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)

The gene for L-lactate dehydrogenase (LDH) (EC 1.1.1.27) of Thermus caldophilus GK24 was cloned in Escherichia coli using synthetic oligonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of the LDH was deduced from the nucleotide sequence. The deduced amino acid sequence agreed with the NH2-terminal and COOH-terminal sequences previously reported and the determined amino acid sequences of the peptides obtained from trypsin-digested T. caldophilus LDH. The LDH comprised 310 amino acid residues and its molecular mass was determined to be 32,808. On alignment of the whole amino acid sequences, the T. caldophilus LDH showed about 40% identity with the Bacillus stearothermophilus, Lactobacillus casei and dogfish muscle LDHs. The T. caldophilus LDH gene was expressed with the E. coli lac promoter in E. coli, which resulted in the production of the thermophilic LDH. The gene for the T. caldophilus LDH showed more than 40% identity with those for the human and mouse muscle LDHs on alignment of the whole nucleotide sequences. The G + C content of the coding region for the T. caldophilus LDH was 74.1%, which was higher than that of the chromosomal DNA (67.2%). The G + C contents in the first, second and third positions of the codons used were 77.7%, 48.1% and 95.5% respectively. The high G + C content in the third base caused extremely non-random codon usage in the LDH gene. About half (48.7%) the codons in the LDH gene started with G, and hence there were relatively high contents of Val, Ala, Glu and Gly in the LDH. The contents of Pro, Arg, Ala and Gly, which have high G + C contents in their codons, were also high. Rare codons with U or A as the third base were sometimes used to avoid the TCGA sequence, the recognition site for the restriction endonuclease, TaqI. Two TCGA sequences were found only in the sequence of CTCGAG (XhoI site) in the sequenced region of the T. caldophilus DNA. There were three segments with similar sequences in the two 5' non-coding regions, probably the promoter and ribosome-binding regions, of the genes for the T. caldophilus LDH and the Thermus thermophilus 3-isopropylmalate dehydrogenase.
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PMID:Nucleotide sequence and characteristics of the gene for L-lactate dehydrogenase of Thermus caldophilus GK24 and the deduced amino-acid sequence of the enzyme. 353 39

Mycoplasma pulmonis has substantial DNase activity exposed on the cell surface. At least part of this activity is attributable to an endonuclease. The activity is destroyed at 56 degrees C and inhibited by either 5 mM EDTA or 10 mM zinc chloride. It can also be eliminated by treatment of intact organisms with trypsin and is regenerated by incubation of the treated organisms in a medium that supports protein synthesis. DNase exposed at the cell surface constitutes 20% of the total DNase activity present in M. pulmonis extracts.
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PMID:Identification and preliminary characterization of external membrane-bound nuclease activities in Mycoplasma pulmonis. 394 Oct 2

The endonucleolytic action of a deoxyribonuclease activity in rabbitpox and vaccinia virus was established by change in sedimentation rate of denatured (3)H-lambda deoxyribonucleic acid substrate. The presence of two deoxyribonuclease activities in pox-virus is confirmed. Exo- and endonuclease activities are unmasked by treatment of purified virus with the detergent Nonidet P-40 and further enhanced by treatment of viral "cores" with trypsin.
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PMID:Virus-associated nucleases: evidence for endonuclease and exonuclease activity in rabbitpox and vaccinia viruses. 501 15

A new Streptococcus sanguis strain Wicky endonuclease was isolated, purified and partially characterized. This nuclease acts preferentially on thermally denatured DNA, is not inhibited by RNA and is activated 3-5 times by trypsin. This activation is accompanied by the reduction of molecular weight of the enzyme. These features distinguish the new S, sanguis nuclease from the 3 previously described S. sanguis endonucleases. With covalently closed circular plasmid DNA, the enzyme causes first the appearance of a single stranded nick, then the second nick on the opposite DNA strand, resulting in plasmid DNA linearization. This nuclease most likely is located at the cell surface. The possible relationship of the described nuclease with ability of S. sanguis cells to take up DNA in genetic transformation is discussed.
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PMID:Surface-located trypsin-activated Streptococcus sanguis strain Wicky endonuclease. 619 73

