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)

Cofilin/ADF affects strongly the structure of actin filaments and especially the intermolecular contacts of the DNase I binding loop (D-loop) in subdomain 2. In G-actin, the D-loop is cleaved by subtilisin between Met47 and Gly48, while in F-actin this cleavage is inhibited. Here, we report that yeast cofilin, which is resistant to both subtilisin and trypsin, accelerates greatly the rate of subtilisin cleavage of this loop in F-actin at pH 6.8 and at pH 8.0. Similarly, cofilin accelerates strongly the tryptic cleavage in F-actin of loop 60-69 in subdomain 2, at Arg62 and Lys68. The acceleration of the loops' proteolysis cannot be attributed to an increased treadmilling of F-actin for the following reasons: (i) the rate of subtilisin cleavage is independent of pH between pH 6.8 and 8.0, unlike F-actin depolymerization, which is pH-dependent; (ii) at high concentrations of protease the cleavage rate of F-actin in the presence of cofilin is faster than the rate of monomer dissociation from the pointed end of TRC-labeled F-actin, which limits the rate of treadmilling; and (iii) cofilin also accelerates the rate of subtilisin cleavage of F-actin in which the treadmilling is blocked by interprotomer cross-linking of the D-loop to the C terminus on an adjacent protomer. This suggests a substantial flexibility of the D-loop in the cross-linked F-actin. The increased cleavage rates of the D-loop and loop 60-69 reveal extensive exposure of subdomain 2 in F-actin to proteolytic enzymes by cofilin.
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PMID:Cofilin induced conformational changes in F-actin expose subdomain 2 to proteolysis. 1536 81

Rhizobium trifolii strains IARI and Rel-1 produced substances with broad and narrow activity spectra, respectively. Reproducible inhibitory zones of various sizes produced by R. trifolii IARI (2 to 14 mm) and R. trifolii Rel-1 (2 to 6 mm) were detected, depending upon the indicator organism used. The maximum production of these substances by both strains of R. trifolii was observed on l-arabinose agar. A preliminary characterization of the antimicrobial substance produced by strain IARI showed resistance to heat (75 to 80 degrees C for 45 min), trypsin, lysozyme, DNase I, and RNase A. On the other hand, the substance produced by strain Rel-1 showed sensitivity to heat (75 to 80 degrees C for 45 min) and trypsin, but resistance to lysozyme, RNase A, and DNase I.
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PMID:Production of Antimicrobial and Bacteriocin-Like Substances by Rhizobium trifolii. 1634 2

Phage varphiX174 A(*) protein cleaves single-stranded DNA and then binds to the 5'-phosphorylated terminus of the cleaved DNA fragment, forming a covalent protein-DNA complex. The bound A(*) protein can religate the termini to form covalently closed single-stranded circles. To determine the nature of the covalent linkage and the amino acid involved, we used A(*) protein to cleave DNA synthesized in vivo with [alpha-(32)P]dATP to form the A(*)-single-stranded DNA complex. The complex was then digested with DNase I and the (32)P-labeled A(*) protein was isolated by electrophoresis on polyacrylamide gels. The isolated complex was digested with either trypsin or Pronase. Incubation of the tryptic digest with snake venom phosphodiesterase gave (32)P-labeled products that migrated on electrophoresis on cellulose plates to the cathode, indicating covalent linkage of (32)P-labeled dAMP residues to a tryptic peptide. High concentrations of snake venom phosphodiesterase released all of the (32)P label as free dAMP. Formic acid/diphenylamine depurination (Burton reaction) of the [alpha-(32)P]dATP-labeled peptide-oligonucleotide complexes caused a transfer of the labeled phosphate from dAMP to the peptide. The phosphorylated peptides were isolated on cellulose plates and shown to be sensitive to bacterial alkaline phosphatase, indicating that a phosphodiester bond linked the peptides to the dAMP. The phosphorylated product of the Pronase digest was identified as free phosphotyrosine by its mobility in three different chromatography systems. Likewise, acid hydrolysis (5.6 M HCl, 110 degrees C, 2 hr) of the phosphorylated tryptic peptides revealed linkage of the phosphate to a tyrosine. Thus, A(*) protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated terminus via a tyrosyl-dAMP phosphodiester bond.
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PMID:Cleavage of single-stranded DNA by the varphiX174 A protein: The A-single-stranded DNA covalent linkage. 1659 85

