Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A neutron structure analysis at 2.2-A resolution has been performed on bovine trypsin covalently inhibited by a transition-state analogue, the monoisopropylphosphoryl (MIP) group. The unique ability of neutron diffraction to locate hydrogen atoms experimentally has allowed the determination of the protonation states of the catalytic site residues (Asp-102 and His-57). Since the bound MIP group mimics the tetrahedral intermediate structure, these correspond to the protonation states at the most crucial step of the hydrolysis. This has resolved a much debated mechanistic issue by showing conclusively that the catalytic base in the transition state of the reaction is His-57, not Asp-102. This finding has important implications for the understanding of the hydrolysis mechanism of the serine proteases. A detailed examination of the stereochemical interaction among the catalytic groups was also conducted to identify their individual roles in the mechanism. Besides functioning as the catalytic group, it was found that His-57 could effectively "steer" the attacking water toward the acyl group during deacylation. Other aspects of protein structure which are observable only by neutron diffraction analysis are also discussed. These include orientation of well-ordered amide side chains, which is made possible by the large scattering difference between nitrogen and oxygen atoms, location and orientation of water molecules, and hydrogen exchange properties of the protein.
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PMID:Direct determination of the protonation states of aspartic acid-102 and histidine-57 in the tetrahedral intermediate of the serine proteases: neutron structure of trypsin. 703 Mar 93

Glucose or sucrose grown cells of Streptococcus mitis ATCC 903 bind spontaneously to the surface of each other producing visible microbial aggregates upon incubation in 10 mM phosphate, citrate-phosphate or tris-maleate buffers. Aggregation was delayed and proceeded at a slower rate when bacteria grown in a culture medium with a low carbohydrate/nitrogen ratio were used. Growth in this culture medium resulted in carbohydrate limitation. The aggregation was highly reproducible and was unaffected by pH in the range of 4.4-7.0 but was decreased at pH 8.0 and completely inhibited at pH 9.0. No inhibition of the reaction was observed when a series of simple and complex carbohydrates were added. There was no significant difference in the rate of aggregation at 20, 30 and 37 degrees C. Aggregation occurred at a demonstrable rate of 0 degrees. Chloramphenicol did not inhibit aggregation. Since inhibition of aggregation was obtained by treatment of bacteria with trypsin or heat it appears that protein of glycoprotein components on the bacterial surface were involved in the reaction.
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PMID:Spontaneous aggregation of streptococcus mitis ATCC 903. 704 18

1. Pigs (twenty-one/diet) were weaned at 2 d of age and given liquid diets (200 g dry matter/l) at hourly intervals during a 26 d experiment. The pigs were fed on a scale based on live weight. The diets contained (g/kg DM): dried skim-milk 730 (diet A), dried whey 508.5, isolated soya-bean protein 218, DL-methionine 3.5 (diet S), and soya-bean oil 270 (diets A and S). Diet T contained equal proportions of diets A and S. Soya-bean supplied 0, 370 and 740 g crude protein (nitrogen x 6.25) kg total crude protein in diets A, T and S respectively. 2. Performance was similar for both diets A and T (P > 0.05). Pigs given diet S scoured severely, and fourteen died. The survivors grew very poorly. Nitrogen retention (g/d per kg live weight) was greater for diet A compared with diet T (P < 0.01), and decreased with age (P < 0.001). 3. Protein digestion was examined in the pigs killed at 28 d of age. The amount of soya-bean protein in the diet did not affect the amount of digesta in the stomach, but soya-bean protein decreased the pH, DM and total N content of the digesta (P < 0.01), and increased, though not significantly (P > 0.05), pepsin activity in the digesta and stomach tissue. Acid secretion into the stomach appeared to be enhanced in pigs given a diet containing soya-bean protein. 4. Amounts of trypsin, chymotrypsin, total N and proportion of non-protein-N in the digesta from the small intestine were similar for both diets A and T. The amounts for both diets were greater in the distal compared with the proximal region of the small intestine (P < 0.05). Chymotrypsin activity in the pancreas was reduced (P < 0.05) in pigs given diet T, although this reduction did not seem to impair digestion in 28-d-old pigs. Trypsin activity in the pancreas was similar for both diets A and T. 5. It seems likely that the neonatal pig does not have the digestive capacity to tolerate the large daily intakes of soya-bean protein when dried skim-milk was totally replaced in the diet (diet S). When half the dried skim-milk was replaced, protein digestion was not impaired in 28-d-old pigs.
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PMID:Artificial rearing of pigs. 11. Effect of replacement of dried skim-milk by an isolated soya-bean protein on the performance of the pigs and digestion of protein. 719 26

