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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Xanthine oxidase (EC 1.2.3.2) was purified from fresh cows' milk by differential centrifugation and hydroxylapatite chromatography in the absence of reducing agents and proteases. The purified isolate possessed an absorbance at 280 nm:absorbance at 450 nm ratio of 4.84; an absorbance (1 cm at 280 nm 1%) of 11.9; an activity:absorbance at 450 nm of 141, a specific activity of 3.59 units/mg; and detectable dehydrogenase activity. The enzyme preparation was obtained in a reversible oxidase form that could be partially converted to xanthine dehydrogenase in the presence of 10mM dithiothreitol or 1% mercaptoethanol. Amino acid analyses revealed that the enzyme was hydrophobic in nature and that lysine constituted its N-terminal residue. The protein contained 22 disulfide and 38 sulfhydryl groups, four of which were detectable in the undenatured protein complex. Discontinuous PAGE in the presence of selected dissociation agents did not result in further resolution. Sodium dodecyl sulfate-PAGE of the purified enzyme revealed a sharp zone with a molecular weight of 151,000 +/- 4000 (i.e., monomer). The purified enzyme exhibited oxidase activity in the presence of 6 M urea and following limited proteolysis by trypsin, chymotrypsin, plasmin, pancreatin, pepsin, and papain. Proteolyzed xanthine oxidase migrated as a single zone in polyacrylamide gels in the presence and absence of dissociating agents such as 1% mercaptoethanol and 6 M urea. Restricted digestion of xanthine oxidase by proteases was indicated by the presence of three major zones with molecular weights ranging from 85,000 to 100,000, 30,000 to 35,000, and 18,000 to 20,000 commonly observed in SDS gels. Amino acid profiles of the principal peptidyl fragments of trypsin-cleaved xanthine oxidase indicated their hydrophobic nature and lysine as the N-terminal residue for all fragments.
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PMID:Characteristics of purified cows' milk xanthine oxidase and its submolecular characteristics. 339 6

A protease activity associated with the micrococcal nuclease-solubilized chromatin from mouse seminiferous tubules has been characterized. Proteolysis of histone H1 and core histones is stimulated in the presence of 3 M urea. The pH optimum of this protease is between pH 8 and 9, and the activity is not inhibited by trypsin or chymotrypsin-active site inhibitors. Leupeptin is an effective inhibitor of the protease at low concentrations. Soluble chromatin from neonatal and prepubertal mice lacks this proteolytic activity until three to four weeks after birth. That the protease activity is localized in the dinucleosomes and higher oligomers but is lacking in mononucleosome populations suggests its association with the linker DNA. Rat testis-soluble chromatin apparently lacks such a protease activity. The developmental expression of this protease and its in situ localization are consistent with a role in histone displacement during mouse spermiogenesis.
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PMID:A protease activity is associated with testicular chromatin of the mouse. 355 31

Partial denaturation of the circular octameric bifunctional enzyme formiminotransferase-cyclodeaminase in increasing urea concentrations leads to sequential dissociation via dimers to inactive monomers. In potassium phosphate buffer, dissociation to dimers in 3 M urea coincides with loss of both activities and a major decrease in intensity of intrinsic tryptophan fluorescence. In the presence of folic acid, these dimers retain the deaminase activity, but with folylpolyglutamates, both activities are protected and the native octameric structure is retained. The protection profiles with polyglutamates are cooperative with a Hill coefficient greater than 2, suggesting that binding of more than one folylpolyglutamate per octamer is required to stabilize the native structure. In triethanolamine hydrochloride buffer, transferase-active dimers that retain the intrinsic tryptophan fluorescence can be obtained in 1 M urea and stabilized at higher urea concentration by the addition of glutamate. Deaminase-active dimers are obtained by the protection of folate in 3 M urea. Proteolysis of the two kinds of dimers by chymotrypsin leads to very different fragmentation patterns, indicating that they are structurally different. We propose that the two dimers retain different subunit-subunit interfaces, one of which is required for each activity. This suggests that the native octameric structure is required for expression of both activities and therefore for "channeling" of intermediates.
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PMID:Dissociation of the octameric bifunctional enzyme formiminotransferase-cyclodeaminase in urea. Isolation of two monofunctional dimers. 359 1

