Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Alpha-chymotrypsin (CT) was modified chemically and physically by the treatments with diisopropyl fluorophosphate, L-(1-tosylamide-2-phenyl) ethylchloromethylketone, hydrogen peroxide and heat. After these treatments, CT lost or decreased both the enzymic activity and ability of releasing histamine from rat mast cells. Ca++ was essential for histamine release by CT, while it enhanced only slightly the enzymic activity. Process of histamine release by CT could be separated into two stages: CT-dependent but not Ca++-dependent, and Ca++-dependent but not CT-dependent. The activated state of mast cells produced by CT decayed rapidly at 37 degrees C in the absence of Ca++, but these cells responded to Ca++ by adding CT once again, suggesting reconstitution of cell membrane structure affected by CT. Isoproterenol, epinephrine, prostaglandin E1, and dibutyryl-cyclic AMP (0.01-0.1 mM) did not inhibit release of histamine induced by CT. Neither theophylline (0.01-0.1 mM) alone nor the combinations of these cyclic AMP-active agents with theophylline inhibited the release of histamine. But, in the presence of papaverine (0.01-0.1 mM) a marked, dose-dependent inhibition was observed. These data suggest that 1) release of histamine by CT from rat mast cells is causally related to its hydrolytic activity, 2) this activity causes a reversible change on mast cell membrane which probably facilitates Ca++-influx through the cell membrane, and 3) there are subtle differences among CT, compound 48/80 and antigens concerning the effect of cyclic AMP-active agents in histamine-releasing mechanisms in mast cells.
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PMID:Mechanism of histamine release by alpha-chymotrypsin from isolated rat mast cells. 5 33

In inside-out red cell membrane vesicles active calcium transport and the formation of the enzyme-phosphate complex (EP) of the calcium pump were simultaneously investigated and the effects of a limited proteolytic digestion examined. In order to visualize the proteolyzed EP forms we have induced the formation of a maximum level EP from [gamma-32P]ATP in the presence of Ca2+ + La3+ and applied a good-resolution acidic discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteolysis of inside-out vesicle membranes by trypsin, Pronase, papain, or chymotrypsin produces a calmodulin-like activation of the calcium pump, abolishes its calmodulin sensitivity, and decreases the original 140-kDa EP complex to a limit polypeptide of 80 kDa. Trypsin digestion produces another major intermediary fragment of 90 kDa, which is still a low-activity calmodulin-sensitive form of the pump. The red cell calcium pump is activated by trypsin both in the absence and presence of Ca2+ during digestion although the rate of activation and the appearance of the 80-kDa polypeptide are enhanced by Ca2+. If proteolytic digestion is carried out by chymotrypsin, a calmodulin-insensitive maximum activation of the calcium pump coincides with the formation of a 125-130-kDa EP-forming polypeptide. Chymotrypsin and carboxypeptidase A have synergistic effects on the formation of this latter high-activity species. Based on these data we suggest a probable molecular arrangement for the functional parts of the red cell membrane calcium pump.
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PMID:Molecular characterization of the in situ red cell membrane calcium pump by limited proteolysis. 242 14

A fluorescent peptide substrate to explore the protease specificity for the amino acid regions C- and N-terminal to the cleavage site has been designed. Intramolecular quenching of indole fluorescence by an N-terminal dansyl group separated by six amino acid residues forms the basis of this assay. For a particular enzyme, specificity can be designed into the peptide sequence by means of the number of residues that separate the two chromophores. In the present instance, the heptapeptide Dns-Gly-Lys-Tyr-Ala-Pro-Trp-Val is used to assay angiotensin converting enzyme (ACE), Astacus protease, carboxypeptidase A, alpha-chymotrypsin, and trypsin, all of which cleave the peptide in accord with their known specificity: Trypsin and Astacus protease hydrolyze only the Lys-Tyr and Tyr-Ala bonds, respectively. alpha-Chymotrypsin primarily cleaves the Tyr-Ala bond while ACE makes three successive dipeptidyl cleavages from the C-terminus. Carboxypeptidase rapidly hydrolyzes first the Trp-Val and then the Pro-Trp bond. For all of the enzymes, catalytic activity (kcat/Km) is in the range from 10(5) to 10(6) M-1 s-1. Hydrolysis causes a fluorescence increase in the 310 to 410 nm region of 8.6- to 13.6-fold depending on the enzyme that is assayed. Assays can be designed based on the increase in tryptophan fluorescence or by individual product analyses using thin-layer or high-performance liquid chromatography. The specificity and sensitivity of such internally quenched fluorescent oligopeptides would seem to be ideal for the assay of specific endoproteases.
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PMID:A fluorescent oligopeptide energy transfer assay with broad applications for neutral proteases. 255 28