1. Four pelleted diets were prepared containing milk or isolated soya-bean protein (ISP) as the major protein source. The milk and ISP were given either as intact proteins or partially (0.650 hydrolyzed with papain before feeding. 2. The diets were given ad lib. to thirty-two pigs from 7-28 d of age. The pigs were slaughtered at 28 d of age. 3. Weight gains, food conversion ratios and nitrogen balances of pigs given diets containing milk protein were better than those of diets containing ISP (231 g/d, 0.80 and 11.5 g/d compared to 209 g/d, 0.88 and 9.00 g/d respectively). 4. Partial hydrolysis of proteins before feeding did not affect the performance of the pigs. 5. Apparent digestibilities of N before the ileum and in the whole tract were 0.78 and 0.94 for the pigs given the ISP diets and 0.86 and 0.97 for the pigs given the milk-protein diets. 6. Retention time of ISP diets in the whole digestive tract was 1475 min and that of the milk-protein diets was 1089 min. 7. pH of digesta in the stomach was 5.0-5.3 for all diets and increased to 6.9-7.1 in the ileum. 8. There were no differences in flows of total N and protein N to the ileum and lower digestive tract between the pigs given the intact-and hydrolyzed-protein diets. 9. Apparent absorptions of N in the stomach, duodenum and jejenum were greater in the pigs given diets containing hydrolyzed proteins than in those given diets containing the intact proteins. 10. Flows of total N and protein N to the ileum tract were greater when the pigs were given the ISP diets than when they were given the milk-protein diets. 11. Hydrolysis of proteins before feeding resulted in a reduced trypsin and chymotrypsin activity in the duodenum and pancreas. 12. Retention of dietary N in the carcass was greater in pigs given the milk-protein diets (0.79) than in those given the ISP diets (0.68).
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PMID:Digestion in the pig between 7 and 35 d of age. 6. The digestion of hydrolyzed milk and soya-bean proteins. 719 57

Protein solubility under different pH conditions, amino acid composition of proteins in the flours, and proteins isolated at a pH of 4.5 (P.I.), as well as trypsin inhibitor and hemagglutinin activities were studied in five Brazilian soybean varieties. The levels of protein and non-protein amino compounds were similar for all varieties. More protein was extracted in slightly alkaline (pH 8.5) water solution than with plain water. Lowering the pH of the extract to 4.5 caused the precipitation of 83% to 86% of the protein. Of the nitrogen remaining in the whey 31% to 39% could be precipitated with 5% trichloroacetic acid. Protein isolated inhibited amino acid patterns similar to those of defatted flours. Trypsin inhibitor activities were higher in the plain water extracts than in the extracts at pH 8.5. Most of the trypsin inhibitory activity was associated with the whey fraction, soluble at a pH of 4.5, while the hemagglutinin activities decreased considerably in the pH 4.5 supernatant, indicating precipitation of the hemagglutinins in this pH.
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PMID:Antinutrient occurrence and some physicochemical properties of the protein fractions of five Brazilian soybean varieties. 719 46