The assumption that a different conformational form was induced in the nuclear estrogen receptor following binding by antiestrogens compared to estrogens was studied by analysing the proteolytic fragments of the receptor following limited digestion with chymotrypsin and trypsin. Nuclei were isolated from MCF-7 cells previously exposed to [3H] 4-OHTAM. The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei. The molecular weights (Mr) were calculated from the sedimentation coefficients (S) determined on a sucrose gradient and from the Stokes radii (Rs) estimated by gel filtration. Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 155,000. This receptor form was degraded by chymotrypsin to a receptor of Mr = 63,000 which could not be further dissociated by 0.4 M KCl and 3 M urea. A similar receptor molecule was released by chymotrypsin from intact nuclei. Digestion of the micrococcal nuclease hydrolysate with trypsin degraded the receptor to a form of a Mr = 67,000 which could not be further dissociated by 0.4 M KCl and 3 M urea. Digestion of intact nuclei with trypsin followed by micrococcal nuclease, solubilized a receptor form of Mr = 80,000 which could be further dissociated with 0.4 M KCl and 3 M urea to a receptor form of Mr = 67,000. This trypsin degraded receptor form seems to be similar in Mr to the chymotrypsin degraded form. On the other hand different receptor fragments of Mr = 33,000 and Mr = 60,000 were excised by chymotrypsin and trypsin respectively from the estradiol ligated estrogen receptor. (Geier et al., J. steroid Biochem. 26 [1987] 35-40.) These results support the assumption of a different conformational form for the antiestrogen ligated receptor, compared to the estrogen ligated receptor since they were differentially susceptible to proteolytic degradation by chymotrypsin.
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PMID:Analysis of the 4-hydroxytamoxifen (4-OHTAM) bound nuclear estrogen receptor from MCF-7 cells by limited proteolysis. 368 14

We use molecular mechanics to calculate the conformational properties of a cyclic urea mimetic of alpha-chymotrypsin proposed, but not yet synthesized, by Cram and co-workers. We find that, in order to bring the structural elements of the catalytic triad into a spatial orientation suitable for proton transfer, the proposed enzyme mimetic must adopt a highly strained conformation. We redesign that part of the molecular architecture holding the catalytic triad in position and suggest two alternative enzyme mimetics. Of these, we find that the mimetic containing a fused ring structure positions the components of the catalytic triad at reasonable distances for proton transfer. We study the effect of these structural alterations on the recognition pattern presented by the enzyme mimetic to the substrate, as illustrated by the molecular electrostatic potential of the artificial enzyme.
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PMID:Computer-aided design of artificial enzymes: cyclic urea mimetics of alpha-chymotrypsin. 379 96

Cell surface fibrils could be released from Streptococcus sanguis 12 but not from strains 12na or N by freeze-thawing followed by brief homogenization. Fibrils were isolated from the homogenate by ultracentrifugation or ammonium sulfate precipitation. Electron microscopy demonstrated the presence of dense masses of aggregated fibrils in these preparations. Under nondenaturing conditions, no proteins were seen in polyacrylamide gel electrophoresis (PAGE). Sodium dodecyl sulfate (SDS)-PAGE analysis revealed a single band stained with Coomassie blue and periodic acid Schiff stain with a molecular weight in excess of 300,000. The protein has been given the name long-fibril protein (LFP). The molecule was susceptible to digestion with subtilisin, pronase, papain, and trypsin, but was unaffected by chymotrypsin or muramidases. Attempts to dissociate the protein into smaller subunits with urea, guanidine, sodium thiocyanate, and HCl were unsuccessful. Gel filtration on a column of Sephacryl S-400 in the presence of 2% SDS resulted in elution of the protein at the void volume. Antibody raised against the LFP excised from an SDS-PAGE gel reacted with long fibrils on the surface of strain 12 and with isolated fibrils by an immunogold labeling technique. Monoclonal antibody reactive with LFP in SDS-PAGE also reacted with fibrils present on the cell. Antisera raised against the fibrils inhibited adherence to saliva-coated hydroxyapatite.
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PMID:Identification and preliminary characterization of a Streptococcus sanguis fibrillar glycoprotein. 379 16

Several trypsin inhibitors with different mobilities on polyacrylamide gel electrophoresis occur in the tubers of taro (Colocasia antiquorum), and they each have a dimeric molecular weight of 40,000. Of all the constituent subunits, molecular weight 20,000, of the taro trypsin inhibitor (TTI), three major subunit components were separated by chromatography on SP-Sephadex C-25 in 8 M urea, and they were named protomers alpha, beta, and gamma in the order of their elution from the SP-Sephadex column. After removal or dilution of the urea, the three protomers could be either reassociated individually or hybridized with each other to form dimeric inhibitors. All of the reassociated dimers were powerful inhibitors of trypsin. Among them, each dimer derived from protomers alpha and gamma was a weak inhibitor of chymotrypsin, whereas the dimer of protomer beta did not inhibit the enzyme. Therefore TTI is presumed to be a mixture of heterogeneous and homogenous dimers whose properties reflect those of their constituent protomers. It was also proved that the major three trypsin inhibitors (TTI-I, TTI-II, and TTI-III) previously isolated from taro tubers are composed of protomers alpha and gamma, i.e., TTI-II is a heterogeneous dimer of protomers alpha and gamma, and TTI-I and TTI-III are homogeneous dimers of protomers alpha and gamma, respectively. The molecular weight of a trypsin-TTI complex saturated with trypsin was found to be 79,000, suggesting the formation of a tetrameric complex.
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PMID:Subunit compositions of trypsin inhibitors from tubers of taro, Colocasia antiquorum var. nymphaifolia? 400 70

