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 study of the regulation of low-abundance blood-brain barrier (BBB) transcripts either in isolated brain microvessels or in endothelial cells in tissue culture (ECL cells) requires isolation of poly(A)+ mRNA. Therefore, we describe here a single-step method for isolation of poly(A)+ mRNA from brain capillaries or ECL cells using proteinase K/sodium dodecyl sulfate cell lysis and oligo-deoxythymidine cellulose affinity chromatography. The yield of poly(A)+ mRNA was approximately 15-19 micrograms/g of brain or choroid plexus, 14-17 micrograms per batch of isolated capillaries in a single bovine forebrain (190 g), and 6-12 micrograms/10(7) ECL cells. Northern blot analysis showed characteristic and undegraded 2.1- and 1.7-kb actin transcripts in brain capillaries and a 2.1-kb actin mRNA in brain and ECL cells. Northern analysis was also used to quantify the glucose transporter type I transcript, which is very rare in basal ECL cells, and this mRNA was shown to be up-regulated by glucose deprivation. This method represents a significant improvement in the mRNA yield for brain capillaries or cultured endothelial cells compared with the conventional two-step method.
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PMID:A one-step procedure for isolation of poly(A)+ mRNA from isolated brain capillaries and endothelial cells in culture. 194 Sep 20

The alkaline plasmid DNA extraction method of Birnboim and Doly was applied for the isolation of human papillomavirus (HPV) from warts. Tissue from common and plantar warts was digested with proteinase K, and the extrachromosomal circular covalently-closed form of HPV-DNA was rapidly extracted by alkaline sodium dodecyl sulphate and phenol-chloroform treatment. Recovery of HPV-DNA from the tissue was sufficient for determination of endonuclease restriction patterns by agarose gel electrophoresis.
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PMID:Human papillomavirus DNA from warts for typing by endonuclease restriction patterns: purification by alkaline plasmid methods. 196 33

We have isolated protransglutaminase E, the zymogen form of epidermal transglutaminase E, from the skin of the adult guinea pig. This zymogen is the source of the large majority of soluble transglutaminase activity of skin. A molecular weight value for protransglutaminase E of 77,800 +/- 700, estimated by sedimentation equilibrium, is in close agreement with the apparent values determined by exclusion chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of the proenzyme with dispase, proteinase K, trypsin, or thrombin produces active enzyme. The enzyme, transglutaminase E, formed by the action of dispase, was observed to exist in the native state as a molecule indistinguishable in size from the zymogen. Under denaturing conditions, however, the enzyme dissociates into two fragments with molecular weights of 50,000 and 27,000. The observation that reducing agents are not needed for this dissociation suggests a noncovalent association of the two peptide chains in the native enzyme. Evidence that the catalytically essential -SH group of the enzyme residues in the Mr 50,000 fragment and that only the Mr 27,000 fragment possesses an unmasked amino terminus provides the basis for a proposed model of zymogen activation. Whether the noncatalytic fragment plays a role in catalysis is not known because separation of the fragments of native enzyme was not achieved.
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PMID:Protransglutaminase E from guinea pig skin. Isolation and partial characterization. 197 27

Tissues stored as paraffin blocks are a potential source of DNA for retrospective clinicogenetic analysis. To assess the feasibility of Southern blot analysis, DNA extracted from paraffin blocks was compared with DNA obtained from fresh-frozen controls of the same tissues. Sections 50-100 microns thick cut from paraffin blocks of 11 normal tissues, 18 lymphoid lesions, and 9 gastric carcinoma samples were deparaffinized and incubated at 45 degrees C for 48 to 72 h in a sodium dodecyl sulfate (SDS)/proteinase K solution. Following organic extraction, alcohol precipitation, restriction endonuclease digestion, and gel electrophoresis, DNA was transferred to nylon membranes. 32P-labelled DNA probes for the immunoglobulin heavy-chain locus and T-cell receptor beta-chain gene were hybridized to the normal tissue and lymphoid samples; the gastric cancers were probed for the HER-2/neu protooncogene. Intact DNA was obtained from the majority of formalin-fixed samples, yielding results qualitatively similar to those from fresh tissues. Degradation is the most significant problem in analyzing DNA extracted from paraffin blocks and compromises accurate quantitation. DNA analysis using paraffin-embedded tissue has potential clinical and research applications and may be a particularly useful way to study gene abnormalities in unusual tumors infrequently available as fresh specimens.
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PMID:Extraction of DNA from paraffin blocks for Southern blot analysis. 198 65