A library of cloned Mycoplasma hyorhinis genomic sequences was constructed by incorporation of EcoRI digestion fragments of mycoplasma DNA into the lambda Charon 4A bacteriophage vector. Immunological screening of recombinant phage plaques identified clones containing genes encoding mycoplasma antigenic structures expressed in an Escherichia coli host. Two such recombinant phage isolates, lambda Ch4A-MhrG1 and lambda Ch4A-MhrG28, were defined and found to contain distinct genomic sequences by analysis of restriction endonuclease fragments. Inoculation of mice with recombinant gene products from lambda Ch4A-MhrG1 yielded antiserum selectively recognizing a Mr 29,500 trypsin-sensitive mycoplasma constituent. This established a means for producing selected immunogenic mycoplasma component in a bacterial host. The cloned genomic sequences of M. hyorhinis encoding expressed mycoplasma antigens represent molecular probes that can be characterized both by specific DNA sequences and by the antigenic structure of corresponding gene products. These genomic fragments define initial physical markers of the M. hyorhinis genome and may be useful in assessing antigenic and molecular genetic relationships within the genus Mycoplasma and among other members of the class Mollicutes.
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PMID:Cloned genomic DNA sequences from Mycoplasma hyorhinis encoding antigens expressed in Escherichia coli. 634 24

A chromatin fraction enriched for Xenopus 5S RNA genes has been isolated by restriction endonuclease digestion and sucrose gradient velocity sedimentation. Soluble chromatin sedimenting at 70-80S contains approximately 50% of the oocyte-expressed 5S RNA genes and only 1.5-3% of total chromatin DNA; this represents a 15- to 30-fold purification of the 5S genes. Such chromatin isolated from somatic cells (blood and cultured kidney cells) retains the transcriptionally-inactive state of the oocyte-expressed 5S genes. Soluble chromatin from somatic cells prepared by micrococcal nuclease digestion also retains the inactive state of the oocyte-type 5S genes. It is likely that the level of chromatin structure responsible for inactivity of the oocyte genes in somatic cells is the nucleosome or short chains of nucleosomes and not supranucleosomal structures. The oocyte-type genes can be rendered transcriptionally active in somatic cell chromatin either by salt extraction of some chromosomal proteins or by treatment with the ion exchange resin Dowex A50W-X2. Alternatively, activation of these genes can be achieved by incubating somatic cell chromatin or nuclei with an extract prepared from Xenopus oocytes. This effect is not specific for 5S RNA genes as the transcription of other small RNAs (including pre-tRNA) is stimulated by the oocyte extract. The activating factor(s) is resistant to micrococcal nuclease, nondialyzable, heat labile and sensitive to trypsin; thus it is highly likely to be a protein or a group of proteins. Partial purification of the activating factor(s) has been achieved by ion exchange chromatography.
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PMID:Control of 5S RNA transcription in Xenopus somatic cell chromatin: activation with an oocyte extract. 686 64

Ribosomal proteins S1 when associated with the 30-S subunit does not interact with 16-S RNA but its binding is determined mostly by protein-protein interactions. These conclusions are based on the following data. 1. Ultraviolet irradiation (lambda = 254 nm) of the 30-S subunit does not result in the covalent cross-linking of S1 with 16-S RNA at irradiation doses up to 150 quanta/nucleotide, whereas the irradiation under the same conditions of S1 . polynucleotide complexes [S1 . poly(U), S1 . poly(A) and S1 . Q beta phage RNA] induces effective formation of polynucleotide-protein cross-links. 2. Mild treatment of 30-S subunits lacking S-1 with RNase A or with cobra venom endonuclease results in removal of 10--20% of the total nucleotide material but does not affect their sedimentation characteristics of their S1 binding capacity. 3. The association of S1 with S1-depleted 30-S subunits is insensitive to aurintricarboxylic acid, which is known as a strong inhibitor of complex formation between S1 and polynucleotides. 4. Mild trypsin treatment of S1-depleted 30-S subunits greatly reduces their S1 binding capacity.
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PMID:Ribosomal protein S1 associates with Escherichia coli ribosomal 30-S subunit by means of protein-protein interactions. 703 93

