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)

Irradiation of intact or EDTA-dissociated L-cell polyribosomes with 254 nm UV light at doses of 1-2 x 10(5) ergs/mm2 extensively crosslinks mRNA to proteins. The crosslinked mRNA-protein complexes can be isolated on the basis of buoyant density in urea-containing CS2SO4 gradients that dissociate non-covalent complexes. Crosslinking of mRNA can also be assayed by phenolchloroform extraction. mRNA recovered from the crosslinked complexes by digestion with proteinase K has the same electrophoretic mobility in polyacrylamide gels as unirradiated mRNA. Therefore, irradiation does not either crosslink RNA molecules to RNA molecules or break phosphodiester bonds. With these methods it has been found that more than 70% of high molecular weight polydisperse mRNA, but only 25-40% of histone mRNA, can be crosslinked to protein. On the basis of buoyant density the histone mRNA-protein complex had a protein content of 26%, whereas the mean protein content of most non-histone mRNA-protein complexes was 65%. It is concluded that most mRNA in polyribosomes is in close contact with proteins, and that histone mRNA can be crosslinked to many fewer proteins that most other mRNAs.
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PMID:Ultraviolet light-induced crosslinking of mRNA to proteins. 42 11

The infective RNA of the calicivirus, vesicular exanthema virus, has been shown to contain a protein which is apparently linked to the RNA by a covalent bond. The protein remained bound to the RNA after boiling with SDS-mercaptoethanol-urea or treating with formamide-dimethylsulphoxide but was removed by incubating with proteinase K. The mol. wt. of the protein was estimated to be about 1o X 1O(3) by electrophoresis in highly cross-linked polyacrylamide gels. The infectivity of the RNA was destroyed by removal of the protein with proteinase K.
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PMID:Presence of a covalently linked protein on calicivirus RNA. 56 87

The location of the protein bound to bacteriophage phi29 DNA has been studied with restriction endonucleases, exonucleases, and polynucleotide kinase. The protein is invariably associated with the two terminal DNA fragments generated by restriction endonucleases. The phi29 DNA prepared with or without proteinase K treatment is resistant to the action of the 5'-terminal-specific exonucleases, lambda-exonuclease and T7 exonuclease. The phi29 DNA is also inaccessible to phosphorylation by polynucleotide kinase even after treatment with alkaline phosphatase. On the other hand, phi29 DNA is sensitive to exonuclease III, and the 3' termini of the DNA can be labeled by incubating with alpha-[32P]ATP and terminal deoxynucleotidyl transferase. The protein remains associated with the phi29 DNA after treatment with various chaotropic agents, including 8 M urea, 6 M guanidine-hydrochloride, 4 M sodium perchlorate, 2 M sodium thiocyanate, and 2 M LiCl. These results are consistent with the notion that the protein is linked covalently to the 5' termini of the phi29 DNA.
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PMID:Bacteriophage phi29 terminal protein: its association with the 5' termini of the phi29 genome. 73 97

Because the ribonucleoprotein forms of the segments of the Uukuniemi virus genome have previously been characterized as circular, we examined the isolated RNAs by electron microscopy under conditions of increasing denaturation. After spreading under moderately denaturing conditions (50 or 60% formamide), 50 to 70% of the molecules were circular. Increasing the formamide concentration to 70 and 85% decreased the number of circular forms, and only linear forms were observed after incubation of the RNA at 60 degrees C for 15 min in 99% formamide. When spread from 4 M urea-80% formamide--another condition known to denature RNA--only 5 to 30% circular molecules were observed. Pretreatment of the RNA with 0.5 M glyoxal at 37 degrees C for 15 min prior to spreading from 50% formamide gave less than 5% cirucular forms. Length measurement of the molecules showed that they were not significantly degraded by any of the methods employed. The circular molecules were destroyed by treatment with pancreatic RNase, but were unaffected by DNase or proteinase K treatment. After complete denaturation of the RNA, the circles could be reformed under reannealing conditions. We conclude that the three size classes of RNA that comprise the Uukuniemi virus genome are circular molecules probably maintained in that form by base pairing between inverted complementary sequences at the 3' and 5' ends of linear molecules.
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PMID:Circular forms of Uukuniemi virion RNA: an electron microscopic study. 85 Mar 4

The molecular weight of phallolysin, the toxic haemolysin from Amanita phalloides, was established by gel chromatography to be 30000 daltons. The isoelectric point (I.P.) was found in Ampholine pH 7-10 at 8.34. In Ampholine pH 7-9 the gel chromatographically homogeneous phallolysin was separated into phallolysin A (I.P. 8.06) and phallolysin B (I.P. 7.49). Sodium dodecylsulphate-polyacrylamide gel electrophoresis indicated a molecular weight of 33000 daltons for phallolysin A. Phallolysin was thermo- and acid-labile. It was relatively stable in alkaline solutions. 8 M urea as well as 0.1% sodium dodecylsulphate caused irreversible denaturation. On the other hand, phallolysin showed resistance to diverse proteases (pepsin, trypsin, alpha-chymotrypsin, subtilisin, pronase E, bromelin, proteinase K) and also alpha-amylase and pancreatin. Treatment with proteinase K did not change the molecular weight and the isoelectric points of phallolysin. Resistance to proteases was not due to inhibition of proteases by phallolysin.
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PMID:Some physico-chemical properties of phallolysin obtained from Amanita phalloides. 117 97