The three-dimensional crystal structure of thermitase complexed with eglin-c in the presence of 100 mM calcium has been determined and refined at 2.0-A resolution to a R-factor of 16.8%. This crystal structure is compared with previously determined structures of thermitase at 0 and 5 mM calcium concentration. In the presence of 100 mM calcium all three calcium binding sites in thermitase are fully occupied. At 100 mM CaCl2 the "weak" calcium binding is occupied by a calcium ion, which is chelated by three protein ligands and four water molecules in a pentagonal bipyramid geometry. Thermitase has, apparently, a monovalent and divalent cation binding position at 2.5-A distance from each other at this site. At low calcium concentrations the monovalent-ion position is occupied by a sodium or potassium ion. The "medium strength" binding site shows in the presence of 100 mM CaCl2 a square antiprism arrangement with eight ligands, of which seven are donated by the protein. At low calcium concentrations we observe a distorted pentagonal bipyramid coordination at this site. The largest difference between these two conformations is observed for ligand Asp-60, which has two conformations with 0.8-A difference in C alpha positions. The "strong" calcium binding site has a pentagonal bipyramid coordination and is fully occupied in all three structures. Structural changes on binding calcium to the weak and "medium strength" calcium binding sites of thermitase are limited to the direct surroundings of these sites. Thermitase resembles in this respect subtilisin BPN' and does not exhibit long-range shifts as have been reported for proteinase K.
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PMID:Calcium binding to thermitase. Crystallographic studies of thermitase at 0, 5, and 100 mM calcium. 199 69

Proteins resistant to proteinase K are rare because of the potency, wide pH optimum, and low peptide bond specificity of this enzyme. Previously, only the prion proteins associated with transmissible spongiform encephalopathies, possibly related proteins in the mollicute Spiroplasma mirum, and proteinase K itself have been reported. We identified a new proteinase K-resistant protein, p40-pr, in two strains of Mycoplasma hyorhinis and in extracts of these organisms. p40-pr's are similar to prion proteins in their resistance to high doses of proteinase K and in the reversal of this resistance by strong denaturing conditions. However, p40-pr's were distinct immunologically, in relative molecular mass, and in their method of extraction. Two immunologically related forms of p40-pr were identified on sodium dodecyl sulfate (SDS) gels and Western immunoblots, a 40-kDa species in boiled samples and a 120-kDa species dissociable by boiling in SDS. Reduction with 2-mercaptoethanol did not affect the mass of p40-pr's or the 120-kDa forms. The development of proteinase K resistance of p40-pr correlated to age-dependent increases in organism protein-lipid ratios. p40-pr-like proteinase K-resistant proteins of 46 to 50 kDa were identified in four of eight additional species of the class Mollicutes but not in S. mirum. However, these mycoplasmal proteins did not react with antibody to the denatured 40-kDa form of M. hyorhinis p40-pr purified by electroelution. The chromatographically purified 46-kDa proteinase K-resistant protein of Mycoplasma orale was an arginine deiminase.
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PMID:Identification and characterization of proteinase K-resistant proteins in members of the class Mollicutes. 199 7

The lipopolysaccharides (LPS) of the 10 species of the genus Bacteroides (sensu stricto) were extracted by the proteinase K method and their resolution compared by several methods of polyacrylamide gel electrophoresis (PAGE). These included sodium dodecyl sulphate (SDS)-PAGE with and without urea, polyacrylamide gradient gels and Tricine [N-Tris (hydroxymethyl) methyl glycine]-SDS-PAGE. The original discontinuous system showed good resolution of LPS from B. thetaiotaomicron, B. caccae and B. ovatus and this was enhanced by urea; B. vulgatus showed a typical ladder pattern associated with repeating polysaccharide units of the O side chains. The LPS profiles of the other species, including B. fragilis, were poorly resolved; the majority of components migrated with the leading edge of the wave front. The resolution of the LPS of these species was marginally improved with gradient gels but the majority of components were separated only within the regions of high polyacrylamide concentration. The Tricine-SDS system was consistently superior to the other methods, with excellent resolution of the LPS profiles of all Bacteroides species.
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PMID:The resolution of bacteroides lipopolysaccharides by polyacrylamide gel electrophoresis. 203 May