Equine (Equus caballus) deoxyribonuclease I (DNase I) was purified from the parotid gland, and its 1295-bp cDNA was cloned. The mature equine DNase I protein consisted of 260 amino acid residues. The enzymatic properties and structural aspects of the equine enzyme were closely similar to those of other mammalian DNases I. Mammalian DNases I are classified into three types--pancreatic, parotid and pancreatic-parotid-based on their tissue distribution; as equine DNase I showed the highest activity in the parotid gland, it was confirmed to be of the parotid-type. Comparison of the susceptibility of mammalian DNases I to proteolysis by proteases demonstrated a marked correlation between tissue distribution and sensitivity/resistance to proteolysis; pancreatic-type DNase I shared properties of resistance to proteolysis by trypsin and chymotrypsin, whereas parotid-type DNase I did not. In contrast, pancreatic-parotid-type DNase I exhibited resistance to proteolysis by pepsin, whereas the other enzyme types did not. However, site-directed mutagenesis analysis revealed that only a single amino acid substitution could not account for acquisition of proteolysis resistance in the mammalian DNase I family during the course of molecular evolution. These properties are compatible with adaptation of mammalian DNases I for maintaining their activity in vivo.
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PMID:Susceptibility of mammalian deoxyribonucleases I (DNases I) to proteolysis by proteases and its relationships to tissue distribution: biochemical and molecular analysis of equine DNase I. 1754 8

Deoxyribonuclease I (DNase I) is known to be a glycoprotein, and two potential N-linked glycosylation sites (N18 and N106) are known for mammalian enzymes. In the present study, N18 and N106 were mutated in order to investigate the biological role of N-linked glycosylation in three mammalian (human, bovine, and equine) DNases I. The enzyme activities of N18Q and N106Q were lower than that of the wild type, and that of the double mutant (N18Q/N106Q) was lower than those of the single mutants, in accord with the sugar moiety contents in the three mammals. In addition, all mutant enzymes were unstable to heat, suggesting that both sites are required for heat stability. Moreover, in human and equine enzymes, the N18Q and N106Q mutant enzymes were less resistant to trypsin, while N18Q/N106Q was the most sensitive to trypsin. As for bovine DNase I, the trypsin resistance of N18Q and N106Q was similar to that of the wild type, but that of N18Q/N106Q decreased in a time-dependent manner. On the other hand, N-linked glycosylation was not related to pH sensitivity. The results of the present study suggest that N18 and N106 are both necessary for (i) full enzymatic activity, (ii) heat-stability, and (iii) trypsin resistance.
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PMID:Two N-linked glycosylation sites (Asn18 and Asn106) are both required for full enzymatic activity, thermal stability, and resistance to proteolysis in mammalian deoxyribonuclease I. 1906 Mar 93

SYNOPSIS: Reports that elasmobranchs (sharks, skates, and rays) may have a low incidence of disease have stimulated interest in understanding the role of their immune system in this apparent resistance. Although research in this area may potentially translate into applications for human health, a basic understanding of the elasmobranch immune system components and how they function is essential. As in higher vertebrates, elasmobranch fishes possess thymus and spleen, but in the absence of bone marrow and lymph nodes, these fish have evolved unique lymphomyeloid tissues, namely epigonal and Leydig organs. As conditions for short-term culture of elasmobranch immune cells have become better understood, the opportunity to examine functional activity of cytokine-like factors derived from conditioned culture medium has resulted in the identification of growth inhibitory activity against a variety of tumor cell lines. Specifically, the medium enriched by short term culture of bonnethead shark (Sphyrna tiburo) epigonal cells (epigonal conditioned medium, ECM) has been shown to inhibit the growth of mammalian tumor cell lines, including fibrosarcoma (WEHI-164), melanoma (A375.S2), B-cell lymphoma (Daudi), T-cell leukemia (Jurkat), pancreatic cancer (PANC-1), ovarian cancer (NIH:OVCAR-3), and three breast carcinoma cell lines (MCF7, HCC38, Hs578T). Of the cell lines tested, WEHI-164, A375.S2, Daudi, and Jurkat cells were among the most sensitive to growth inhibitory activity of ECM whereas PANC-1 and NIH:OVCAR-3 cells were among the least sensitive. In addition, ECM demonstrated preferential growth inhibition of malignant cells in assays against two different malignant/non-malignant cell line pairs (HCC38/HCC38 BL and Hs 578T/Hs 578Bst). Separation of protein components of ECM using SDS-PAGE resulted in a very reproducible pattern of three major bands corresponding to molecular sizes of approximately 40-42 kD, 24 kD, and 17 kD. Activity is lost after heating at 75 degrees C for 30 min, and can be diminished by treatment with proteinase K and protease. Activity is not affected by treating with trypsin, DNase I or RNase A.
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PMID:Elasmobranch immune cells as a source of novel tumor cell inhibitors: Implications for public health. 1934 8

