Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This paper addresses the problem of detecting weak incorporation of BrdUrd and the related efficiency of the denaturation protocols used to unmask this thymidine analog. Evidence is presented that measuring the distribution of BrdUrd-tagged fluorescence intensities by image cytometry generates a standard deviation threshold that discriminates between positive and negative MRC5 cells in vitro. A comparison of the thresholding by standard deviation (SDT) with the usual thresholding by the nuclear total fluorescence intensity (FIT) demonstrated that SDT has a significantly higher sensitivity (99.4-100%, depending on the denaturation protocols) than FIT (94.7 and 74.3%, respectively), although both tests have a high specificity (93% and 100%, respectively) for detecting S cells. Since detecting the S cells is not only dependent on the test used, but also on the denaturation protocols, a quality index (QI) was derived from the standard deviation and the mean value of the non-specific fluorescence of negative cell population versus BrdUrd fluorescence of positive cell population. The following DNA denaturation protocols have been assessed according to QI: acidic denaturation, thermal denaturation in formamide, and thermal denaturation in distilled water. Each denaturation procedure was preceded or not by incubation in either proteinase K or Triton X-100. The results showed that thermal denaturation in formamide, especially when preceded by proteinase K incubation, revealed the largest difference between negative and positive cells. This work also demonstrated that image cytometry of BrdUrd-labelled cells can be suitable for clinical application because of the high sensitivity provided and the small samples needed.
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PMID:Detection of S cells and evaluation of DNA denaturation protocols by image cytometry of fluorescent BrdUrd labelling. 218 16

Smooth (S)-lipopolysaccharide (LPS) preparations from reference and field strains of several biovars of Brucella abortus, B. melitensis, and B. suis were prepared by (i) the hot phenol-water method, (ii) hot sodium dodecyl sulfate extraction and proteinase K digestion, or (iii) dimethyl sulfoxide extraction. These S-LPS-enriched fractions were further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining after periodate oxidation. Immunoblots were developed by using either monoclonal antibodies specific for Brucella A or M antigens or polyclonal polyspecific or monospecific sera from rabbits, cattle, and goats. The specificity of monoclonal antibodies reactive with Brucella unique (A or M) epitopes was demonstrated by enzyme-linked immunosorbent assay, LPS latex agglutination, or agglutination inhibition. The most-represented subunits of S-LPS ranged in Mr from 30,000 to 70,000 relative to marker proteins. According to A or M immunodominance, two sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns were clearly distinguished among biovars, whatever the fraction tested: a close succession of regularly spaced narrow bands for A greater than M strains and regularly spaced triplets of bands including either (i) a first thin band followed by two thick bands for B. abortus M greater than A strains or (ii) one thick band between two thin bands for B. melitensis or B. suis M greater than A strains. Moreover, A and M specificities were reaffirmed by sandwich enzyme immunoassay and latex agglutination inhibition with monoclonal antibodies and polyclonal sera.
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PMID:Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of smooth-lipopolysaccharide heterogeneity among Brucella biovars related to A and M specificities. 222 39

A monoclonal antibody, prepared against whole cells of Clostridium tyrobutyricum, recognized a surface antigen extracted by heat treatment or by hot phenol-water treatment. This antigen, after analysis by polyacrylamide gel electrophoresis and immunoblotting, has been shown to present a regularly-spaced ladder pattern similar to those shown by the lipopolysaccharide of many gram-negative bacteria. The proteinase K has been shown to have no effect on the recognition of this epitope by the monoclonal antibody. On the contrary, the inhibition of the antigen reactivity to the monoclonal antibody after a mild periodate oxidation suggests the involvement of a carbohydrate moiety in the epitope. Moreover, the SDS-PAGE analysis of phenol-water extracts has shown an additional compound, detected by silver staining but not recognized by the monoclonal antibody.
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PMID:Demonstration of a surface antigen of Clostridium tyrobutyricum by use of immunoblotting with a monoclonal antibody. 232 79

Modification of the alkaline lysis at elevated temperature technique is proposed isolation of plasmid DNA from lactobacilli. Modification consists of colorimetric control of culture phase during the biomass growth, pH control at the probes treatment with lysozyme and alkaline solution of natrium dodecylsulfate by adding the indicator bromcrezolpurple into the medium for biomass growth. The high concentration of lysozyme is used (10 mkg.ml-1). Lactobacilli are lysed at 2 min incubations of the probes with the lytic solution in the boiling water bath. The treatment of the probes by proteinase K, by the mixture of chloroform:phenol:isoamyl spirit (25:24:1 vol/vol/vol) and by diethylpirocarbonate increased considerably the quality of the obtained DNA preparations. The modified technique is suitable for isolation of the plasmid DNA from lactobacilli of different species, enterococci, streptococci and other lactic bacteria. The connection of antibiotic resistance marker and the plasmid profile of lactobacilli under different conditions with the presence of the plasmid DNA- protein complex is discussed.
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PMID:[Optimization of the method of isolation of microamounts of plasmid DNA from lactobacilli]. 236

