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)

Fatty acid synthase from the uropygial gland was inactivated by treatment with pyrenebutyl methanephosphonofluoridate by specific modification of the "active serine" at the thioesterase domain. Treatment of fatty acid synthase with 3-(4-maleimidylphenyl)-7-diethylamino-4-methylcoumarin resulted in the loss of the condensation activity and overall synthase activity. Acetyl-CoA and malenyl-CoA protected the enzyme from inactivation by this reagent suggesting that the pantetheine thiol was modified. In support of this conclusion was the finding that modification of the primer-binding thiol with iodoacetamide prior to the modification with the coumarin derivative resulted in no change in the binding of the coumarin to the enzyme. Furthermore, the presumptive active site peptide isolated after proteolysis released its attached coumarin upon treatment with alkali under beta-elimination reaction conditions. Graphical analysis of the binding data suggested that binding of one coumarin derivative/subunit of the synthase would result in complete loss of the synthase activity. When the synthase was modified with the coumarin and pyrene derivatives, fluorescence resonance energy transfer occurred from the pyrene at the thioesterase site to the coumarin attached to the pantetheine thiol. Dissociation of the enzyme to monomers did not decrease the efficiency of transfer, but limited trypsin treatment, which released the thioesterase domain, abolished the fluorescence resonance energy transfer. These results suggested that the energy transfer occurred between intrasubunit sites. The distance between the pyrene at the thioesterase active site and the coumarin attached to pantetheine thiol on the same subunit of fatty acid synthase was estimated from the efficiency of energy transfer to be 37 A.
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PMID:Measurement of distance between the active serine of the thioesterase domain and the pantetheine thiol of fatty acid synthase by fluorescence resonance energy transfer. 391 10

Dissociation of the ovotestes of Helix pomatia requires the presence of carbohydrates as well as trypsin, but is independent of cation content. Dispersed cells obtained from the ovotestes reaggregate rapidly into histotypically organized tissue. The role of cellular adhesiveness is facilitated by centrifugation, but pseudopodial activity of the cells is suggested as another factor in reaggregation.
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PMID:Molluscan cells: dissociation and reaggregation. 564 65

Regulatory subunits (R subunits) of mouse lung cAMP-dependent protein kinases undergo age-dependent changes in endogenous proteolysis, with the greatest amount of the major Mr = 37,000 proteolytic fragment detectable during fetal and neonatal development. Homogenization of lung in the presence of various protease inhibitors does not affect this age-related difference, suggesting that the observed quantitative change in R subunit proteolysis occurs in vivo. Mechanisms were sought to account for this age-dependent change. The production of a Mr = 37,000 proteolytic fragment can be stimulated in lung extracts by the addition of exogenous calcium and is due to the action of an endogenous Ca2+-stimulated protease. Neonatal lung extracts show more Ca2+-stimulated proteolysis of R subunits than adult extracts, although only slight age-related differences in either the Ca2+-stimulated protease or its specific endogenous inhibitor were observed. Age-dependent differences in R subunits which may affect sensitivity to proteases were also examined. Analysis of the two-dimensional patterns of adult and neonatal 8-N3-[32P]cAMP-labeled R subunits before or after limited proteolysis with trypsin suggests that the R subunits are structurally similar. Differences are found, however, in the relative proportions of adult and neonatal Type I R subunits (RI) in the holoenzyme or dissociated forms. An increased proportion of neonatal R subunits exist in the dissociated state, whereas adult R subunits exist primarily in the holoenzyme form. Dissociated R subunits from mouse lung are more susceptible than the holoenzyme to limited proteolysis by the partially purified lung Ca2+-stimulated protease. Dissociation of the holoenzyme in vivo may be a major factor in the age-dependent proteolytic changes observed in mouse lung protein kinases.
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PMID:Developmental changes in the endogenous Ca2+-stimulated proteolysis of mouse lung cAMP-dependent protein kinases. 632 Jul 33

