.05). The improvement of corpus atrophy correlated well with the high serum level of pepsinogen I (P = 0.005), but showed no correlation with the levels of anti-parietal cell antibody. CONCLUSIONS: : These results suggest that gastric atrophy and intestinal metaplasia are reversible events in some patients. (+info)
Construction, expression and characterization of a chimaeric mammalian-plant aspartic proteinase.
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Aspartic proteinases are a well-characterized class of proteinases. In plants, all nascent aspartic proteinases possess a 100-amino-acid, plant-specific sequence (PSS) within their C-terminal lobe, presumed to possess a targeting role in vivo. In this study, the PSS domain from the Arabidopsis thaliana aspartic proteinase was inserted into porcine pepsinogen at the identical location found in nascent plant aspartic proteinases, to create a chimaeric mammalian-plant enzyme. Based on enzymic activity, this chimaeric enzyme demonstrated increases in pH stability above 6 and temperature stability above 60 degrees C compared with commercial pepsin. Differential scanning calorimetry of the chimaeric enzyme illustrated an approx. 2 degrees C increase in denaturation temperature ( T (m)), with increases in co-operativity and similar enthalpy values. Kinetic analysis indicated an increase in K (m) and decreased k (cat) compared with pepsin, but with a catalytic efficiency similar to the monomeric plant aspartic proteinase from wheat. Using oxidized insulin B-chain, the chimaeric enzyme demonstrated more restricted substrate specificity in comparison with commercial pepsin. This study highlights the use of a chimaeric enzyme strategy in order to characterize unique protein domains within enzyme families, and, for the first time, a putative structure-function role for the PSS as it pertains to plant aspartic proteinases. (+info)