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 immunophysical characteristics of 29 Serratia marcescens strains isolated from hospitalized patients in three different cities were studied. Their outer membrane antigens were compared by solid-phase radioimmunoassay inhibition, and their proteinase K-treated, whole-cell lysates were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. The strains had a limited number of unique outer membrane lipopolysaccharide (LPS) and capsular polysaccharide (K) antigens. By solid-phase radioimmunoassay inhibition, these strains could be divided into four distinct LPS and five K antigenic groups. By SDS-PAGE, the LPS groups could be further divided into three distinct SDS-PAGE core polysaccharide profiles and five distinct O-side-chain polysaccharide profiles. Immunoblot analysis with rabbit antiserum confirmed the limited heterogeneity of these isolates. Of the strains tested, no PAGE profile was unique to blood or nonblood isolates or to organisms collected from a given hospital. Variability of O and core PAGE profiles was not a function of organism growth cycle. Five representative Serratia strains were tested by SDS-PAGE and immunoblot analysis and in a bactericidal assay with normal human serum. We found that (i) the normal human serum had antibodies to the LPS of each of the strains, (ii) the anti-LPS antibody measured by immunoblot did not correlate with the level of bactericidal activity in the normal human serum, (iii) three of four sepsis isolates were serum sensitive, (iv) two Serratia strains serum sensitive in log-phase growth became serum resistant in late stationary-phase growth and under limiting nutrient conditions, and (v) no LPS PAGE profile distinguished serum-sensitive from serum-resistant strains.
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PMID:Immunophysical characterization of human isolates of Serratia marcescens. 240 11

A panel of monoclonal antibodies (MAbs) was prepared to analyze the antigenic structure of the tick-borne encephalitis (TBE) virus glycoprotein E. Nineteen different epitopes were identified and characterized with respect to serological specificity, functional activity, structural properties, and topological relationships. Except for 3 isolated epitopes (i1, i2, and i3), these cluster to form three non-overlapping domains termed A, B, and C. The structural properties of epitopes were assessed by analyzing the effect of different treatments (SDS denaturation, reduction and carboxymethylation, performic acid oxidation, exposure to pH 5.0, CNBr, and trypsin cleavage) on the antigenic reactivities of each epitope. Only 3 epitopes of domain A as well as i2 were sensitive to SDS alone, whereas all others were SDS resistant. Reduction and carboxymethylation, however, destroyed the antigenic reactivity of all epitopes of domain B and also that of two SDS-resistant epitopes of domain A, indicating the role of disulfide bridges in stabilizing the conformation of these epitopes. Deglycosylation by N-Glycanase abolished the SDS resistance of domain C, providing evidence of the role of the carbohydrate side chain in stabilizing these epitopes. A conformational change induced by acid pH was revealed by differences in protease (proteinase K) cleavage maps before and after acid pH treatment. The conformational change involved the epitopes of domain A and occurred between pH 6.0 and 5.5 with the the threshold at pH 7.0.
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PMID:Epitope model of tick-borne encephalitis virus envelope glycoprotein E: analysis of structural properties, role of carbohydrate side chain, and conformational changes occurring at acidic pH. 246 73

In an attempt to identify antigenic differences between Treponema pallidum subsp. pallidum (T. pallidum) and Treponema pallidum subsp. pertenue (T. pertenue) a gene bank of T. pertenue was constructed in lambda vector EMBL3. Clones carrying the T. pertenue gene encoding a 190 kDa protein, TyF1, were selected and the DNA was expressed in E. coli. TyF1 was shown to be closely related, but slightly different from the previously cloned T. pallidum antigen TpF1. TyF1 and TpF1 are high molecular weight antigens of about 190 kDa, which dissociate into 19 kDa subunits after heat treatment in presence of SDS. The difference between the two proteins is most obvious after treatment with proteinase K, which yields a 115 kDa component from TyF1 and a 95 kDa component from TpF1, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The structural genes encoding TyF1 and TpF1 were sequenced and the predicted amino acid sequences differed in a single amino acid residue at position 40, which is arginine in TyF1 and glutamine in TpF1. Similarities TyF1 and TpF1 with the previously described 4D antigen are discussed. The antibody response to TyF1 and TpF1 seems higher in syphilis patients than in yaws patients. The possibility of using the difference between these T. pallidum and the T. Pertenue antigens for serological discrimination of syphilis and yaws is discussed.
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PMID:Treponema pallidum subspecies pallidum (Nichols) and Treponema pallidum subspecies pertenue (CDC 2575) differ in at least one nucleotide: comparison of two homologous antigens. 247 12