Chymotrypsin, trypsin, carboxypeptidase A and B, elastase and enterokinase activities were measured in buffer solutions and in human duodenal juice after incubation with wheat bran, cellulose, guar gum, pectin, psyllium and lignin. The different types of dietary fiber led to inhibition of enzymatic activity in most experiments, e.g., lignin could totally ablish the activity of isolated trypsin and chymotrypsin. Only in enterokinase was there no influence. Inhibition depended on incubation time; the effect was proportional to fiber concentration and inversely related to enzyme level. Treatment of fiber with hydrochloric acid (pH 1.5) and heat (95 degrees C) destroyed inhibitory activity in some experiments. The effect of lignin on one enzyme (trypsin) was reduced by the addition of another enzyme (chymotrypsin). It is concluded that dietary fiber could affect digestion by inhibiting proteolytic pancreatic enzymes.
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PMID:Effect of dietary fiber on proteolytic pancreatic enzymes in vitro. 282 29

The mechanism-based inactivations of a number of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties, are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated here, inactivated all the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or beta-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1); the inhibited enzyme was quite stable to reactivation (kdeacyl = 2 X 10(-5) s-1), while enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation was decreased dramatically in the presence of reversible inhibitors or substrates, and ultraviolet spectral measurements indicate that the isocoumarin ring structure is lost upon inactivation. Chymotrypsin A gamma is totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and approximately 1 equiv of protons is released upon inactivation. These results indicate that these compounds react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the first mechanism-based inhibitors reported for many of the enzymes tested, and 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor.
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PMID:Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor. 389 37

The interaction of serine protease (esterases) with 6-chloro-2-pyrones was investigated. Time-dependent inactivation of chymotrypsin, alpha-lytic protease, pig liver elastase, and cholinesterase was found with 3- and 5-benzyl-6-chloro-2-pyrone, as well as 3- and 5-methyl-6-chloro-2-pyrone. No inactivation was observed with the unsubstituted 6-chloro-2-pyrone. The substituted pyrones did not inactivate papain or carboxypeptidase A, as well as a number of other nonproteolytic enzymes. The substituted chloropyrones, therefore, show considerable selectivity toward serine proteases. Analogues in which the 6-chloro substituent is replaced by H or OH do not inactivate. The presence of the halogen is, therefore, essential for inactivation. Chymotrypsin catalyzes the hydrolysis of 3-benzyl-6-chloro-2-pyrone. At pH 7.5, (E)-4-benzyl-2-pentenedioic acid is the major product, and 2-benzyl-2-pentenedioic anhydride is a minor product. The ration of hydrolysis product found to the number of enzyme molecules inactivated varies from 14 to 40. The enzyme inactivated with the 3-benzyl compound does not show a spectrum characteristic of the pyrone ring. This suggests that inactivation by 3-benzyl-6-chloro-2-pyrone occurs in a mechanism-based fashion after enzymatic lactone hydrolysis. When the enzyme is inactivated with the 5-benzyl compound, absorbance due to the pyrone ring is observed. We suggest that inactivation occurs through an active site directed mechanism involving a 1,6-conjugate addition of an active site nucleophile to the pyrone ring.
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PMID:Novel inactivators of serine proteases based on 6-chloro-2-pyrone. 641 Nov 20

It was shown that folate and its derivatives have a profound effect on stabilizing thymidylate synthase in vitro and in vivo, as a consequence of ternary formation between the folate, dUMP, or FdUMP, and the synthase. The degree to which complex formation is affected can be revealed qualitatively by circular dichroism and quantitatively by equilibrium dialysis using the Lactobacillus casei synthase. In contrast to the pteroylmonoglutamates, the pteroylpolyglutamates bind to thymidylate synthase in the absence of dUMP, but even their binding affinity is increased greatly by this nucleotide or its analogues. Similarly, treatment of the synthase with carboxypeptidase A prevents the binding of the pteroylmonoglutamates and reduces the binding of the polyglutamates without affecting dUMP binding. The latter does not protect against carboxypeptidase inactivation but does potentiate the protective effect of the pteroylpolyglutamates. To determine the region of the synthase involved in the binding of the glutamate residues, Pte[14C]GluGlu6 was activated by a water soluble carbodiimide in the presence and absence of dUMP. This folate derivative behaved as a competitive inhibitor of 5,10-CH2H4PteGlu, in contrast to methotrexate which was non-competitive. Separation of the five cyanogen bromide peptides from the L. casei synthase revealed 80% of the radioactivity to be associated with CNBr-2 and about 15% with CNBr-4. Chymotrypsin treatment of CNBr-2 yielded two 14C-labeled peaks on high performance liquid chromatography, with the slower migrating one being separated further into two peaks by Bio-gel P2 chromatography. All three peptides came from the same region of CNBr-2, encompassing residues 47-61 of the enzyme. From these studies it would appear that the residues most probably involved in the fixation of PteGlu7 are lysines 50 and 58. In contrast, methotrexate appeared to bind to another region of CNBr-2.
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PMID:Studies on identifying the binding sites of folate and its derivatives in Lactobacillus casei thymidylate synthase. 641 23

