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)

Bifunctional alkylating agents are known to produce cross-links between DNA and protein and between paired DNA strands. The possibility of discriminating these two classes of cross-links in L1210 cells treated with haloethylnitrosoureas or nitrogen mustard was explored with the alkaline elution technique. Two classes of cross-links were demonstrated, based on sensitivity to proteinase K; the proteinase-sensitive cross-links appear to be DNA-protein cross-links, and the proteinase-resistant class may include interstrand cross-links. Proteinase-sensitive cross-links form more rapidly than do proteinase-resistant cross-links in cells treated with chloroethylnitrosoureas, perhaps because these agents can chloroethylate protein sulfhydryl or amino groups followed by rapid reaction of these chloroethylated groups with DNA. Although both types of cross-links produced by nitrogen mustard disappeared or were repaired after 24 hr, the removal of cross-links produced by chloroethylnitrosoureas either did not occur or was incomplete in 24 hr. In addition to cross-links, cells treated with haloethylnitrosoureas exhibited DNA strand breaks; a method is suggested for estimating the apparent frequencies of strand breaks and cross-links in the DNA.
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PMID:DNA-protein cross-linking and DNA interstrand cross-linking by haloethylnitrosoureas in L1210 cells. 15 Sep 40

Poliovirus type I, vaccine strain (LSc, 2ab), which is a temperature- and actinomycin D-sensitive mutant derived from type I Mahoney strain, was grown in HeLa cells in the presence of 32P and a low concentration of actinomycin D. Seven and a half h p.i., genome 32P-RNA was recovered from the purified virion. Analysis of RNase TI digests of the RNA by two-dimensional gel electrophoresis revealed that three possible point mutation sites exist in the large and unique oligonucleotides in the fingerprint. Neither a capping structure nor a nucleotide such as pppNp, ppNp or pNp, was detected by ion exchange column chromatography at pH 5.0 after digestion of virion RNA with RNase T2. Instead, a 32P-labelled compound, which could be digested with Pronase or proteinase K, was eluted at the void volume of the column. Proteinase K digests of the 32P-labelled compound contained pUp or pU as a single labelled c"mpound, when genome RNA was digested with RNase T2 or nuclease P1, respectively, before digestion with the proteinase. Our data locate possible point mutation sites on the genome of a mutant strain (LSc, 2ab) of type I poliovirus and show that a protein (VPg) is covalently bound to the 5'-terminus of RNA. The protein (VPg) of LSc, 2ab strain migrates faster than capsid protein VP4 (mol. wt. 7000 to 8000) in a polyacrylamide gel and is thus similar to the VPg of the wild-type virus.
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PMID:Possible point mutation sites in LSc, 2ab poliovirus RNA and a protein covalently linked to the 5'-terminus. 23 Feb 98

Brain, spleen, and selected lymph nodes from sheep with clinical signs of scrapie were analyzed for presence of proteinase K-resistant protein (PrP-res). Diagnosis of scrapie on the basis of detection of PrP-res was compared with diagnosis on the basis of histologic evaluation of the brain from clinically affected or exposed sheep. Proteinase K-resistant protein was found in every brain that was histologically positive for scrapie, and in addition, was found in the brain of several clinically positive sheep that were not diagnosed as scrapie-positive by histologic evaluation. Proteinase K-resistant protein was also found in 87% of the spleens and lymph nodes from sheep that had PrP-res detected in brain homogenates. Therefore, analysis of sheep brain, spleen, or lymph nodes for PrP-res provided a diagnostic approach that was superior to histologic examination alone for detection of naturally scrapie agent-infected sheep.
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PMID:Diagnostic implications of detection of proteinase K-resistant protein in spleen, lymph nodes, and brain of sheep. 135 64