The dissociation of the trypsin-sensitive basic tails of the core histones in core chromatin has been followed as a function of [NaCl] using proton NMR spectroscopy. The tails dissociate in a highly cooperative all or none manner over the salt concentration range 0.2-0.6 M, that is, below the salt concentration required to dissociate the complete molecule. Assuming that each basic tail dissociates independently, the total number of salt linkages involved in binding the tails to DNA is 103. This equals the number of basic side chains in the tails of an octamer. The standard free energy of dissociation, delta G degree, in 1 M NaCl at 297 K is 3.6 kcal/mol. Temperature had no effect on the extent of dissociation up to 45 degrees C. However, between 45 and 65 degrees C, where the premelting transition in the core chromatin occurs, the tails dissociated completely. Dissociation of the tails was associated with a conformational transition in the DNA consistent with loss of supercoiling. From this, and the results of a previous study, it can be shown that the structured, trypsin-resistant domain of each core histone octamer makes 100 salt linkages to DNA. Thus, in 10 mM salt, each core octamer makes a total of 203 salt linkages to DNA.
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PMID:Differential dissociation of histone tails from core chromatin. 650 40

Cytochrome P-450scc consists of two domains linked with a short loop of the polypeptide chain; under hydrolysis by trypsin the domains retain their associated state due to rigid noncovalent interactions. A partial separation of the domains by gel-chromatography on Sephadex G-200 with retention of a haem group in domain I has been achieved after incubation of the trypsin-modified cytochrome P-450scc in 50 mM phosphate buffer (pH 7.2)/1 M NaCl/0.3% sodium cholate/0.3% Tween 80. The separation of domains I and II to individual fragments of the haemoprotein polypeptide chain has been achieved by chromatography under denaturation conditions on the activated thiopropyl-Sepharose via a selective covalent immobilization of domain II. Dissociation of a complex of domains I and II has been effectuated in the presence of 7 M guanidine. Structural characteristics of individual domains have been investigated. It is established that domain I containing a haem group is the N-terminal moiety, and domain II, the C-terminal moiety of the polypeptide chain of cytochrome P-450scc. The pathways of limited trypsinolysis of the native cytochrome P-450scc have been determined. The peptides containing cysteine residues localized on the surface of domain II and responsible for the interaction of haemoprotein with activated thiopropyl-Sepharose have been isolated in a homogeneous form and their amino-acid sequences have been assessed.
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PMID:The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria. Localization of haem group and domains in the polypeptide chain. 651 66

1. Dissociation of the hamster cheek pouch epithelium by a method combining trypsin attack and EDTA exposure, yielded a mixture f highly viable isolated cells (basal, spinous, and granular cells). 2. Such heterogeneous cell population was reproducibly separated into 4 fractions by pycnic sedimentation: in order of increasing density, Fraction I and Fraction II predominantly contained cells of the basal layer (93% and 76%, respectively), Fraction III basal, spinous and granular cells in equivalent concentration, Fraction IV mainly granular cells (69%). Horny squamae sedimented at the bottom of the tube. 3. Individual cells of the whole population and those of the population fractions separated by density gradient, were analyzed and charcterized as regards morphology, viability, volume, dry mass, density and DNA synthesizing ability. 4. A positive correlation was found between morphology, dry mass and density of the cells, showing that differentiation occurs by increments in mass and density. The weights of the basal, spinous and granular cells were distributed within ranges well defined and scantily overlapping, and were positively correlated with cell differentiation. Also cell density increased with differentiation degree. On the contrary, volume distribution of the various types of cells showed differences of minor importance, indicating for an increase of solids concentration in the more differentiated cells. 5. DNA synthesis took place only in basal cells, the density of which was generally very low.
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PMID:A quantitative cytochemical study of isolated epithelial cells of the hamster cheek pouch. 677 73

The N-acetylgalactosaminyltransferase activity and its associated endogenous acceptor present in neural retina cells from embryonic chicks can be separated into three distinct forms which migrate to different densities upon centrifugation in sucrose gradients. Two of these forms, termed L and H, are also present in preparations of plasma membranes. Dissociation of tissues into single cells by trypsin results in populations lacking the cell-surface forms of transferase activity towards endogenous acceptor. This loss can be prevented by including 1 mM Ca2+ in the trypsin-dissociation medium. Reacquisition of cell-surface activity among trypsin dispersed cells occurs with time in culture. In the presence of cycloheximide, the less dense particulate form, L. Appears first, with a concomitant decrease in the cytosol form. Upon removal of the block to protein synthesis, L is transformed into the denser from, H. Our studies suggest that peak H of transferase/acceptor polypeptide. The possible involvement of the transferase/acceptor in adhesion among neural retina cells is discussed.
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PMID:An N-acetylgalactosaminyltransferase and its acceptor in embryonic chick neural retina exist in interconvertible particulate forms depending on their cellular location. 679 87

