Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The pH of the hamster sperm acrosome was estimated by a method based on the distribution of monoamines between membrane enclosed volumes maintaining pH gradients. A fluorescent amine, 9-aminoacridine, was used to permit both microscopic and fluorometric measurements of amine distribution. Cauda epididymal hamster sperm incubated with 9-aminoacridine accumulated the amine in the acrosomal volume. In the presence of NH4Cl or the ionophore Nigericin (compounds which discharge pH gradients) 9-aminoacridine fluorescence disappeared from the acrosome. Amine distribution between the acrosome and external volume was estimated by fluorometric measurement of sperm filtrates in the presence and absence of NH4Cl and Nigericin. These values, together with an estimated acrosomal volume of 0.4mu3 were used to calculate an acrosomal pH of less than 5. In addition, an acrosomal pH of 5 or less was obtained with 14C-methylamine. We suggest that such an acidic acrosomal pH of 5 or less could serve to inhibit the activation or autoactivation of the acrosomal zymogen proacrosin to acrosin, a trypsin-like enzyme involved in fertilization.
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PMID:The pH of the hamster sperm acrosome. 2 69

An immunological study of anthranilate synthetase (ASase) has been initiated using quantitative precipitation, enzyme neutralization, and immunodiffusion methods. Cross-reactivity of anthranilate synthetase-anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase (ASase-PRTase) from Escherichia coli, Klebsiella aerogenes, and Salmonella typhimurium and ASase from Serratia marcescens and Pseudomonas putida was detected with antibodies to ?E. coli trypsin-treated ASase. Cross-reactivity of antigens was also obtained with S. marcescens anti-ASase. Indices of dissimilarity verified the overall structural similarity of ASase-PRTase from E. coli, K. aerogenes, and S. typhimurium and the divergence from S. marcescens ASase. Further divergence of these enzymes from ASase in B. subtilis and P. putida was apparent. Precipitation of ASase components I and II (ASase CoI and ASase CoII) was obtained using anti-ASase or antiserum fractionated to contain component-specific antibodies. Anti-ASase inhibited enzyme activity to binding to determinants on both subunits. Anti-ASase CoI inhibited the ammonia-dependent reaction and interfered with amide transfer from glutaminyl-ASase CoII. Anti-ASase CoII inhibited the glutamine reaction by blocking amide transfer. Enzyme neutralization experiments indicate more conservation of determinants at the active site region of ASase CoII compared to ASase CoI in the enterobacteria. A particulate form of ASase-PRTase in E. coli, K. aerogenes, and S. typhimurium could be distinguished by quantitative precipitation and immunodiffusion.
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PMID:Immunological study of anthranilate synthetase. 5 Mar 16

Tertiary amine local anesthetics cause a time- and dose-dependent, reversible increase in insulin binding sites in cultured chick embryo fibroblasts. Incubation of fibroblasts with 0.2 mM dibucaine for 3 h at 37 degrees C results in a twofold to threefold increase in insulin binding, with an increase in average number of binding sites (Ka = 3.0 X 10(7) M-1) from 9 X 10(3) per cell. Trypsin or ethyleneglycoltetraacetic acid (EGTA) alone increases insulin binding twofold to threefold, but fails to further increase 125I-insulin binding in cells pretreated with dibucaine. Transformation of chick embryo fibroblasts with Rous sarcoma virus causes a threefold to fivefold increase in insulin binding, which is not further increased by incubation with dibucaine. As demonstrated by transmission electron microscopy, dibucaine and trypsin also induce changes in the cytoskeleton of chick embryo fibroblasts, characterized by disorganization and disappearance of microfilament and microtubule bundles. These alterations are accompanied by gross morphologic changes, including rounding of cells and appearance of numerous ruffles and blebs on the cell surface. These observations are consistent with the hypothesis that expression of surface receptors in cultured chick embryo fibroblasts is related to the organization and disorganization of cytoskeletal structures.
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PMID:Alteration of insulin binding and cytoskeletal organization in cultured fibroblasts by tertiary amine local anesthetics. 23 24