Treatment of the purple membrane with carboxypeptidase A, Pronase, or papain, results in the cleavage of amino acids from the carboxyl terminus of bacteriorhodopsin, a maximum of about 17 amino acids being released with papain. Protease-treated bacteriorhodopsin, after denaturation, refolds to the native structure, binds retinal as tightly as the intact protein and, on reconstitution into vesicles, gives full proton translocating activity. The CD spectrum of papain-treated purple membrane shows exciton coupling characteristic of the intact purple membrane. The trimeric bacteriorhodopsin in papain-treated purple membrane dissociates into monomers in Triton X-100 which, after removal of the detergent, reassociate to form the oligomeric structures. Chymotrypsin cleaves papain-treated bacteriorhodopsin between amino acids 71 and 72 as has been previously found for intact bacteriorhodopsin. The resulting fragments C-1 (amino acids 72-231) and C-2 (amino acids 1-71) reassociate, bind retinal, and regenerate the native chromophore, as previously demonstrated for the corresponding fragments from the intact protein. We conclude that the COOH-terminal peptide in bacteriorhodopsin is not required for the correct refolding of denatured bacteriorhodopsin to the native tertiary and quarternary structure, for chromophore regeneration or for light-driven proton translocation.
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PMID:Removal of the carboxyl-terminal peptide does not affect refolding or function of bacteriorhodopsin as a light-dependent proton pump. 670

Lysosomal acid hydrolases were surveyed in elicited and non-elicited rat peritoneal macrophages to determine the types of enzymes present and optimal assay conditions. Adherent peritoneal cells (primarily macrophages) were cultured 24 hours prior to use. Intracellular distribution of enzymes was determined by differential centrifugation of whole cell homogenates into nuclear, cytoplasmic, and lysosomal fractions. The acid glycosidase, acid phosphatase, acid protease, and lysozyme were largely sedimentable in the lysosomal fraction. Much enzyme activity was latent, being activated by addition of Triton X-100. Chymotrypsin-like protease activity in cell fractions was apparently due to low level mast cell contamination. Elicited macrophages had elevated total cell protein as compared to non-elicited cells, but changes in intracellular enzyme levels were selective depending on the enzyme and the stimulus used to elicit macrophages. Thioglycollate-elicited cells showed elevations of most acid hydrolases compared to non-elicited cells, whereas enzyme levels in zymosan-elicited cells were similar to those in non-elicited cells. All elicited cells showed marked decreases in total cellular alpha-D-mannosidase and alpha-L-fucosidase compared to non-elicited cells. Intracellular lysozyme levels also varied between different rat strains. Cultured macrophages exhibited increasing intracellular levels and extracellular secretion of acid hydrolases, especially extracellular lysozyme (10-25 mug/10(6) cells/day), over 72 hours. No significant intra- or extracellular elastinolytic activity was detected.
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PMID:Selective modification of rat peritoneal macrophage lysosomal hydrolases by inflammatory stimuli. 710 74

We have examined the effect of alpha-chymotrypsin on isolated mast cells from different sources. The enzyme induced a dose-dependent secretion of histamine from purified and non-purified populations of rat peritoneal mast cells. The release was non-cytotoxic and was inhibited by metabolic blockers and extremes of temperature. The process was relatively slow, being essentially complete within 20 min, and was unaffected by phosphatidylserine. A substantial component of the secretion persisted in the absence of extracellular Ca2+. The release was suppressed by extremes of pH and a variety of anti-allergic compounds and serine esterase inhibitors. In addition to the secretion of preformed mediators, alpha-chymotrypsin also induced the metabolism of arachidonic acid, resulting in the release of prostaglandin D2 in a dose-related manner from purified rat peritoneal mast cells. alpha-Chymotrypsin exhibited a marked tissue and species selectivity in its action and tissue mast cells of the rat, guinea pig and human were generally resistant to the enzyme except at cytotoxic concentrations. On the basis of these results, the possible role of endogenous serine esterases in mast cell activation is discussed.
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PMID:Some studies on the effects of alpha-chymotrypsin on mast cells from the rat and other species. 872 May 91


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