The kinetics of hydrolysis of "inverse substrates," p-amidinophenyl alkanoates, catalyzed by urokinase, plasmin, kallikrein, and trypsins from various sources were studied. Dissociation constants of acyl enzyme-ligand complexes, which are a characteristic parameter of the reaction with "inverse substrates," were analyzed with a view to comparing the spatial requirements of active sites. It was concluded that the spatial restraint of the active site as regards coexistence of the acyl residue and specific ligand is strictest for hog pancreatic kallikrein and this restraint decreases in the following order: human urokinase; bovine plasmin, bovine and hog trypsins; Streptomyces fradiae trypsin; and Streptomyces griseus trypsin.
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PMID:Comparative studies on the structure of active sites. Behavior of "inverse substrates" toward trypsin and related enzymes. 681 67

General aspects of the mechanism of antithrombin action were elucidated by a comparison of the inactivation of trypsin by antithrombin with the inactivation of coagulation proteinases by the inhibitor. Bovine antithrombin and bovine trypsin were shown to form an inactive equimolar complex. A non-complexed, proteolytically modified form of antithrombin, electrophoretically identical with that formed in the reaction with coagulation proteinases, was also produced in the reaction with trypsin. In the absence of heparin, the inactivation of trypsin by antithrombin was 20 times faster than the inactivation of thrombin; the second-order rate constant was 1.5 x 10(5)m(-1).s(-1) at 25 degrees C and pH 7.4. However, the inhibition of thrombin was accelerated about 30 times more efficiently by small amounts of heparin than was trypsin inhibition. Dissociation of the antithrombin-trypsin complex at pH 7.4 followed first-order kinetics with a half-life for the complex of about 80h at 25 degrees C. The complex was rapidly and quantitatively dissociated at pH 11, resulting in the liberation of a modified two-chain form of the inhibitor, cleaved at the same Arg-Ser bond as in modified antithrombin released from complexes with thrombin, Factor Xa and Factor IXa. This supports the previous proposal that this bond is the active-site bond of antithrombin. Antisera specific for thrombin-modified antithrombin reacted with purified antithrombin-trypsin complex, indicating that the inhibitor was present in the complex in a form immunologically identical with thrombin-modified antithrombin. The results thus suggest a common mechanism, but different kinetics, for the inhibition of trypsin and coagulation proteinases by antithrombin.
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PMID:Mechanism of inactivation of trypsin by antithrombin. 681 49

In this report we examined the effects of trypsin treatment of immune guinea pig T lymphocytes on their binding to syngeneic macrophages. Immune T cells positively selected by culture for 1 wk with antigen and treated with trypsin showed dramatic binding to macrophages in the absence of additional antigen. This binding was rapid and greatest at 1 hr after the addition of trypsin-treated T cells to macrophages. Thereafter, the bound T cells dissociated from the macrophages and did not rebind. Dissociation of macrophage-bound T cells appeared to be an active process that could be inhibited by blocking T cell protein synthesis. Macrophage binding by trypsin-treated T cells was dependent on prior in vitro stimulation; similar treatment of immune T cells taken directly from the animal did not augment binding, and treatment of T cells cultured without antigen resulted in only modest binding. In addition, the trypsin-induced binding phenomenon showed elements of genetic restrictions. Trypsin treatment of immune T cells selected by culture for 1 wk with antigen resulted in preferential binding to syngeneic macrophages. In addition, immune F1 T cells selected by culture with antigen-pulsed parental macrophages preferentially bound to macrophages of the haplotype used for selection. Evidence is presented suggesting that the trypsin-induced binding was not simply due to antigen carry-over from the trypsinized lymphocytes and favoring the possibility that this binding may be antigen-independent. These results suggest that antigen-activated T cells express covert interaction sites for macrophages and that these sites are actively covered-up by other membrane components.
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PMID:Evidence for covert cellular interaction sites expressed by activated T lymphocytes. 698 Sep 35


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