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)

Hydrolysis of serum albumin by proteinase K was strongly (greater than 7-fold) stimulated by urea and dodecylsulfate in a dose-dependent manner. With an oligopeptide as substrate, however, proteinase K was inactivated by dodecylsulfate. This indicates that the apparent activation of proteinase K by urea and dodecylsulfate is caused primarily by denaturation of the protein substrates. Although dodecylsulfate inhibited ribonuclease activity in the test-tube completely, it could not prevent RNA degradation during isolation of polysomal RNA, to which ribonuclease had been added, because of the reversible nature of the dodecylsulfate inhibition. Complete protection of RNA, however, was achieved by a combination of dodecylsulfate and proteinase K. The combined action of the detergent and proteinase K was also effective in degrading "masked" proteins in a poly(adenosine diphosphoribose) preparation which could not be attacked by the proteinase alone.
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PMID:Stimulation of proteinase K action by denaturing agents: application to the isolation of nucleic acids and the degradation of 'masked' proteins. 123 99

Ehrlich ascites tumor cell putative nuclear pre-messenger RNA (pre-mRNA) was isolated under conditions minimizing RNA degradation by ribonucleases, aggregation, and non-specific protein-RNA interaction. Isolated under these conditions, it sedimented 10 to 12 S; proteinase K, a powerful proteolytic enzyme with a broad action spectrum, gave similar sedimentation values and polyacrylamide gel electrophoresis revealed the major component migrating ahead of 16S E. Coli rRNA marker. Cesium chloride buoyant density analysis of pre-mRNA revealed 2 components (1.51 and 1.68 g/cm3). Therefore, pre-mRNA appeared to be smaller than some previous reports.
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PMID:Characterization of putative nuclear pre-messenger RNA of Ehrlich ascites tumor cells. 124 Dec 93

The study described here was carried out to further characterize reference strains of Serpulina (Treponema) hyodysenteriae representing serotypes 8 and 9. Results obtained from restriction fragment length polymorphism analysis, enteropathogenicity testing, and endotoxin profiles confirmed their identifications. Electron microscopy indicated that both strains were covered with a thin layer of capsule-like material. Immunoblot analysis indicated that an antigen in the 19-kDa region of proteinase K-digested whole cells reacted only with homologous antiserum. The serotype-specific antigens were sensitive to periodate oxidation but resistant to proteinase K digestion and migrated in the same region as purified lipopolysaccharides. Immunoblotting with proteinase K-digested whole cells appeared as useful as immunodiffusion with extracted lipopolysaccharide for the serological classification of S. hyodysenteriae. Immunogold labeling of whole cells and purified periplasmic flagella showed strong cross-reactions between S. hyodysenteriae and Serpulina innocens. Outer membrane preparations of strains representing serotypes 8 and 9 contained four major proteins which reacted with antisera against both species, and one major protein with a molecular mass of 46 kDa which reacted only with antisera against S. hyodysenteriae, irrespective of the serotype. Our findings suggest that periplasmic flagella and some outer membrane proteins are antigens common to both S. hyodysenteriae and S. innocens, whereas a 46-kDa outer membrane protein may be a species-specific antigen of S. hyodysenteriae. Finally, we propose immunoblotting as an alternative method to immunodiffusion for the serotyping of S. hyodysenteriae.
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PMID:Molecular characterization of Serpulina (Treponema) hyodysenteriae isolates representing serotypes 8 and 9. 128 Jun 46

Binding of 125I-labelled fibronectin and vitronectin to streptococci of group A (S. pyogenes), group B (S. agalactiae) and group C (S. dysgalactiae and S. zooepidemicus) isolated from various human infections and bovine mastitis, and S. uberis bovine isolates, was studied. Binding of vitronectin and fibronectin was common among both human groups A and C, and bovine group C streptococci. S. agalactiae strains of human and bovine origin as well as S. uberis bovine isolates bound low levels of both proteins. The binding of radiolabelled fibronectin and vitronectin to selected groups A and C streptococcal strains was specific, time-dependent and occurred with both live and heat-killed (80 degrees C for 15 min) cells. Binding declined rapidly after treatment of cells with trypsin or proteinase K, while pepsin digestion at pH 5.5 affected vitronectin but not fibronectin binding.
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PMID:Comparative studies on binding of vitronectin and fibronectin to groups A and C streptococci. 128 49