A novel method for quantifying biomolecules immobilised onto gold and silver nanoparticles is reported; fluorescent-labelled antibodies and DNA are hydrolysed on the surface of the nanoparticles by the addition of trypsin and DNase I, respectively, resulting in the release of the quantifiable fluorescent label into the bulk solution.
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PMID:Quantitation of biomolecules conjugated to nanoparticles by enzyme hydrolysis. 1943 94

Staphylococcus aureus uses the SaeRS two-component system to control the expression of many virulence factors such as alpha-hemolysin and coagulase; however, the molecular mechanism of this signaling has not yet been elucidated. Here, using the P1 promoter of the sae operon as a model target DNA, we demonstrated that the unphosphorylated response regulator SaeR does not bind to the P1 promoter DNA, while its C-terminal DNA binding domain alone does. The DNA binding activity of full-length SaeR could be restored by sensor kinase SaeS-induced phosphorylation. Phosphorylated SaeR is more resistant to digestion by trypsin, suggesting conformational changes. DNase I footprinting assays revealed that the SaeR protection region in the P1 promoter contains a direct repeat sequence (GTTAAN(6)GTTAA [where N is any nucleotide]). This sequence is critical to the binding of phosphorylated SaeR. Mutational changes in the repeat sequence greatly reduced both the in vitro binding of SaeR and the in vivo function of the P1 promoter. From these results, we concluded that SaeR recognizes the direct repeat sequence as a binding site and that binding requires phosphorylation by SaeS.
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PMID:In the Staphylococcus aureus two-component system sae, the response regulator SaeR binds to a direct repeat sequence and DNA binding requires phosphorylation by the sensor kinase SaeS. 2017 98

Metarhizium anisopliae is an entomopathogenic fungus well characterized for the biocontrol of a wide range of plagues. Its pathogenicity depends on the secretion of hydrolytic enzymes that degrade the host cuticle. To identify proteins involved in the infection process and in host specify, immunoproteomic analysis was performed using antiserum produced against crude extract of M. anisopliae cultured in the presence of Rhipicephalus (Boophilus) microplus and Dysdercus peruvianus cuticles. Spots detected using antisera produced against M. anisopliae cultured in cuticles and spore surface proteins, but not with antiserum against M. anisopliae cultured in glucose, were identified so as to give insights about the infection process. An MS/MS allowed the identification of proteases, like elastase, trypsin, chymotrypsin, carboxypeptidase and subtilisin (Pr1A, Pr1I and PR1J), chitinases, DNase I and proline-rich protein. Chymotrypsin and Pr1I were inferred as host specific, being recognized in D. peruvianus infection only. This research represents an important contribution to the understanding the adaptation mechanisms of M. anisopliae to different hosts.
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PMID:Metarhizium anisopliae host-pathogen interaction: differential immunoproteomics reveals proteins involved in the infection process of arthropods. 2094 40

We report a simple staining-free gel electrophoresis method to simultaneously probe protease and nuclease. Utilizing gold nanoparticles (Au-NPs) dual-functionalized with DNA and peptide, the presence and concentration of nuclease and protease are determined concurrently from the relative position and intensity of the bands in the staining-free gel electrophoresis. The use of Au-NPs eliminates the need for staining processes and enables naked eye detection, while a mononucleotide-mediated approach facilitates the synthesis of DNA/peptide conjugated Au-NPs and simplifies the operation procedures. Multiplex detection and quantification of DNase I and trypsin are successfully demonstrated.
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PMID:Staining-free gel electrophoresis-based multiplex enzyme assay using DNA and peptide dual-functionalized gold nanoparticles. 2258 8


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