Cholesterol absorption by small intestinal brush border membrane vesicles from taurocholate mixed micelles is a second-order reaction. From a comparison of reaction rates and order before and after proteinase K treatment of brush-border membrane vesicles, it is concluded that cholesterol absorption is protein-mediated. It is shown that the desorption of cholesterol from taurocholate mixed micelles is by a factor of about 10(4) faster than that from egg phosphatidylcholine bilayers. When brush border membrane vesicles are stored at room temperature, intrinsic proteinases are activated and proteins are liberated from the brush border membrane. These proteins collected in the supernatant catalyze cholesterol and phosphatidylcholine exchange between two populations of small unilamellar phospholipid vesicles. One of the active proteins present in the supernatant is purified by a two-step procedure involving gel filtration on Sephadex G-75 SF and affinity chromatography on a Nucleosil-phosphatidylcholine column. The protein thus obtained is pure by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has an apparent molecular weight of slightly less than 14,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a value of 11,500 determined by gel filtration on Sephadex G-75 SF.
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PMID:Cholesterol-transfer protein located in the intestinal brush-border membrane. Partial purification and characterization. 203 43

Porphyromonas gingivalis W50, W83, A7A1-28, and ATCC 33277 were investigated for their abilities to lyse sheep, human, and rabbit erythrocytes. All of the P. gingivalis strains studied produced an active hemolytic activity during growth, with maximum activity occurring in late-exponential-early-stationary growth phase. The enzyme was cell bound and associated with the outer membrane. Fractionation of P. gingivalis W50 localized the putative hemolysin almost exclusively in the outer membrane fraction, with significant hemolytic activity concentrated in the outer membrane vesicles. Ca2+ and Mg2+ ions significantly increased the expression of hemolytic activity. Hemolytic activity was inhibited by proteinase K, trypsin, the proteinase inhibitors Na-P-tosyl-L-lysine chloromethyl ketone and benzamidine, the metabolic inhibitor M-chlorophenyl-hydrazone, and iodoacetate. KCN and sodium azide (NaN3) only partially inhibited P. gingivalis hemolytic activity, while antiserum to whole cells of each of the P. gingivalis strains had a significant inhibitory effect on hemolytic activity. The P. gingivalis W50 hemolysin was inhibited by cysteine, dithiothreitol, and glutathione at concentrations of at least 10 mM; at low concentrations (i.e., 2 mM), dithiothreitol did not completely inhibit hemolytic activity. Heating to temperatures above 55 degrees C resulted in an almost complete inhibition of hemolytic activity. The effect of heme limitation (i.e., iron) on hemolysin production indicated that either limitation or starvation for heme resulted in significantly increased hemolysin production compared with that of P. gingivalis grown in the presence of excess heme.
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PMID:Hemolytic activity in the periodontopathogen Porphyromonas gingivalis: kinetics of enzyme release and localization. 203 55

Proteinase R and T purified from Tritirachiam album limber were characterized in comparison with proteinase K using circular dichroism (CD), enzyme activity, thermal melting, and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). CD analysis suggested that these three proteins possess some beta-sheet structure, with little alpha-helix except for proteinase R which showed about 14% alpha-helix. SDS-PAGE and gel filtration in 0.1% SDS indicated that proteinase T and K are resistant to SDS-induced unfolding similar to subtilisin. Thermal denaturation experiments showed the melting temperature for proteinase T to be 67 degrees and that for proteinase K to be 65 degrees in the absence of Ca2+, with higher melting temperatures in the presence of Ca2+. However, the enzyme activities of proteinase T and R were significantly lower than those of proteinase K.
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PMID:Comparative study on proteinase R, T, and K from Tritirachiam album limber. 207 93


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