An acid DNase (DNase II) from porcine spleen was purified by sequential chromatography over carboxymethyl-cellulose, blue dextran-Sepharose, hydroxylapatite, and sulfoxyethyl-cellulose. The purified enzyme shows two polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr 35,000 (alpha chain) and 10,000 (beta chain). The sum of the two molecular weights is that of the native enzyme (45,000). Thus, the DNase II molecule is an alpha,beta dimer. The two polypeptides are not joined by disulfide bonds, but can be cross-linked chemically with dimethyl suberimidate. They are dissociable in 8 M urea, after which they can be isolated by gel filtration on Sephadex G-100, eluting with 1 M acetic acid. Once dissociated, the two polypeptides cannot be reassociated to regenerate DNase II activity. The sum of the amino acid compositions of the two polypeptides is that of the native enzyme, and both contain carbohydrate. The beta chain is devoid of histidine, half-cystine, valine, and methionine. The NH2-terminal amino acid of the alpha chain is leucine, while that of the beta chain cannot be identified by either dansylation or Edman degradation. Alkylation of an essential histidine residue of DNase II occurs on incubation of the enzyme with [2-14C] ICH2COOH (Oshima, R. G., and Price, P. A. (1973) J. Biol. Chem. 248, 7522-7526). Radioactivity is found only in the alpha chain. After hydrolysis of the alpha chain with trypsin, chymotrypsin, and thermolysin, radioactive peptides were isolated by gel filtration on Sephadex G-25 and reversed-phase high performance liquid chromatography. Sequence analyses of the radioactive peptides show alkylation of 1 of 9 histidines in the entire amino acid sequence of DNase II. The sequence around this histidine, determined by manual microsequencing and by the release of amino acids with carboxypeptidases A and B, is Ala-Thr-Glu-Asp-His-Ser-Lys-Trp.
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PMID:The subunit structure and active site sequence of porcine spleen deoxyribonuclease. 403 Jul 66

The reagent p-fluorobenzenesulfonyl chloride modifies the protein side chains of tyrosine, lysine, and histidine and the alpha-NH2 group. The p-fluorobenzenesulfonyl (Fbs-) group, identified by the 19F nuclear magnetic resonance signal, exhibits a different 19F chemical shift for each functional group modified. The Fourier-transformed spectra of the Fbs- group displayed the expected nine-line multiplet in Fbs- amino acids and simple Fbs- peptides but not in the Fbs- proteins, where the resolution was less. Lysozyme, RNase, DNase, and chymotrypsin react with this reagent and each Fbs- protein exhibits a distinctive pattern of 19F NMR signals due to the label, suggesting that the reaction of the reagent varies with the reactivity of the side chains in a protein. The three major 19F signals of the unfolded Fbs-RNase in 8 M urea are due to the Fbs- label on the imidazolium, alpha-NH2, and epsilon-NH2 groups. Based upon results from amino acid and 19F NMR analyses of the tryptic-chymotryptic peptides of Fbs-RNase, portions of the imidazolium and epsilon-NH2 resonances were assigned to the Fbs- label on His-105 and Lys-41, respectively, while the alpha-NH2 resonance was entirely due to the Fbs- label on the alpha-NH2 of Lys-1. Because Fbs-RNase has an unchanged, near-ultraviolet circular dichroism spectrum and because it retains approximately 80% of the RNase activity, the conformation of Fbs-RNase is probably not altered from the folded conformation of the native enzyme. Upon unfolding in 8 M urea or heating at 70 degrees C, Fbs-RNase gave a 19F NMR spectrum differing from that of the folded Fbs-RNase. In the presence of uridylic acid, Lys-41 was the only residue protected from modification by the reagent with a concomitant reduction of the epsilon-NH2 resonance, and the RNase thus modified was fully active. Hence, 19F NMR analysis of protein, via the reaction with p-fluorobenzenesulfonyl chloride, provided not only information about the protein conformation but also direct measurements of the modification status.
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PMID:The use of p-fluorobenzenesulfonyl chloride as a reagent for studies of proteins by fluorine nuclear magnetic resonance. 406 17

The rate of production of thiol groups by alkaline cleavage of disulfide bonds in the black-eyed pea (Vigna unguiculata) trypsin and chymotrypsin inhibitor (BTCI) was determined from thiol quantitation with 5, 5'-dithiobis-(2-nitrobenzoic acid), and increase in absorption at 240 nm. Rate constants were estimated at pH's 12.8, 13.0, 13.5, and 25 degrees C. At pH 13.0, the second-order rate constant was obtained as a function of temperature. The energy of activation was Ea = 10.7 kcal/mole, while the change in activation free energy was delta G not equal to = 21.6 kcal/mole. The results obtained fit the hydrolysis mechanism, in which S-S split occurs through a nucleophilic attack of a hydroxide ion on the disulfide bond. Urea (8M), unexpectedly, showed an effect of attenuation on the hydrolysis rate of disulfide bonds in BTCI.
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PMID:Alkaline cleavage of disulfide bonds in the black-eyed pea trypsin and chymotrypsin inhibitor. 406 66


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