Cryptosporidiosis is a diarrheal disease of humans, calves, and other mammals caused by the coccidian parasite Cryptosporidium parvum. Immune bovine serum and two surface-reactive antisporozoite mAb with neutralizing activity were used to identify sporozoite surface Ag by radioimmunoprecipitation/SDS-PAGE and immunoblotting. When isolated sporozoites were incubated with mAb 18.44, 12 to 25 times the ID50 for mice was completely neutralized. This mAb binds diffusely to the sporozoite surface and recognizes a sporozoite surface Ag that eluted in the void volume of a Bio Gel A column with an exclusion limit of 500,000 daltons. The Ag recognized by mAb 18.44 was not radiolabeled with 125I or [35S] methionine, migrated with the dye front in SDS-PAGE, and was insensitive to proteinase K digestion, suggesting a non-protein composition. mAb 17.41 significantly neutralized 25 times the ID50 of sporozoites for mice. This mAb binds multifocally to the sporozoite surface and recognizes [35S] methionine-labeled sporozoite surface Ag of 28,000 m.w., 55,000 m.w., and 98,000 m.w. Immune bovine serum immunoprecipitated [35S] methionine- or 125I-labeled sporozoite Ag ranging from less than 14,300 m.w. to greater than 200,000 m.w., including surface Ag of 28,000 m.w. and 55,000 m.w. The results indicate that two different molecules capable of inducing neutralizing antibody are exposed on the surface of C. parvum sporozoites.
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PMID:Neutralization-sensitive epitopes are exposed on the surface of infectious Cryptosporidium parvum sporozoites. 247 30

The binding of C3 and C9 on serum sensitive (FA635) and serum resistant (FA638) transformants of serum sensitive Neisseria gonorrhoeae strain F62 was examined. Previous studies showed that these transformants have Protein IAs which are minimally different by proteinase K cleavage and primary structural and peptide mapping and bear LPS which vary slightly on SDS-PAGE. Binding of C3 and C9 on FA635 exceeded binding on FA638 in NHS and in adsorbed NHS. Monoclonal antibody 4G5, which binds to PI on FA638 but not FA635, increases C9 binding on FA638 to levels 3-3.5 fold greater than on FA635 but does not result in killing. The majority of additional 125IC9 deposited on FA638 following presensitization with 4G5 is released from the bacterial surface by trypsin. These results extend our earlier results with N. gonorrhoeae by showing that, although PI monoclonals can lead to substantial deposition of non-bactericidal C5b-9, this C5b-9 is not fully inserted into the gonococcal outer membrane.
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PMID:Complement binding on serum-sensitive and serum-resistant transformants of Neisseria gonorrhoeae: effect of presensitization with a non-bactericidal monoclonal antibody. 250 12

Changes in lipopolysaccharide (LPS) which occur when serum susceptible gonococci are converted to resistance by incubation with cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) have been investigated. Transfer of radioactivity to bacterial LPS from CMP-NANA labelled with 14C in the NANA moiety was detected by fluorography following lysis, proteinase K digestion and SDS-PAGE. Incorporation of radioactivity was inhibited by cytidine 5'-monophosphate (CMP). Both the radioactivity of the LPS and the resistance of gonococci to fresh human serum were largely lost after incubation with neuraminidase. No evidence was obtained to suggest that CMP-NANA is an inducer of new protein synthesis as well as a substrate for the sialylation of LPS. Little radioactivity was incorporated into components other than LPS. Sialylated, serum resistant gonococci were less able than serum susceptible gonococci to absorb the bactericidal activity of fresh human serum. Hence, we conclude that serum resistance conferred on gonococci by CMP-NANA is due to transfer of sialyl groups to surface LPS sites and this inhibits their reaction with bactericidal antibody in human serum.
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PMID:Sialylation of lipopolysaccharide and loss of absorption of bactericidal antibody during conversion of gonococci to serum resistance by cytidine 5'-monophospho-N-acetyl neuraminic acid. 250 53

Freshly isolated monocytes in suspension express 2000 to 4000 high affinity receptors for IFN-gamma. Because monocytes change phenotypically as they migrate out of the circulation and adhere to extracellular matrix, modulation of the expression of IFN-gamma receptors may occur. In order to determine if adherence alone modulates the receptor for IFN-gamma, we have studied receptor expression in adherent human peripheral blood monocytes. Elutriation-purified monocytes were allowed to adhere to polystyrene overnight at 37 degrees C. These cells now expressed 1 to 2 x 10(5) low affinity (Ka = 10(8) liters/M) receptors for [125I]rIFN-gamma. Binding to this receptor was specific and saturable. The expression of these receptors occurred rapidly (within 3 h) after adherence and was not inhibited by cycloheximide treatment. Binding to the receptor was abrogated by treating cells with trypsin, but was enhanced after treatment with alkaline protease or proteinase K. mAb against the high affinity receptor did not block binding to the low affinity receptor on adherent cells. The low affinity receptor transduced a signal to the cell as measured by the IFN-gamma-induced enhancement in FcR for human IgG1. The structure of the receptor on adherent cells was investigated by chemical cross-linking techniques. A receptor-[125I]rIFN-gamma complex was observed by SDS-PAGE to have a Mr of 180,000 to 200,000. Reduction of this complex with 2-ME resulted in the loss of the high Mr complex and the appearance of a doublet of lower Mr of 68,000 and 82,000. In contrast, cross-linking of monocytes in suspension yielded a complex of 110,000 to 120,000 Mr, which was unchanged upon reduction. Upon adherence, human monocytes express large numbers of a novel receptor for rIFN-gamma which is capable of stimulating the cell. This receptor appears to be composed of at least two components which are disulfide linked and structurally differs from the high affinity receptor on nonadherent monocytes.
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PMID:Characterization of a novel low affinity receptor for IFN-gamma on adherent human monocytes by radioligand binding studies and chemical cross-linking. 252 81