The effects of protein modification procedures on the biologically most important properties of cholera toxin, i.e. the toxic activity, the GM1 receptor-binding capacity and the antigenic (antibody-fixing) properties, have been studied quantitatively using microgram amounts or less of toxin protein. Most of the 24 group-specific reagents used had either no inhibitory effect on the toxic or the combination of GM1-binding and antibody-fixing properties of cholera toxin, or they had a concomitant inhibitory effect on these activities. Separate testing of GM1- and antibody-binding revealed a close, but not absolute, structural association between these properties, Amino group reactive substances were particularly effective in decreasing the GM1-binding activity, while leucine aminopeptidase had no effect. This suggests that lysine residues may be involved in binding toxin to the acidic GM1 receptor. Sodium dodecylsulphate and mercaptoethanol, which caused dissociation of the subunits of cholera toxin as indicated by polyacrylamide gel electrophoresis, abolished toxicity without inhibiting the concomitant GM1- and antibody-binding properties of the toxin. Similar differential effects were also obtained with three reagents which did not seem to change the aggregation state of the toxin. These substances all had specificity for arginine, suggesting that arginyl residues of the toxin molecule may be involved in a 'toxic site' distinct from the receptor-binding site(s). A selective effect on the toxic site was also found by treating the toxin with carboxypeptidase or trypsin in the presence of urea; in the absence of urea no enzymic effect on any toxin property was noted.
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PMID:Protein reagent modification of cholera toxin: characterization of effects on antigenic, receptor-binding and toxic properties. 120 72

The mechanism of CO2 action in changing coccidian oocyst wall permeability was indirectly studied by substituting NO, NO2, N2O, H2S, SO2, CH4, NH3, and 8M urea in place of CO2 in an established excystation procedure. Changes in oocyst wall permeability of Eimeria stiedai, E. bovis, and E. tenella were determined by incubation in test gases and cysteine HCl followed by attempted activation of sporozoites by trypsin and bile and staining of intraoocyst components with methylene blue. The gases CH4, NO2, and N2O were negative for all 3 tests, as were SO2, NH3, and 8M urea which, in addition, were toxic to the oocysts. Both H2S and NO were capable of mimicking the action of CO2 and are related chemically to the reducing agent, and thus tend to underscore its importance in excystation. It now appears that the role of CO2 is that of an allosteric effector enhancing the action of the reducing agent.
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PMID:The effects of selected gases on excystation of coccidian oocysts. 126 26

Enzymatic semisyntheses of growth hormone releasing factor (GRF), a 44-residue peptide amide hormone, from C-terminal acid precursors, are compared. A recombinant alpha-amidating enzyme was used to convert the glycine-extended precursor, GRF(1-44)-Gly-OH, to GRF(1-44)-NH2 in an essentially quantitative fashion. Trypsin was used to convert the precursors, GRF(1-43)-OH and GRF(1-44)-OH, to GRF(1-44)-NH2 (60 and 15% conversion, respectively) in a 75% v:v N,N'-dimethylacetamide solution containing a large excess of leucine amide. Carboxypeptidase Y catalyzed transpeptidations of the precursors, GRF(1-44)-OH and [Ala44]-GRF(1-44)-OH, to GRF(1-44)-NH2 in aqueous leucine amide solutions were also attempted. The trypsin catalyzed direct amidation of [Ala15]-GRF(1-29)-OH in concentrated ammonium acetate/ammonia buffer (95% 1,4-butanediol cosolvent) to form the superactive analog, [Ala15]-GRF(1-29)-NH2 (ca. 25% conversion at equilibrium), is also described.
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PMID:Comparison of enzymatic semisyntheses of peptide amides: human growth hormone releasing factor and analogs. 182 39

The large subunit of Escherichia coli carbamoyl phosphate synthetase (a polypeptide of 117.7 kDa that consists of two homologous halves) is responsible for carbamoyl phosphate synthesis from NH3 and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus (demonstrated by NH2-terminal sequencing). UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of [14C]UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradiation with [14C]UMP. The [14C]UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. In the presence of SDS, the labeled large subunit is cleaved by trypsin or by V8 staphylococcal protease at a site located 15 or 25 kDa, respectively, from the COOH terminus (shown by NH2-terminal sequencing), and only the 15- or 25-kDa fragments are labeled. Similarly, upon cleavage of the aspartyl-prolyl bonds of the [14C]UMP-labeled enzyme with 70% formic acid, labeling was found only in the 18.5-kDa fragment that contains the COOH terminus of the subunit. Thus, UMP binds to the COOH-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain. 198 78