Outer membrane extracts of Treponema hyodysenteriae were used to evaluate the antibody responses in immunized or convalescent pigs. Western blot (immunoblot) analysis identified antibodies in sera reactive with 14- to 19-kilodalton (kDa) antigens. Reactivity against these antigens could be removed only by absorption of sera with butanol-water-extracted endotoxin from the homologous strain of T. hyodysenteriae. Treatment of the outer membrane extracts with 0.1 M sodium meta-periodate, but not with proteinase K, abolished reactivity with both outer membrane and endotoxin antigens (14 and 19 kDa). These results indicate that swine vaccinated with the outer membrane extract of T. hyodysenteriae develop antibody responses to outer membrane antigens qualitatively similar to those of swine convalescing from active infection, especially antibodies against low-molecular-mass antigens. The nature of the 14- to 19-kDa antigens appears consistent with that of treponemal endotoxin and lipopolysaccharide.
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PMID:Characterization of the major outer membrane antigens of Treponema hyodysenteriae. 246 Apr 6

Bacterial restriction endonucleases were used to produce DNA cleavage patterns that could be useful as tools to study the relatedness among Anaplasma marginale isolates. Bovine erythrocytes infected with A. marginale were lysed, washed, and embedded in agarose. The embedded erythrocytes and bacterial pathogens were partially digested by sequential infiltration of the agarose with acetone, lysozyme, sodium dodecyl sulfate, and proteinase K. The unfragmented genomic DNA was left supported and protected in a porous matrix. The DNA was digested in situ in agarose under the following conditions: (i) brief treatment with phenol, (ii) brief washing with distilled water, and (iii) adjustment of restriction enzyme digestion mixture to compensate for the volume of the agarose. The cleaved DNA was electrophoresed horizontally to produce a DNA cleavage pattern. Of 19 restriction enzymes screened, 12 produced distinct DNA bands from the genomes of each of the five A. marginale isolates examined. The DNA cleavage pattern produced from each isolate with a given restriction enzyme was reproducible. However, the DNA cleavage patterns produced from different isolates with a given restriction enzyme were not necessarily identical. This procedure could be modified for general bacterial DNA isolation, in situ agarose digestion, and manipulations.
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PMID:Isolation and restriction endonuclease cleavage of Anaplasma marginale DNA in situ in agarose. 283 4

The apical plasma membrane of epithelial cells of frog and toad urinary bladder is subject to large modifications during the induction of water permeability by the antidiuretic hormone. A better characterization of the apical membrane is necessary for a clear understanding of the mechanisms of hormone action. Towards this end, apical material was extracted by enzymatic treatment and by incubation with detergent. Proteolytic enzyme alone had little effect under our conditions. A pretreatment with several glycosidases (alpha-mannosidase or endo-beta-N-acetylglucosaminidase H) increased the hydrolytic action of papain, elastase, proteinase K or Staphylococcus aureus V8 protease and allowed the detection of a major 76 kD in SDS gel electrophoresis. The n-octyl-beta-D-glucopyranoside (0.2%) led to the extraction after 150 mn of 1 to 5 micrograms proteins per cm2 of amphibian urinary bladder apical surface. The extracted proteins migrated as several bands on SDS gels. One of them probably corresponds to the 76 kD fragment obtained after proteolysis. The absence of alteration of the water permeability after extraction and the good preservation of the ultrastructure are evidence for the localisation of the 76 kD at the apical membrane surface. This protein may be the best candidate as antigen to raise antibodies against the apical surface of amphibian urinary bladder epithelial cells.
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PMID:Apical material extracted from amphibian urinary bladder epithelium by enzymes and detergent treatment. 293 6

