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)

In this paper we describe an enhanced method for the large scale production of high quality 13C/15N labelled NTPs. High amounts of labelled RNA was obtained from E. coli cells grown in 13C/15N enriched medium and treated with chloramphenicol. Total RNA was extracted from spheroplasted cells in the presence of SDS and proteinase K and subsequently degraded to NMPs by nuclease P1 and high concentrations of nuclease S1 in a low salt buffer. To avoid non-specific degradation of the RNA, nuclease digestion was performed in a short term reaction on native, not heat-denatured RNA. CMP, AMP, GMP and UMP were chromatographically separated and converted to the corresponding NTPs by a mixture of kinases in the presence of a coupled redox system based on thioredoxin and dithiothreitol. The quality of the 13C/15N labelled NTPs was tested by in vitro transcription.
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PMID:A method for production of 13C/15N double labelled RNA in E. coli, and subsequent in vitro synthesis of ribonucleotide 5' triphosphates. 754 14

Hydroxymethylacylfulvene (HMAF, MGI 114) is a novel antitumor drug and a potent pro-apoptotic agent that has the potential to alkylate cellular nucleophiles. The objective of these studies was to characterize drug uptake and cellular targets for drug binding in human leukemia CEM cells. The uptake of [14C]HMAF had two components: a rapid phase (0-10 min) and a slow phase. At 10 microM drug (37 degrees), the rapid and slower phase amounted to 0.86 and 0.13 pmol/min/10(6)cells, respectively. HMAF uptake was inhibited 82% by low temperature (4 degrees) at 4 hr. Cell-associated HMAF localized to nuclear (50%), cytoplasmic (37%), and membrane fractions (10%). Continued drug uptake appeared to be driven by covalent binding to cellular macromolecules. Approximately 1/4 and 2/3 of cell-associated HMAF formed covalent adducts after 10 min and 4 hr, respectively, as found by perchloric acid precipitation. Drug adducts were not readily reversible; 77% of the covalently bound radiolabel was retained by the cells 20 hr after drug treatment. Combinations of DNase, RNase, and proteinase K with perchloric acid precipitation showed that approximately 60, 30, and 10% of the covalently bound drug was associated with the protein, DNA, and RNA fractions, respectively. Incubation of 100 microM [14C]HMAF (24 hr) with purified DNA, serum albumin, thioredoxin, and thioredoxin reductase resulted in 6, 22, 14, and 11 pmol [14C]HMAF/microg DNA or protein, respectively. Results indicate that multiple targets for HMAF binding may contribute to the pro-apoptotic and antiproliferative action of the drug.
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PMID:Drug uptake and cellular targets of hydroxymethylacylfulvene (HMAF). 1042 61

The prion protein (PrP) is an essential, and probably the only, component of the infectious agent responsible for the transmissible spongiform encephalopathies. In its cellular (PrP(C)) form, it is a soluble, alpha-helix-rich protein of yet unknown function attached to the outer membrane of neurons through a glycosylphosphatidyl inositol anchor. In its pathogenic, "scrapie" form (PrP(Sc)), it appears as an aggregate showing no detectable covalent modifications but displaying a profoundly altered conformation enriched in beta-sheet structure. Reduction of the single disulfide bridge in the prion protein with millimolar concentrations of dithiothreitol results in transformation of the alpha-helix-rich to the beta-sheet-rich conformation, with concomitant decrease in solubility. We report here that thioredoxin coupled with thioredoxin reductase and NADPH efficiently reduces recombinant Syrian hamster (29-231) prion protein under physiologically relevant conditions. The reduced prion protein immediately becomes insoluble and precipitates, although it does not gain significant resistance to proteinase K. The thioredoxin/thioredoxin reductase system is approximately 7000 times more efficient than dithiothreitol.
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PMID:Thioredoxin converts the Syrian hamster (29-231) recombinant prion protein to an insoluble form. 1116 30

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis decays steadily when exposed to agents that induce oxidative modification of cysteine residues (Cu(2+), benzofuroxan, disulfides, arsenite, oxidized ascorbate). Inactivation takes place with a concomitant loss of cysteine sulfhydryl groups and dimerization of large subunits of the enzyme. 40% activity loss induced by the vicinal thiol-reagent arsenite is caused by modification of a few neighbor residues while the almost complete inactivation achieved with disulfides is due to extensive oxidation leading to formation of mixed disulfides with critical cysteines of the protein. In most cases oxidative inactivation is also accompanied by an increased sensitivity to proteolysis by trypsin, chymotrypsin or proteinase K. Both enzymatic activity and resistance to proteolysis can be restored through treatment with several thiols (cysteamine, cysteine, dithiothreitol and, more slowly, reduced glutathione). Redox effectors which are thought to regulate the chloroplast activity (NADPH, ferredoxin and thioredoxin) do not reactivate the oxidized enzyme. When ribulose-1,5-bisphoshate carboxylase/oxygenase is incubated with cystamine/cysteamine mixtures having different disulfide/thiol ratio (r), inactivation takes place around r=1.5 while proteolytic sensitization occurs under more oxidative conditions (r=4). It is suggested that oxidative modification may happen in vivo under exceptional circumstances, such as senescence, bleaching or different kinds of stress, leading to enzyme inactivation and triggering the selective degradation of the carboxylase that has been repeatedly observed during these processes.
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PMID:Redox regulation of enzymatic activity and proteolytic susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis. 2431 20