Limited proteolysis experiments were performed with outer membranes from Comamonas acidovorans to probe the topology of its major protein component, the anion-selective porin Omp32. Proteinase K treatment above a critical temperature of 42 degrees C cleaved the surface-exposed regions of the porin, yielding membrane-embedded fragments which were separated by SDS polyacrylamide gel electrophoresis or reversed phase chromatography. The identification of the proteinase K-sensitive sites was performed by microsequencing. This allowed us to determine six surface-exposed sites of the porin, all located in nonconserved primary structure regions. These results along with the previously determined amino acid sequence and in conjunction with some structural constraints applicable to porins allowed us to propose a chain-folding model of the Omp32 porin. The features of our model are compared with the structure of the Rhodobacter capsulatus porin, recently established by X-ray crystallography (Weiss et al., 1991) and they are used to elucidate the structural basis of the anion selectivity.
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PMID:Topology of the anion-selective porin Omp32 from Comamonas acidovorans. 137 89

Circular dichroic spectroscopy has been used to study the effect of pH, guanidinium hydrochloride concentration and temperature on the conformation of the fungal subtilisin-like proteinase K and the bacterial DY. The ellipticity of the bands in the far ultraviolet region remains almost unchanged in the pH range 3.0-11.0 (PMS-proteinase K) and 5.0-10.0 (PMS-subtilisin DY). The same ranges of pH stability were determined from the pH dependence of the near ultraviolet dichroic spectra. Hence the changes in the tertiary and secondary structure occur in parallel. Proteinase K is considerably more stable at acidic and somewhat more stable at alkaline pH than subtilisin DY. At neutral pH proteinase K is more resistant to denaturation by guanidinium hydrochloride than is subtilisin DY. The midpoints of the denaturation curves were 6.2 M and 3.2 M guanidinium, respectively. The thermal unfolding of proteinase K occurred at a higher temperature than for subtilisin DY, the transition midpoints being 65 degrees and 48 degrees, respectively. Thus proteinase K is overall a much more robust molecule than subtilisin DY, showing greater resistance to all three forms of denaturation. The differences in the stability of the two proteinases can be partly explained by differences in their calcium binding sites.
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PMID:Spectroscopic studies on proteinase K and subtilisin DY. Relation to X-ray models. 148 40

Skeletal muscle actin was lightly digested by proteinase K, which cleaved the peptide bond between Met-47 and Gly-48, producing a C-terminal 35 kDa fragment. Proteinase K-cleaved actin (proK-actin) did not polymerize into F-actin upon addition of salt. In the presence of phalloidin, however, it polymerized slowly into F-actin (proK-F-actin), indicating that the cleaved actin did not dissociate into the individual cleaved fragments but retained the global structure of actin. Electron microscopy showed that proK-F-actin had the typical double-stranded structure of a normal actin filament and formed the arrowhead structure when decorated with HMM. Heavy meromyosin ATPase was weakly activated by proK-F-actin: Vmax = 0.24 s-1, and Kapp = 2.8 microM, while Vmax = 7.6 s-1, and Kapp = 13 microM by F-actin. Correspondingly, in vitro this proK-F-actin slid very slowly on HMM attached to a glass surface at an average velocity of 0.47 microns/s, or 1/12 of that of intact F-actin. The fraction of sliding filaments was less than 50%. Assuming that the nonmotile filaments attached to HMM were not involved in ATPase activation, the sliding velocity correlated with the ATPase activity activated by proK-F-actin.
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PMID:Muscle actin cleaved by proteinase K: its polymerization and in vitro motility. 149 Oct 13

