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)

The lipopolysaccharides of Neisseria meningitidis and Neisseria gonorrhoeae were examined by electrophoresis after whole-cell lysis and proteinase K digestion. The banding patterns observed from clinical isolates and laboratory strains demonstrated lipopolysaccharide which included a small number of smooth high-molecular-weight molecules as well as the previously reported lower-molecular-weight rough lipopolysaccharide.
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PMID:Lipopolysaccharide banding patterns of Neisseria meningitidis and Neisseria gonorrhoeae. 642 61

Addition of pyruvate to growth medium failed to induce the changes in Neisseria gonorrhoeae that have been reported previously. Addition of 3.8 mM sulfite or 1.3 mM sulfite plus 14 mM pyruvate restored the medium's reactivity in a test for cysteine and its ability to induce changes in N. gonorrhoeae. The induced changes that were restored were (i) increased colonial opacity and roughness, (ii) increased sensitivity to killing by normal human serum, and (iii) electrophoretic changes that may represent changes in lipopolysaccharide. Further characterization of the electrophoretic changes showed that the bands were resistant to treatment with proteinase K, that they were not affected by EDTA and urea, and that they were not dependent upon the stage of growth.
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PMID:A role for sulfite in inducing surface changes in Neisseria gonorrhoeae. 643 4

Coxiella burnetii morphological cell types were fractionated into large-cell variant cell walls, two fractions of small-cell variant cell walls, and one fraction of small-cell variant whole cells. Based on the contents of peptidoglycan (PG)-constituents and the yields of the sodium dodecyl sulfate-insoluble PG-protein complex (PG-PC) from cell walls, the fraction of large-cell variant cell walls contained significantly less PG than did the fraction of small-cell variant cell walls. The yields of PG-PC from the fractions of large-cell variant cell walls and small-cell variant cell walls were 2 and 32%, respectively. These results indicated that the PG of the large-cell variant cell walls may be partially digested by PG-lytic enzymes or incompletely synthesized, whereas the small-cell variant cell walls appeared to have intact PG. Proteins associated with PG-PC were resistant to proteolysis by trypsin, protease VI, and proteinase K. Saturated and unsaturated fatty acids were detected in whole cells and cell walls but not in PG-PC, which contained a 3-deoxy-D-mannooctulosonic acid-like component that is also present in phase I lipopolysaccharide. Immunogenicity of the fractions was tested by measuring the temporal sequence of phase II and phase I antibody responses in vaccinated rabbits. Both phase II and phase I antibody responses were demonstrated with all fractions except the sodium dodecyl sulfate supernatant of the small-cell variant cell walls, whereas PG-PC elicited a pure phase II antibody response up to 29 days postvaccination. The immunogenicity of these fractions may reflect a quantitative difference in antigen concentration or may be due to a qualitative difference in phase II and I determinants.
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PMID:Biochemical and immunological properties of Coxiella burnetii cell wall and peptidoglycan-protein complex fractions. 650 Dec 33

Human promyelocytic leukemia cells, when differentiated into macrophages by treatment with phorbol myristate acetate, secrete a cytolytic factor. Enhanced production was achieved when the cultures were treated with bacterial lipopolysaccharide (LPS). Production of the factor was inhibited when cultures were treated with dactinomycin immediately after LPS treatment. Tritirachium alkaline proteinase treatment inactivated the factor, indicating that it has an essential protein moiety. The molecular weight was found to be approximately 40,000 by Sephacryl S-200 gel filtration. The factor was stable at 56 degrees C for 30 minutes, but 80% of the activity was inactivated at 70 degrees C in 30 minutes. The factor was destroyed (96%) by dialysis against 0.01 M HCl (pH 2) for 14 hours. The cytolytic factor had little activity on normal fibroblasts, but it was able to significantly kill transformed cells in vitro.
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PMID:Production of a cytotoxin from phorbol myristate acetate-treated human promyelocytes. 658 36