The specific recognition by mitochondria of the precursor of porin and the insertion into the outer membrane were studied with a radiolabeled water-soluble form of porin derived from the mature protein. High-affinity binding sites had a number of 5-10 pmol/mg mitochondrial protein and a ka of 1-5 X 10(8) M-1. Binding was abolished after trypsin pretreatment of mitochondria indicating that binding sites were of protein-aceous nature. Specifically bound porin could be extracted at alkaline pH but not by high salt and was protected against low concentrations of proteinase K. It could be chased to a highly protease resistant form corresponding to mature porin. High-affinity binding sites could be extracted from mitochondria with detergent and reconstituted in asolectin-ergosterol liposomes. Water-soluble porin competed for the specific binding and import of the precursor of the ADP/ATP carrier, an inner membrane protein. We suggest that (i) binding of precursors to proteinaceous receptors serves as an initial step for recognition, (ii) the receptor for porin may also be involved in the import of precursors of inner membrane proteins, and (iii) interaction with the receptor triggers partial insertion of the precursor into the outer membrane.
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PMID:High-affinity binding sites involved in the import of porin into mitochondria. 296 May 20

The fungal proteinase K (EC 3.4.21.14) is a very potent unusually stable member of the subtilisin family. Its X-ray structure determined at 0.15-nm resolution shows two bound Ca2+ ions. Ca1 is in near-ideal pentagonal bipyramidal configuration with Asp200 carboxylate and Pro175 peptide C = O in an apical, and Val177 peptide C = O and four water molecules in an equatorial position, whereas Ca2 displays incomplete octahedral coordination with the carboxylate of Asp260, the peptide C = O of Val16 and the two water molecules. Scatchard analysis of the titration of Ca2+-free proteinase K with Ca2+ yields a single dissociation constant (7.6 +/- 2.5) x 10(-8) M associated with the tightly bound Ca1 whereas Ca2 is so weakly bound that it cannot be titrated. If proteinase K is depleted of Ca2+ by treatment with EDTA, followed by gel filtration, its enzymatic activity drops within 6 h to 20% of its original value, without autolysis. Addition of excess Ca2+ immediately raises the residual activity to 28%, but full activity is not achieved. Removal of Ca2+ triggers a conformational change of the substrate recognition site because there is a direct connection, via secondary structure hydrogen bonds, between the Ca1 binding site and the substrate-recognition site. This is indicated further by circular dichroism and fluorescence-spectroscopic data, and by reversed-phase FPLC, carried out in the presence and absence of Ca2+, but the overall structure of the enzyme is not affected. Depletion of Ca2+ also influences binding of longer peptide inhibitors of the chloromethane type, it increases the rate of autolysis after about 48 h, it reduces the thermal stability (measured by activity tests from 65 degrees C to 46 degrees C), and it enhances the deactivation by 8 M urea which inactivates to only 65%, whereas sodium dodecyl sulfate totally inactivates at a concentration of 12.5%.
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PMID:The enzymatic activity of proteinase K is controlled by calcium. 316 26

The crystal and molecular structure of proteinase K was determined by X-ray diffraction data to 0.15-nm resolution. The enzyme belongs to the subtilisin family with an active-site catalytic triad Asp39--His69--Ser224 but is a representative of a subgroup with a free Cys73 close to and 'below' the active His69. Besides this Cys72, proteinase K has two disulfide bonds, Cys34--Cys123 and Cys178--Cys249, which contribute to the stability of the tertiary structure consisting of an extended central parallel beta-sheet decorated by six alpha-helices, three short antiparallel beta-sheets, 18 beta-turns and involving several internal, structurally important water molecules. Proteinase K exhibits two Ca2+-binding sites, one very strong and the other weak, which were the sites of the heavy atoms (Pb2+, Sm3+) used to solve the crystal structure. The weak binding site is liganded to the N and C termini, Thr16 and Asp260, and is only incompletely coordinated by oxygen ligands. The strong binding site is coordinated in the form of a pentagonal bipyramid with the side chain carboxylate of Asp200 and the C = O of Pro175 as apex, and C = O of Val177 and four water molecules in the equatorial plane. Upon removal of this Ca2+, proteinase K loses activity which is interpreted in terms of a local structural deformation involving the substrate-recognition site (Ser132--Gly136), probably associated with a cis----trans isomerization of cis Pro171. Several water molecules are located in the active site. One, W335, is positioned in the 'oxyanion hole' and is displaced by the C = O of the scissile peptide bond of the substrate, as indicated by crystallographic studies with peptide chloromethane inhibitors. Based on these experiments, a reaction mechanism is proposed where the peptide substrate forms a three-stranded antiparallel pleated sheet with the recognition site of proteinase K consisting of Ser132--Leu133--Gly134 on one side and Gly100--Ser101 on the other, followed by expulsion of the oxyanion hole water W335 and hydrolytic cleavage by the Asp39--His69--Serr224 triad. These latter residues display low thermal motion corresponding to well-defined geometry and are hardly accessible to solvent molecules, whereas the recognition-site amino acids are more flexible and partially exposed to solvent.
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PMID:Three-dimensional structure of proteinase K at 0.15-nm resolution. 320 85


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