The polymerase chain reaction with prior reverse transcription of RNA into cDNA was applied to hepatitis C virus RNA detection in human serum samples of different origin. In order to eliminate false negative results, the following steps were optimized: RNA extraction, reverse transcription, and oligonucleotide primer selection. We compared different RNA extraction methods using guanidinium salt/detergent and proteinase K digestion/phenol extraction, and tested virus particle enrichment with polyethylene glycol precipitation and ultracentrifugation. RNA extraction with guanidinium salt/detergent was the most efficient method. Ultracentrifugation of single samples did not improve hepatitis C virus RNA detection. Polyethylene glycol precipitation performed poorly. Recombinant thermostable reverse transcriptase produced cDNA from fewer samples than did Moloney murine leukaemia virus reverse transcriptase. Nested oligonucleotide primers from the 5'-terminal non-coding region of the hepatitis C virus genome amplified cDNA from more samples than did primers from the coding regions. Thirty six anti-hepatitis C virus antibody positive samples were tested; nested primers (nucleotides 6 to 327 and 15 to 288) yielded 21 amplificates, whereas primers from the coding region produced 16 amplificates (nucleotides 4684-5276) and 5 amplificates (nucleotides 5166-5270), respectively. The most efficient combination of steps was RNA extraction with guanidinium salt solution, reverse transcription with Moloney murine leukaemia virus reverse transcriptase and nested polymerase chain reaction primed with primers from the 5'-terminal non-coding region of the hepatitis C virus genome. Other combinations produced more false negative results. Three different groups of anti-hepatitis C virus antibody positive individuals had markedly different viraemia patterns: Hepatitis C virus RNA was detected in the sera of only 10% of anti-hepatitis C virus antibody positive blood donors, but in 90% of anti-hepatitis C virus antibody positive patients with clinically manifest hepatitis C, and 90% of anti-hepatitis C virus antibody positive haemophiliacs who had received plasma products in the past which had not been virus-inactivated. No hepatitis C virus RNA could be detected in the sera of 450 anti-hepatitis C virus antibody negative blood donors with elevated serum alanine aminotransferase catalytic concentrations.
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PMID:Improved detection of hepatitis C virus RNA by reverse transcription and polymerase chain reaction. 128 41

We have studied the antigenic (immunotype) and physical characteristics of the lipooligosaccharide (LOS) of epidemiologically related Neisseria meningitidis case (36) and carrier (76) isolates associated with a virulent clone of meningococci (ET-5 complex). LOS immunotypes were determined by dot blotting using immunotype specific monoclonal antibodies and physical characteristics were determined from silver stained SDS-PAGE following proteinase K digestion. The genetic similarity of the different isolates was confirmed by analysis of the restriction fragment length polymorphisms. An association between LOS immunotype expression and invasive disease was found; 97% of case isolates expressed the L3,7,9 immunotype, of which 13% additionally expressed the L1,8,10 determinant. The LOS immunotypes of carrier strains were much more heterogeneous. The predominant immunotype was L1,8,10 (70%) and only 24% expressed L3,7,9 alone. Genotypically related case isolates from Norway (6) and Austria (18) expressed the L3,7,9 immunotype with similar frequency to the U.K. isolates. The combination of LOS immunotype and capsule expression appears to be related to the virulence of these meningococcal strains.
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PMID:The lipooligosaccharide immunotype as a virulence determinant in Neisseria meningitidis. 128 98

The surface topography of a 190-residue COOH-terminal colicin E1 channel peptide (NH2-Met 333-Ile 522-COOH) bound to uniformly sized 0.2-micron liposomes was probed by accessibility of the peptide to proteases in order (1) to determine whether the channel structure contains trans-membrane segments in addition to the four alpha-helices previously identified and (2) to discriminate between different topographical possibilities for the surface-bound state. An unfolded surface-bound state is indicated by increased trypsin susceptibility of the bound peptide relative to that of the peptide in aqueous solution. The peptide is bound tightly to the membrane surface with Kd < 10(-7) M. The NH2-terminal 50 residues of the membrane-bound peptide are unbound or loosely bound as indicated by their accessibility to proteases, in contrast with the COOH-terminal 140 residues, which are almost protease inaccessible. The general protease accessibility of the NH2-terminal segment Ala 336-Lys 382 excludes any model for the closed channel state that would include trans-membrane helices on the NH2-terminal side of Lys 382. Lys 381-Lys 382 is a major site for protease cleavage of the surface-bound channel peptide. A site for proteinase K cleavage just upstream of the amphiphilic gating hairpin (K420-K461) implies the presence of a surface-exposed segment in this region. These protease accessibility data indicate that it is unlikely that there are any alpha-helices on the NH2-terminal side of the gating hairpin K420-K461 that are inserted into the membrane in the absence of a membrane potential. A model for the topography of an unfolded monomeric surface-bound intermediate of the colicin channel domain, including a trans-membrane hydrophobic helical hairpin and two or three long surface-bound helices, is proposed.
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PMID:Constraints imposed by protease accessibility on the trans-membrane and surface topography of the colicin E1 ion channel. 128 5