A nitrocellulose filter binding assay was applied to isolate and to analyze the fraction of chicken DNA fragments associated with residual nuclear polypeptides resistant to SDS/proteinase K treatment and phenol extraction. It is shown that the DNA-polypeptide complexes retained on nitrocellulose filters are located on a non-random sub-set of DNA sequences. (a) Southern analysis reveals that the fractions of DNA fragments from chicken erythrocytes and from hen oviduct cells associated with the resistant polypeptides have a lower sequence complexity than unfractionated DNA. Moreover, the retained DNA fractions from different cell types of the same species are highly homologous. (b) All DNA fragments of the transcriptionally active and inactive ovalbumin gene map in the DNA fraction passing the filters indicating that the tight DNA-polypeptide complexes are not remnants of transcription complexes. (c) By use of a genomic sub-set library prepared from DNA retained on filters, clones were isolated with sequences mapping specifically in the DNA fraction associated with the tight DNA-polypeptide complexes. The results are consistent with fixed covalent DNA-polypeptide complexes in the chicken genome whose location is essentially identical in different cell types of the same species and apparently determined by DNA signal-sequences.
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PMID:Site-specific location of covalent DNA-polypeptide complexes in the chicken genome. 254 4

The superoxide-forming NADPH oxidase of resting macrophages can be activated in a cell-free system by certain anionic amphiphiles, most notably SDS. Activation requires the cooperation of membrane-associated and cytosolic components. We now report that at least two cytosolic factors are required for SDS-elicited activation of NADPH oxidase of guinea pig macrophages. Treatment of cytosol with ammonium sulfate at 37% saturation led to the partition of the two factors in the supernatant and precipitate fractions (termed components sigma 1 and sigma 2, respectively). Although each fraction by itself was inactive, recombining them resulted in complete recovery of the original ability of native cytosol to support SDS-elicited superoxide production by octyl-glucoside solubilized macrophage membranes. Both components are proteins, as shown by their susceptibility to trypsin and proteinase K, and were inactivated by heating at 60 degrees C. sigma 2, but not sigma 1, was inactivated by treatment with the covalent sulfhydryl reagent N-ethylmaleimide. On high-performance gel filtration, sigma 1 was found to have a molecular mass of 30 to 52 kDa, whereas sigma 2 eluted with molecules of 150 to 440 kDa. Component sigma 1 was partially purified from the ammonium sulfate supernatant fraction of cytosol by hydrophobic interaction chromatography followed by gel filtration. A material behaving like sigma 1 was also found to be present in the cytosol of guinea pig thymus cells, lymph node lymphocytes and brain and of the mouse myeloma cell line MOPC 315. However, sigma 2 appears to be strictly phagocyte specific. The molecular characteristics of sigma 1 components from nonphagocytic cells were similar to those of macrophage sigma 1, as shown by their presence in the supernatant, after treatment of cytosol with ammonium sulfate at 37% saturation, a molecular mass close to 30 to 52 kDa and a similar behavior on hydrophobic interaction chromatography. These findings raise the possibility that cytosolic component sigma 1 might be the bearer of a cellular function, more general than the one suggested by its role in the activation of NADPH oxidase of phagocytes.
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PMID:Activation of the superoxide-forming NADPH oxidase of macrophages requires two cytosolic components--one of them is also present in certain nonphagocytic cells. 255 80

There are no published methods on plasmid isolation from Peptostreptococcus spp., therefore two methods of plasmid isolation from this genera were analysed: the boiling and alkaline-SDS methods. Plasmid DNA was not recovered by the boiling method, however, with the alkaline-SDS method, cryptic plasmid DNA was detected in two P. asaccharolyticus and one P. magnus strains. To achieve optimum lysis, Peptostreptococcus cells were treated with lysozyme (2 mg/ml) for 15 min. at 37 degrees C followed by proteinase K (0.2 mg/ml) for 1 h at 37 degrees C. In addition we report, the occurrence of clindamycin or metronidazole-resistant peptostreptococci, but these phenotypes were not correlated with plasmid carriage.
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PMID:Plasmid analysis and antimicrobial susceptibilities of Peptostreptococcus species. 259 60


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