1. Pigs (sixteen/diet) were weaned at 2 d of age and given liquid diets (200 g dry matter/l) during a 26 d experiment. The pigs were fed on a scale based on live weight. Dried skim-milk was the only source of protein in diet U and was partially or totally replaced by a soya-bean isolate (diet B) or a concentrate (diets C and D). Soya-bean protein provided 500, 700 or 350 g/kg total crude protein (nitrogen x 6.25) in diets B, C and D respectively. 2. Performance was similar for diets B and D, but poorer than that of pigs given diet U. The apparent digestibility and retention of N of these diets was similar. Pigs given diet C scoured severely and twelve died. 3. Protein digestion was studied in pigs given diets U, B and D, killed at 28 d of age, at the termination of the feeding experiment. The dry matter content and proportion of N in the digesta in the stomach were reduced in pigs given soya-bean protein. Pepsin concentrations in digesta and stomach tissue were unchanged. 4. The concentrations of trypsin and chymotrypsin in the pancreas were greater in pigs given the soya-bean protein concentrate compared with milk protein, but only the increase in trypsin was significant (P less than 0.01). Digesta from the small intestine of pigs given the soya-bean-protein isolate contained less chymotrypsin (P less than 0.05). There were no differences in the proportion of non-protein-N in the total N in the digesta, suggesting that proteolysis of the milk and soya-bean proteins were equally by 28 d of age.
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PMID:Artificial rearing of pigs. 12. Effect of replacement of dried skim-milk by either a soya-protein isolate or concentrate on the performance of the pigs and digestion of protein. 720 50

Anti-rat glomerular basement membrane (GBM) rabbit serum was produced by immunizing rabbits with the supernatant substance of trypsin-digested rat GBM. Nephritis was induced in rats by a single intravenous administration of 0.25 ml of anti-serum and changes in pathohistological and biochemical parameters during the process of the disease were investigated in comparison with those of Masugi nephritis and the modified type of Masugi nephritis previously reported. In light microscopic studies, histological changes seen in the kidneys closely resembled those of typical human glomerulonephritis. Changes such as hypercellularity, adhesion between capillary wall and Bowman's capsule, crescent formation and hyalinization in glomeruli and interstitial infiltration were the most pronounced on the 30th day after the anti-serum injection. In immunofluorescent studies, a linear fixation of rabbit IgG was observed along the GBM from the 1st day and the staining of a certain intensity was preserved throughout the experimental periods. A linear staining with anti-rat IgG serum was recognized from the 10th day. The fixation of fibrinogen was also seen in not only the glomerular capillary walls, but also in Bowman's space after the 10th day. Proteinuria significantly increased from the 1st day, reached a peak of 12 times the control level, and thereafter gradually decreased. The patterns of progress of urinary alkaline phosphatase and N-acetyl-beta-glucosaminidase activities were much the same as those seen in cases of proteinuria and the levels at their peak times were about 10 and 3 times control levels, respectively. Plasma urea nitrogen level transiently increased on the 5th day and then reverted to the control level by the 30th day. Plasma cholesterol levels were significantly high from the 5th to the 20th days. It is concluded that glomerular damages in this model are more severe, so-called, "nephritic type" and continue for longer periods than in cases of Masugi nephritis, however, do not differ in degree and duration from findings in the modified type of Masugi nephritis.
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PMID:[Pharmacological studies on experimental nephritic rats (11). Changes in pathohistological and biochemical parameters in anti-rat GBM rabbit serum-induced nephritis (author's transl)]. 728 45