We have reported a method for detection of Chlamydia trachomatis by polymerase chain reaction (PCR) with two oligonucleotides based on sequences within the major outer membrane protein gene from C. trachomatis serovar L2. In the previous report, in addition to treatment of the mixture of first-voided urine (FVU) sediment and 1 ml of urine with proteinase K. DNA purification by phenol extraction was necessary for preparation of template DNA for PCR. In this study, FVU sediment was suspended in 1 ml of Chlamydiazyme dilution buffer and a part of the suspension was treated with proteinase K for DNA extraction. The DNA extraction solution could be used as template for PCR without purification of DNA by phenol extraction. One hundred FVU specimens obtained from male urethritis patients were examined with the two methods (PCR and IDEIA) for detection of C. trachomatis. In 33 of 100 specimens, the DNA fragments of C. trachomatis was amplified by the PCR and in 32 of 100, the chlamydial antigen was detected by IDEIA. The positive and negative coincidence rate of the PCR to IDEIA were 93.8% (30.32) and 95.6% (65/68) respectively, resulting in a high overall coincidence rate at 95%. Thus, the improved method with PCR using FVU as a specimen is proved to be a useful, non-invasive diagnostic tool for diagnosis of chlamydial urethritis.
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PMID:[Comparison of polymerase chain reaction and IDEIA Chlamydia in detection of Chlamydia trachomatis from first-voided urine of male urethritis patients]. 129 26

We previously reported that novel Mg(2+)-ATPases were induced in rat liver peroxisomes by clofibrate administration and that these activities consisted of at least two types of enzymes, N-ethylmaleimide (NEM)-sensitive and -resistant. Here we present evidence that neither of these major peroxisomal ATPases is associated with the 70-kDa peroxisomal membrane protein (PMP70), because: (i) proteinase K treatment of peroxisomes resulted in inactivation of only NEM-sensitive ATPase, whereas disappeared PMP70 completely; (ii) NEM-sensitive ATPase activity was barely immunoprecipitated with anti-PMP70 IgG; (iii) the solubilized ATPases behaved differently from PMP70 on native PAGE; and finally (iv), the major peroxisomal ATPases were separated from PMP70 on gel filtration chromatography.
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PMID:Major ATPases on clofibrate-induced rat liver peroxisomes are not associated with 70 kDa peroxisomal membrane protein (PMP70). 129 80

The purification of cell wall antigens of Chlamydia psittaci by affinity chromatography on polymyxin B agarose is described. Chlamydial cell wall antigens were prepared using different methods: heat treatment, ultrasonication and sodium deoxycholate treatment. The antigens were subsequently purified by gel chromatography. The highest amount of cell wall antigens was obtained by heat treatment of the chlamydiae at 90 degrees C and pH 8.5. The purified antigens showed molecular weights of 450 kDa to 700 kDa. Treatment of heat-extracted antigens with trypsin, pronase E or proteinase K did not destroy antigenic activity or alter the molecular weight. On the other hand, potassium metaperiodate treatment led to a significant decrease of both. Chlamydial cell wall antigens extracted by heat treatment and purified by affinity chromatography were used as ELISA-antigen. Using this ELISA and the complement fixation test (CFT), a total of 576 bovine sera was tested. 92 of these sera reacted positively in the ELISA and only 13 in the CFT. In addition, the prepared ELISA was compared with a commercial ELISA. 12 out of 15 sera tested were positive when using the ELISA described and 10 were positive when using the commercial ELISA.
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PMID:Purification of Chlamydia psittaci antigen by affinity chromatography on polymyxin B agarose for use in the enzyme-linked immunosorbent assay (ELISA). 130 87


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