Cultured human monocytes released cytostatic activity upon in vitro activation with lymphokines and lipopolysaccharide. This activity was mainly due to the presence of two different cytostatic factors, termed CstF I and II, which were separated by ion-exchange chromatography. At neutral pH, CstF I bound to the weak anion exchanger DEAE-Sephacel but not to the weak cation exchanger CM-Sepharose, whereas CstF II bound to CM-Sepharose but not to DEAE-Sephacel. The molecular weights of CstF I and II as determined by gel filtration were 55,000 and 40,000, respectively. Upon chromatofocusing, CstF I behaved as if it had an isoelectric point of 5.3. Neither CstF I nor CstF II bound specifically to concanavalin A-Sepharose, indicating the absence of carbohydrate residues containing alpha-D-mannopyranosyl, alpha-D-glucopyranosyl, or sterically related components. Both factors were susceptible to inactivation by proteinase K, demonstrating their protein nature. CstF II was purified more than 3,000-fold upon chromatography on CM-Sepharose and Sephacryl S-200. Ion-exchange chromatography and chromatofocusing of CstF I removed 97% of the proteins in the monocyte supernatant, but only 15% of the activity was recovered, resulting in a fivefold purification of CstF I.
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PMID:Physicochemical characterization of cytostatic factors released from human monocytes. 714 97

Smooth, rough, and neutral forms of lipopolysaccharide (LPS) from Pseudomonas aeruginosa were used to assess the appropriate conditions for effective enzyme-linked immunosorbent assay (ELISA) of LPS. Each of these forms of well-defined LPS was tested for the efficiency of antigen coating by various methods as well as to identify an appropriate type of microtiter plate to use. For smooth LPS, the standard carbonate-bicarbonate buffer method was as efficient as the other sensitivity-enhancing plate-coating methods compared. The rough LPS, which has an overall hydrophobic characteristic, was shown to adhere effectively, regardless of the coating method used, to only one type of microtiter plate, CovaLink. This type of plate has secondary amine groups attached on its polystyrene surface by carbon chain spacers, which likely favors hydrophobic interactions between the rough LPS and the well surfaces. Dehydration methods were effective for coating microtiter plates with the neutral LPS examined, which is composed predominantly of a D-rhamnan. For the two dehydration procedures, LPS suspended in water or the organic solvent chloroform-ethanol was added directly to the wells, and the solvent was allowed to dehydrate or evaporate overnight. Precoating of plates with either polymyxin or poly-L-lysine did not give any major improvement in coating with the various forms of LPS. The possibility of using proteinase K- and sodium dodecyl sulfate-treated LPS preparations for ELISAs was also investigated. Smooth LPS prepared by this method was as effective in ELISA as LPS prepared by the hot water-phenol method, while the rough and neutral LPSs prepared this way were not satisfactory for ELISA.
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PMID:Appropriate coating methods and other conditions for enzyme-linked immunosorbent assay of smooth, rough, and neutral lipopolysaccharides of Pseudomonas aeruginosa. 749 23

Using flow cytometry and fluorescein-labelled lipopolysaccharide (LPS) from Salmonella minnesota R595 (FITC-ReLPS), we studied the role of membrane proteins in the recognition of LPS by human polymorphonuclear granulocytes (PMN) in the absence of serum. Treatment of PMN with trypsin, pronase E or proteinase K reduced both the binding of FITC-ReLPS to PMN at 4 degrees and the response of PMN to LPS at 37 degrees, as measured by luminol-enhanced chemiluminescence. Neuraminidase treatment enhanced both activities. Trypsin treatment of PMN after the binding of FITC-ReLPS effectively reduced fluorescence when cells were kept at 4 degrees, while further incubation of FITC-ReLPS-labelled PMN at 37 degrees rendered fluorescence insensible to trypsin. These results indicate a protein structure of the LPS binding site, association of FITC-ReLPS with the cell membrane at 4 degrees and subsequent internalization at 37 degrees. The binding of FITC-ReLPS was not inhibited by the anti-CD14 monoclonal antibody (mAb) 3C10, which recognizes a functional epitope of CD14. Furthermore, binding of FITC-ReLPS was observed to PMN obtained from a patient with paroxysmal nocturnal haemoglobinuria who lacked membrane-bound CD14. Stimulation of PMN with tumour necrosis factor (TNF) or LPS enhanced the binding of FITC-ReLPS at 4 degrees. This was not observed after activation of PMN devoid of granules (cytoplasts), indicating that the binding of LPS at the cell surface is enhanced by mobilization of LPS-binding proteins from intracellular granules. These studies provide evidence that LPS binding and activation of PMN involves protein structures at the cell surface different from CD14, and that granules constitute a pool of LPS-binding proteins that can be translocated to the cell surface upon stimulation.
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PMID:Modulation of lipopolysaccharide binding to human granulocytes. 753 36