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

1. A ribonuclease isolated from porcine thyroid cytosol using phenol: sodium dodecylsulfate treatment was associated with RNA and identical to latent alkaline ribonuclease. 2. Distribution of activity between aqueous and phenolic phases depended on pH, RNA, and ribonuclease inhibitor. 3. The ribonuclease was totally resistant to urea, guanidinium: HCl, chloroform:isoamyl alcohol, ethanol, heating at 100 degrees C for 10 min or at 80 degrees C plus 100 mM NaCl. It was highly resistant to hydrolysis by proteinase K except in the presence of detergent. 4. The extreme stability and other properties of latent alkaline ribonuclease could be the result of its association with RNA.
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PMID:Porcine thyroid cytosolic, latent alkaline ribonuclease: resistance to protein denaturants. 149 76

The electrophoretic analysis of lipid A-associated protein (LAP), obtained from S. sonnei, in polyacrylamide gel in the presence of sodium dodecyl sulfate and urea has revealed the heterogeneity of the preparation; it has found to contain three main components with molecular weights of 43, 38 and 18 KD and some minor components with molecular weights of 49, 45 35, 30, 29, 27, 5, 21 and 14 KD. The electrophoretic mobility of the main protein components in the isolated preparation of LAP coincides with that of endotoxin components. The dissociation of proteins and lipopolysaccharide in the process of boiling the endotoxin in 2% sodium dodecyl sulfate is indicative of the noncovalent binding of these components. LAP contained in the endotoxin, in contrast to isolated LAP, is resistant to trypsin and proteinase K. The enzyme immunoassay (EIA) system with the use of LAP as a component of its solid phase has been developed, which makes it possible to carry out the quantitative determination of antibodies to this protein. The EIA system shows high sensitivity in the determination of anti-LAP IgG antibodies: in hyperimmune rabbit sera their titer is 1:250,000-1:800,000. As shown by the method of competitive EIA, the antigenic affinity of LAP of different origin corresponds to the degree of taxonomic propinquity of microorganisms: the maximal degree of cross reactions is observed between LAP obtained from S. sonnei, S. flexneri and Escherichia coli, while their affinity to Salmonella typhi is considerably less; remote microbial species (Bacterium bifidum and Sarcina marcescens) give practically no cross reactions.
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PMID:[Shigella endotoxin protein--its electrophoretic and serological properties]. 188 99

We report here that at least seven low Mr GTP-binding proteins (range 21.5 to 29 kDa) are associated with the membranes of zymogen granules from rat pancreas. GTP binding proteins of similar Mr but in different relative proportions were found in the cytosolic fraction. Treatment of intact granules with either trypsin or proteinase K caused the complete digestion of all the GTP-binding proteins, indicating that the proteins are located on the cytoplasmic face of the granule membrane. All the GTP-binding proteins were relatively resistant to extraction by 1.0M NaCl, 6.0M urea and 0.2M Na2CO3 (pH 11.0) but partitioned into the detergent phase of Triton X 114 extracts indicating that the proteins are tightly associated with the granule membrane. By analogy with the function of other small Mr GTP-binding proteins in regulation of membrane fusion events in eukaryotic cells, we suggest that these low Mr GTP-binding proteins in the pancreatic acinar cell may be involved in regulated secretion.
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PMID:Low molecular weight GTP-binding proteins associated with zymogen granule membranes from rat pancreas. 189 70

Chronic experimental hyperglycemia mediated by galactose has been shown to induce browning and cross-linking of rat tail tendon collagen that could be duplicated in vitro by nonenzymatic galactosylation. To investigate the nature of these changes, Sprague-Dawley rats were placed on a 33% galactose diet without and with sorbinil for 6 and 12 mo. Collagen-linked fluorescence and pentosidine cross-links increased with age and galactosemia in tail tendons (P less than 0.001) and skin but were essentially unresponsive to aldose reductase inhibition (ARI). In contrast, tendon breaking time in urea, a likely parameter of cross-linking, was markedly improved (P less than 0.001) by ARI. Fluorescence that was inhibited by sorbinil treatment was increased in pepsin and proteinase K digest of aortic tissue from galactosemic rats (P less than 0.001), but impaired enzymatic digestibility was not observed. Systolic blood pressure as potential consequence of aortic stiffening was not increased in galactosemia. These data suggest that fluorescence in skin and tendon might be in part due to advanced glycosylation and pentosidine formation because these were not decreased by ARI. However, they also suggest that nonfluorescent cross-links may also be forming because, in contrast to fluorescence, tail tendon breaking time was partly corrected by ARI. Thus, it appears that extracellular matrix changes in chronic galactosemia are complex, being partly attributable to advanced glycosylation and partly to polyol-pathway activation.
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PMID:Tissue-specific effects of aldose reductase inhibition on fluorescence and cross-linking of extracellular matrix in chronic galactosemia. Relationship to pentosidine cross-links. 190 47


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