We previously reported that the adenovirus type 5 E3 14.5-kilodalton protein (14.5K) forms a complex with E3 10.4K and that both proteins are required to down-regulate the epidermal growth factor receptor in adenovirus-infected human cells. Both proteins are also required to prevent cytolysis by tumor necrosis factor of most mouse cell lines infected by adenovirus mutants that lack E3 14.7K. The E3 14.5K amino acid sequence suggests that 14.5K is an integral membrane protein with an N-terminal signal sequence for membrane insertion. Here we show that 14.5K was found exclusively in cytoplasmic membrane fractions. Radiochemical sequencing of 14.5K indicated that the N-terminal signal sequence is cleaved predominantly between Cys-18 and Ser-19. With a mutant that does not express 10.4K, cleavage occurs predominantly between Phe-17 and Cys-18, indicating that the presence or absence of 10.4K affects the signal cleavage site. 14.5K was extracted into the detergent phase with Triton X-114, it remained associated with membranes after extraction with Na2CO3 at pH 11.5, and it was partially protected by membranes from proteinase K digestion; these observations indicate that 14.5K is an integral membrane protein. Proteinase K digestion followed by immunoprecipitation with antipeptide antisera directed against the N or C terminus of mature 14.5K indicated that 14.5K is oriented in the membrane with its N terminus in the lumen and its C terminus in the cytoplasm. Thus, 14.5K is a type I bitopic membrane protein. Previous studies indicated that 10.4K is also an integral membrane protein oriented with its C terminus in the cytoplasm. Altogether, these findings suggest that cytoplasmic membranes are the site of action when 10.4K and 14.5K down-regulate the epidermal growth factor receptor and prevent tumor necrosis factor cytolysis.
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PMID:The adenovirus E3 14.5-kilodalton protein, which is required for down-regulation of the epidermal growth factor receptor and prevention of tumor necrosis factor cytolysis, is an integral membrane protein oriented with its C terminus in the cytoplasm. 153 70

The proteinaceous insect cuticle is an effective barrier against most microbes, but entomopathogenic fungi can breach it using extracellular proteases. We report here the isolation and characterization of a cDNA clone of the cuticle-degrading protease (Pr1) of Metarhizium anisopliae. The cDNA sequence revealed that Pr1 is synthesized as a large precursor (40.3 kDa) containing a signal peptide, a propeptide and the mature protein predicted to have a molecular mass of 28.6 kDa. The primary structure of Pr1 has extensive similarity with enzymes of the subtilisin subclass of serine endopeptidases and the serine, histidine and aspartate components of the active site in subtilisins are preserved. Proteinase K demonstrated the closest sequence similarity to Pr1 (61%) but Pr1 was twofold more effective than proteinase K at degrading isolated cuticles of Manduca sexta and 33-fold more effective at degrading structural proteins bound to the cuticle by covalent bonds. We postulate that the additional positively charged residues on the surface of the Pr1 molecule, as determined using proteinase K, may facilitate electrostatic binding to cuticle proteins which is a prerequisite for activity. Northern-blot analysis of RNA and nuclear run-on assays demonstrated transcriptional control of the expression of Pr1 during nutrient deprivation and during the formation of infection structures. Southern-blot analysis demonstrated that genes with significant homologies to Metarhizium Pr1 were present in the entomopathogens Aspergillus flavus and Verticillium lecanii but not Zoophthora (= Erynia) radicans.
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PMID:Molecular cloning and regulatory analysis of the cuticle-degrading-protease structural gene from the entomopathogenic fungus Metarhizium anisopliae. 155 99

The differential cleavage of surface proteins of Borrelia burgdorferi IRS strains by several proteases was examined. Proteinase K, trypsin, chymotrypsin and thermolysin all cleaved the outer surface protein B (OspB) to undetectable levels by Coomassie Brilliant Blue staining, whereas some residual protein was detected by immunoblotting with polyclonal and monoclonal antibodies. Not even antigenic fragments were detectable by immunoblotting with 1A8 monoclonal antibody reactive with OspB. Less effective or ineffective was the cleavage of OspB by V8 protease and proteinase A, respectively. The outer surface protein A was cleaved only by proteinase K. The effect of trypsin on borreliae viability and adhesion to cultured cells was also studied. The trypsin treatment of borreliae did not impair the viability of organisms which continued to synthesize the cleaved OspB. The attachment of B. burgdorferi to HEp-2 cells was reduced by 41% after treatment with trypsin, whereas preincubation of borreliae with monoclonal antibody 1A8 and guinea pig immune serum reduced the adhesion of borreliae to the cells by 32% and 87%, respectively.
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PMID:Differential cleavage of surface proteins of Borrelia burgdorferi by proteases. 160 94

Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted.
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PMID:Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction. 169 90


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