Two heat-sensitive R.BamHI mutants, T157I and P173L, and one cold-sensitive R.BamHI mutant, T114I, were isolated after chemical mutagenesis of the bamhIR gene that codes for the restriction endonuclease BamHI (R.BamHI). The thermosensitivity of T114I, T157I and P173L is revealed by the 10(2)-10(3) lower plating efficiency at the non-permissive temperature of strains bearing these alleles. The conditional-lethal phenotype can be rescued by introduction of the cognate bamhIM gene into the same cell. The mutant enzymes induce the SOS response in vivo and display reduced phage restriction activity. The P173L protein, when expressed at 30 degrees C and purified, shows reduced thermostability at 65 degrees C. T157I and P173L mutants yield different intermediates during partial trypsin digestion. The conditional-lethal BamHI mutants could be used to deliver in vivo DNA cleavage and for further isolation of relaxed-specificity mutants.
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PMID:Isolation of temperature-sensitive mutants of the BamHI restriction endonuclease. 760 13

A number of inhibitors with different specificities were used to probe the involvement of proteinases in the mechanism of cytotoxic T lymphocyte (CTL)-mediated lysis. N-Acetyl-L-tyrosine ethyl ester (ATEE) and N-benzoyl-L-arginine ethyl ester (BAEE) are reversible substrate inhibitors of proteinases with trypsin-like and chymotrypsin-like specificities, respectively. BAEE did not prevent either the chromium release or DNA fragmentation induced in mouse tumor target cells by a mixed lymphocyte population. In contrast, ATEE inhibited both processes. The irreversible proteinase inhibitor 3,4-dichloroisocoumarin (DCI) also blocked both chromium release and DNA fragmentation, but at significantly lower concentrations than ATEE. More importantly, chromium release was more susceptible to inhibition by DCI than DNA fragmentation. Addition of a combination of the endonuclease inhibitor aurintricarboxylic acid plus DCI resulted in virtually complete inhibition of both DNA fragmentation and chromium release when the drugs were added at the beginning of the incubation period. In contrast addition of DCI 15 or 30 min following initiation of the lytic cycle abolished the affect of DCI on fragmentation, but not lysis. A model which suggests a dual role for the proteinases in CTL-mediated target cell death is presented. First, proteinases are involved in the initiation of DNA fragmentation. Second, they have an ongoing function in membrane damage.
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PMID:Proteinases are involved in both DNA fragmentation and membrane damage during CTL-mediated target cell killing. 773 79

We have partially characterized the granules of the human NK cell line, YT-INDY, and assessed granule-mediated lysis and DNA fragmentation of assorted targets. Biochemical studies demonstrated significant quantities of granzyme B (asp-ase) and a heretofore undescribed chymase but no tryptase (i.e., granzyme A or 3) or distinct met-ase. YT-INDY expressed mRNA for granzyme B, perforin and CCPX. The existence of perforin was confirmed by immunoblot. The granules lysed both human and murine NK-sensitive and NK-resistant targets. YT-INDY and NK3.3, two human cytotoxic cells, were also lysed. EGTA reduced lysis by only 50%, suggesting that a perforin-independent lytic pathway is associated with the granules. In addition, 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF), an inhibitor that selectively blocked the chymase and 3,4-dichloroisocoumarin (DCI), an inhibitor that inactivated both chymase and asp-ase activities, marginally affected lysis. By gel electrophoresis and 125I-labeled deoxyuridine release assay, only murine cells (SP2/0 and YAC-1) underwent DNA fragmentation, and cleavage was completely inhibited by DCI, whereas EGTA, AEBSF and aurintricarboxylic acid (ATA) had no effect. The results, therefore, underscore the central role of granzyme B in granule-mediated DNA fragmentation, emphasize that the protease acts via an ATA-resistant endonuclease pathway and stress that nucleolysis does not invariably accompany granule-mediated cytolysis. Finally, ATA inhibited the asp-ase activity of isolated but not granule-associated granzyme B. ATA, therefore, is not a specific endonuclease inhibitor and results obtained with ATA should be viewed cautiously.
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PMID:Human granzyme B is essential for DNA fragmentation of susceptible target cells. 808 28


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