Hydrolytic enzymes were measured in gut contents from four sudden death victims. Pancreatic amylase and total protease activities decreased distally from the small bowel to the sigmoid/rectum region of the large intestine, showing that considerable breakdown or inactivation of the enzymes occurred during gut transit. To determine whether pancreatic enzymes were substrates for the gut microflora, mixed populations of bacteria were grown in a 3-stage continuous culture system on a medium that contained pancreatic extract as the sole nitrogen source. The multichamber system (MCS) was designed to reproduce in vitro, the low pH, high nutrient, fast growth conditions of the caecum and right colon and the neutral pH, low nutrient, slow growth conditions of the left colon. Results showed that pancreatic amylase was resistant to breakdown by intestinal bacteria compared with the peptide hydrolases in pancreatic secretions. Leucine aminopeptidase, trypsin and to a lesser degree, chymotrypsin, were easily degraded by gut bacteria, but pancreatic elastase was comparatively resistant to breakdown. Protein degradation in the MCS, as determined by enzyme activities, protein concentration and ammonia and phenol production, increased concomitantly with system retention time over the range 24-69 h. These results suggest that intestinal bacteria play an important role in the breakdown of hydrolytic enzymes secreted by the pancreas and that this process and protein fermentation in general, is likely to occur maximally in individuals with extended colonic retention times.
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PMID:Influence of retention time on degradation of pancreatic enzymes by human colonic bacteria grown in a 3-stage continuous culture system. 248 Mar 41

Glutamine synthetase (L-glutamate: ammonia ligase [ADP forming]) [EC 6.3.1.2] has been purified from a Gram-positive, acid-fast bacterium, Mycobacterium phlei, by simple procedures with 57% recovery. The enzyme resembled that from Mycobacterium smegmatis in the subunit size (56,000), molecular weight (670,000), amino acid composition, the amino acid sequence of the NH2-terminal, and the secondary structure. The enzyme activity was regulated by adenylylation of each subunit in the dodecameric molecule. M. phlei glutamine synthetase possesses two useful characteristics: high thermostability and resistance to protease digestion. The enzyme was not inactivated on exposure to 60 degrees C for 2 h or 37 degrees C for 72 h, or after incubation with 1% trypsin or chymotrypsin at 37 degrees C for 12 h, pH 7.8. With saturating substrate levels, the Arrhenius plot was nonlinear and concave downward with an intersection point at 45 degrees C, and the activation energies were calculated to be 3.2 and 9.6 cal/mol from the slopes. The specific activity of the highly adenylylated enzyme (E10.7) was remarkably lower than that of the slightly adenylylated enzyme (E2.5); however, both enzymes show similar profiles of the Arrhenius plot. These results indicate that the adenylylation of the enzyme does not affect its activation energies.
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PMID:Physical and chemical characterization of glutamine synthetase purified from Mycobacterium phlei. 256 61

Two monoclonal antibodies (MAb I and IV) have been prepared which showed high and specific reactions towards bovine heart mitochondrial coupling factor B (FB). Both have been identified as sub-type IgG1 of mouse immunoglobulins. MAb I reacts with purified and functionally active FB, alkylated or oxidized forms of FB and even with peptides formed on digestion of FB with trypsin. When used together, MAb I and IV reacted with FB in immunoblots of normal and urea treated samples of mitochondria, submitochondrial particles, ammonia-EDTA extracted particles, and H+-ATPase. Both MAbs inhibited FB-stimulated ATP-dependent reverse electron flow activity when FB was incubated with the antibody either before or after its addition to FB-deficient AE-particles. Reactivity of MAb I towards FB declined upon exposure of FB to guanidine HC1 while reactivity of MAb IV remained unaltered.
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PMID:Monoclonal antibodies to mitochondrial coupling factor B. 257 11


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