The complement protein C3, when activated by limited proteolysis, forms a short-lived reactive intermediate fragment, 'nascent' C3b, which is known to bind covalently to certain surfaces. The characteristics of the covalent binding reaction have been studied by using Sepharose-trypsin as a combined proteolytic activator and binding surface for C3. Binding of C3 to Sepharose-trypsin is saturable, with a maximum of 25-26 molecules of C3b bound per molecule of trypsin. A minimum life-time of about 60 microseconds for the reactive intermediate has been calculated from binding of C3 at saturation. Initial binding efficiencies of over 30% can be obtained at physiological pH and ionic strength. The efficiency of C3 binding to Sepharose-trypsin decreases as pH increases and also shows a slight decline at high ionic strength. The covalent binding of C3 to Sepharose-trypsin can be inhibited by a range of oxygen and nitrogen nucleophiles. Activation of C3 in the presence of radioactive forms of four such nucleophiles, phenylhydrazine, methylamine, glycerol and glucosamine results in apparent covalent incorporation of the nucleophile into the C3d fragment of C3. The quantity of radioactive nucleophile bound can be predicted from the observed potency of the nucleophile as an inhibitor of the binding of C3 to Sepharose-trypsin. The radioactive nucleophiles may be considered as 'active-site' labels for C3.
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PMID:The covalent-binding reaction of complement component C3. 730 16

T200 glycoprotein, a major cell surface component of murine hematopoietic cells, is a phosphorylated transmembrane glycoprotein. Two distinct regions of the molecule can be defined by radiolabeling with a variety of metabolic precursors or by lactoperoxidase-catalyzed iodination, in combination with protease treatments, immunoprecipitation techniques, and peptide "mapping" analysis. A relative protease-resistant domain, which is exposed on the cell surface and contains the antigenic site recognized by a monoclonal anti-T200 antibody known to react with the exterior cell surface, contains most if not all of the mannose-containing oligosaccharide units of the glycoprotein and all of the amino acid residues labeled by lactoperoxidase-catalyzed iodination of intact viable cells. This protease-resistant fragment migrates with an apparent molecular weight of approximately 100,000 in sodium dodecyl sulfate-polyacrylamide gels. The remaining portion of the molecule contains a region, extensively digested by trypsin, which is exposed on the cytoplasmic side of the plasma membrane and contains phosphoserine residues which can be labeled with 32PO4 in vivo. A 125I-labeled tryptic peptide derived from this region of the molecule was obtained if membrane preparations from cells disrupted by nitrogen cavitation were labeled by lactoperoxidase-catalyzed iodination.
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PMID:Disposition of T200 glycoprotein in the plasma membrane of a murine lymphoma cell line. 735 47

Treatment of human C3 with hydroxylamine or hydrazine at physiological pH and ionic strength totally abrogates the intrinsic ability of this protein to sustain classical pathway induced hemolysis of sheep red blood cells. Concomitant with the loss of this function the appearance of a single sulfhydryl group can be followed by titration with the sulfhydryl-specific reagents p-(chloromercuri)benzoate, [1-14C]iodoacetamide, 2,2'-dipyridyl disulfide, and 5,5'-dithiobis(2-nitrobenzoic acid). These reagents have also been used to follow the appearance of a free sulfhydryl group on conversion of C3 to C3b with bovine trypsin. Autoradiography of the electrophoretogram of separated alpha-, alpha'-, and beta-polypeptide chains of inactivated, [1-14C]carboxamidomethylated C3 samples has shown that the reactive sulfhydryl group is present in the alpha chain of C3 and in the alpha' chain of C3b, respectively. Digestion of the radiolabeled protein with porcine elastase has localized this sulfhydryl group to a 28 000-dalton fragment of the alpha chain with immunochemical and functional reactivities of the C3d domain. Autoradiographic analysis of a hydrolysate prepared from radioalkylated C3 and subjected to high-voltage paper electrophoresis has shown the labeled amino acid to be [1-14C]-S-(carboxymethyl)cysteine. The susceptibility of native C3 to rapid and irreversible inactivation by nitrogen nucleophiles with the parallel appearance of a cysteinyl residue may indicate the presence of an internal thiol ester. The relationship of the proposed thiol ester to the ability of nascent C3b to acylate cell surface components and carbohydrate polymers is discussed within the context of a transesterification reaction.
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PMID:Third component of human complement: appearance of a sulfhydryl group following chemical or enzymatic inactivation. 740 85


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