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)

beta protein, a key component of Red-pathway of phage lambda is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli. To facilitate studies on structure-function relationships, we overexpressed beta protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of beta protein in solution. A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis. Stoichiometric measurements indicated a site-size of 1 monomer of beta protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that beta protein formed stable nucleoprotein complexes with 36-mer, but not with 27- or 17-mer DNA. Interestingly, the interaction of beta protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgCl2, and the binding was abolished by 250 mM NaCl. The Kd of beta protein binding to 36-mer DNA was on the order of 1.8 x 10(-6) M. Photochemical cross-linking of native beta protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain. Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end. These findings provide a basis for further understanding of the structure and function of beta protein.
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PMID:Characterization of the DNA-binding domain of beta protein, a component of phage lambda red-pathway, by UV catalyzed cross-linking. 898 71

An aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646 was purified 196-fold by a combination of Mono-Q, Reactive Green 19 agarose affinity, and hydroxyapatite chromatographies. The purified enzyme runs as a single band of 140 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass was estimated to be 163 +/- 3.8 kDa by gel filtration, indicating that this enzyme is a monomeric protein. The binding of the enzyme to Reactive Green 19 agarose was Mg2+ dependent. The binding capacity was estimated to be about 0.2 mg of Reactive Green agarose per ml in the presence of 10 mM MgCl2. This enzyme can catalyze the reduction of a wide range of aryl carboxylic acids, including substituted benzoic acids, phenyl-substituted aliphatic acids, heterocyclic carboxylic acids, and polyaromatic ring carboxylic acids, to produce the corresponding aldehydes. The Km values for benzoate, ATP, and NADPH were determined to be 645 +/- 75, 29.3 +/- 3.1, and 57.3 +/- 12.5 microM, respectively. The Vmax was determined to be 0.902 +/- 0.04 micromol/min/mg of protein. Km values for (S)-(+)-alpha-methyl-4-(2-methylpropyl)-benzeneacetic acid (ibuprofen) and its (R)-(-) isomer were determined to be 155 +/- 18 and 34.5 +/- 2.5 microM, respectively. The Vmax for the (S)-(+) and (R)-(-) isomers were 1.33 and 0.15 micromol/min/mg of protein, respectively. Anthranilic acid is a competitive inhibitor with benzoic acid as a substrate, with a Ki of 261 +/- 30 microM. The N-terminal and internal amino acid sequences of a 76-kDa peptide from limited alpha-chymotrypsin digestion were determined.
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PMID:Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646. 917 90

Cadmium is a highly toxic metal entering cells by a variety of mechanisms. Its toxic action is far from being completely understood, although specific interaction with the cellular calcium metabolism has been indicated. Metal ions that influence intracellular Ca2+ concentrations or compete with Ca2+ for protein binding sites may exert an effect on actin filaments, whose assembly and disassembly are both regulated by a number of calcium-dependent factors. Cadmium is such a metal. Much evidence demonstrates that cadmium interferes with the dynamics of actin filaments in various types of cells. Here we show that, at high (0.8-1.0 mM) concentrations, CdCl2 causes actin denaturation. At such Cd2+ concentrations, actin precipitates (really actin, as shown by SDS-PAGE, see Fig. 1B) in the form of irregular, disordered clots, clearly appreciable by electron microscopy. Denaturation seems to be reversible since, after Cd2+ removal by dialysis, the polymerizability of sedimented actin is restored almost completely. On the other hand, at concentrations ranging from 0.25 to 0.6 mM, CdCl2 is more effective as an actin polymerizing agent than both MgCl2 and CaCl2. The Cd-related increase in the actin assembly rate is ascribable to an enhanced nucleation rather than to an increased monomer addition to filament growing ends. The latter, in contrast, appears quite slow. Critical concentration measurements revealed that the extent of polymerization of both Mg- and Cd-assembled actin are very close (C(c) ranges from 0.25 to 0.5 microM), while Ca-polymerized actin shows a polymerization extent markedly lower (C(c) = 4.0 microM). By both the fluorescent Ca2+ chelator Quin-2 assay and limited proteolysis of actin by trypsin and alpha-chymotrypsin, the real substitution of G-actin-bound Ca2+ by Cd2+ has been appreciated. The increase in Quin-2 fluorescence after addition of excess CdCl2 indicates that, in our experimental conditions, Ca2+ tightly-bound to actin is partially (60-70%) replaced by Cd2+, forming Cd-actin. Electrophoretic patterns after limited proteolysis reveal that the trypsin cleavage sites in the segment 61-69 of the actin polypeptide chain are less accessible in Cd-actin than in Ca-actin, although the cation-dependent effect is less pronounced in Cd-actin than in Mg-actin. Our results are consistent with some of the consequences on microfilament organization observed in Cd2(+)-treated cells; however, considering the positive effect of Cd2+ on actin polymerization in solution we have noticed that this was never observed in vivo. A different indirect effect of Cd2+ on some cellular event(s) influencing cytoplasmic actin polymerization appears to be reasonable.
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PMID:Actin assembly by cadmium ions. 920 70

Using 32P-labeled 2-azidoadenosine 5'-triphosphate (2N3ATP) and 8-azidoadenosine 5'-triphosphate (8N3ATP), we have identified a site on human interferon alpha2 (IFN-alpha2) that binds adenine nucleotides. The results from saturation and competition experiments demonstrated the specificity of the nucleotide interaction. Half-maximal saturation of IFN-alpha2 was observed at 10 microM 2N3ATP or 35 microM 8N3ATP. ATP effectively decreased photoinsertion of both photoaffinity analogs of ATP. Photoinsertion of 8N3ATP was enhanced by MgCl2, independent of the ionic strength, and exhibited an optimum pH between 7.0 and 7.5. Immobilized-Al3+ affinity chromatography and HPLC were used to purify the modified peptides from IFN-alpha2 that had been photolabeled with 8N3ATP and digested with trypsin or chymotrypsin. Overlapping-sequence analysis localized the sites of photoinsertion to the region corresponding to Lys121-Tyr135 in the amino acid sequence of IFN-alpha2, which almost perfectly overlaps a nuclear-localization signal (R120KYFQRITLYLKEKKY135).
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PMID:Identification of an adenine-nucleotide-binding site on interferon alpha2. 928 95


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