Sera from patients suffering from Mediterranean spotted fever (i.e. an infection due to Rickettsia conorii) were studied by immunoblot to investigate cross-reactivity. A prevalence of IgM antibodies to Proteus OX 19, Proteus OX 2, to the Rickettsia typhus group, to Legionella pneumophila serovars 4 and 5, to L. bozemanii Wiga and to L. micdadei Tatlock was found. Western blot confirmed that the antibodies were directed against the lipopolysaccharide as demonstrated by proteinase K digestion of the antigens. Cross-adsorptions showed that there is a common cross-reacting epitope among L. bozemanii Wiga, R. typhi and Proteus OX 19 but cross-reacting antibodies to L. micdadei and OX 2 were distinct and independent. This IgM cross-reaction could lead to a misdiagnosis.
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PMID:Immunoblot cross-reactions among Rickettsia, Proteus spp. and Legionella spp. in patients with Mediterranean spotted fever. 754 Dec 70

Monoclonal antibodies (mAbs) against Actinobacillus pleuropneumoniae serotype 2 (reference strain Shope 4226 and field isolate F46) were produced. Twelve hybridoma clones were selected against both strains, and all the antibodies secreted were found to be reactive with whole-cell antigen of the homologous strain in ELISA, whereas only one mAb was reactive in slide agglutination test. The predominant antibody classes were IgG2b and IgG3, although IgG1 and IgM were also obtained. Immunoblot assay showed that mAbs could recognize a ladder band profile which is in accordance with the O-antigen of lipopolysaccharide. Most of the epitopes involved were resistant to proteinase K and also to boiling in the presence of sodium dodecyl sulfate and reducing conditions, but they were sensitive to periodic acid. The 12 mAbs recognized neither reference strains of the remaining A. pleuropneumoniae serotypes nor other taxonomically related Gram-negative organisms. The suitability of mAbs for serotyping of field isolates was also examined, and a high correlation (97.4%) was found between the results previously established by indirect hemagglutination with polyclonal rabbit sera and those obtained by ELISA with mAbs. The panel of mAbs described in this study was found to be extremely useful for identifying field isolates belonging to serotype 2 and could be used as a complementary serotyping method.
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PMID:Characterization of monoclonal antibodies to O-antigen of lipopolysaccharide of Actinobacillus pleuropneumoniae serotype 2 and their use in the classification of field isolates. 754 Dec 71

Monoclonal antibody (MAb) 4E8C12 has been previously reported to recognise low mol. wt proteins from enterohaemorrhagic Escherichia coli (EHEC) serotypes O157:H7 and O26:H11. Crude lipopolysaccharide (LPS) preparations from proteinase K-digested bacterial suspensions reacted in Western blots with MAb 4E8C12, as did highly purified LPS from O157:H7 strains. The material recognised by this antibody was, therefore, LPS. The LPS epitope was identified by a whole-cell ELISA in several EHEC, verotoxin producing E. coli (VTEC) and verotoxin-negative strains in addition to E. coli serotypes O157:H7 and O26:H11. Acriflavine and bile salts enhanced the production or availability of the epitope at the cell surface and in culture supernates. These data indicate that the presence of the epitope did not correlate with the virulence of these organisms.
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PMID:Further characterisation of a monoclonal antibody reactive with Escherichia coli O157:H7. 756 87


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