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)

A high molecular weight (HMW) fraction of the 150,000 g supernatant of rat brain homogenates contains protein-tRNA complexes which are able to incorporate [3H]Arg and [3H]Lys into tRNA. The aminoacylation of tRNA(Arg) was found to be dependent on ATP and inhibited by RNase. Conversely, the aminoacylation of tRNA(Lys) did not require exogenous ATP and was resistant to RNase and ATPase. In HMW fractions of regenerating rat sciatic nerves, the charging of both tRNA(Arg) and tRNA(Lys) was resistant to RNase and ATPase and did not require exogenous ATP. Because sciatic nerves are rich in axoplasm and tRNAs are known to be present in axons, we tested the hypothesis that degradative enzyme-resistant, ATP-tRNA complexes were of axonal origin. In HMW fractions from rat liver (containing no axons), both tRNA(Arg) and tRNA(Lys) were sensitive to RNase and required exogenous ATP for charging. But, in similar fractions of axoplasm obtained from the giant axon of squid, both tRNAs were insensitive to RNase and ATPase and did not require exogenous ATP for charging. These results suggest that tRNAs in axons are present in protected HMW complexes and contain endogenous stores of ATP. The presence of ATP in the HMW complexes was demonstrated by the luciferase-luciferin assay for ATP. The nature of the protection of tRNAs from RNases was examined by dissociating proteins from HMW complexes by boiling, treating with proteinase K, or overhomogenizing the tissue. These procedures failed to render brain tRNA(Lys) susceptible to RNase. But phenol-extracted, ethanol-precipitated brain tRNA(Lys) was sensitive to RNase, suggesting that the protection of tRNA(Lys) may be by a protease- and heat-resistant polypeptide or by a nonproteinaceous mechanism.
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PMID:Evidence that axonal tRNAs are resistant to RNase and ATPase and can be aminoacylated in the absence of exogenous ATP. 153 73

The mammalian cell antigen reactive with the autoantibody anti-Jo-1 has been shown to contain tRNAHis. The RNA sequence of this human and mouse cell tRNA was determined in a search for unusual features that might be related to antigenicity. The 5' terminal nucleotide is unique among other sequenced tRNAs in that it is a methylated guanine. The presence of the hypermodified base queuine, which occurs in the wobble position of the anticodon of tRNAHis from several species, was not detected in the tRNAHis immunoprecipitated by anti-Jo-1 from either human HeLa or mouse Friend erytholeukemia cell extracts. The binding of protein(s) appears to confer antigenicity on tRNAHis since either proteinase K treatment or phenol extraction resulted in the loss of immunoprecipitability. However, we have not succeeded in identifying an antigenic protein, and we find that the antigenic complex is not resolved from purified tRNAHis by Sephacryl S-200 column chromatography. Immunofluorescence studies indicate that the antigenic form of tRNAHis is located preferentially in the mammalian cell cytoplasm. The results presented here are discussed in light of an earlier report (1) on the nature of the Jo-1 antigen.
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PMID:A mammalian tRNAHis-containing antigen is recognized by the polymyositis-specific antibody anti-Jo-1. 618 8

Quick-blot, a method for selectively immobilizing either mRNA or DNA on nitrocellulose, is described in detail. Essential elements of the procedure for immobilizing DNA include tissue lysis, proteinase K treatment, solubilization of nucleic acids in hot 12.2 molal NaI, passage through a nitrocellulose filter, and acetylation of residual protein with acetic anhydride. Advantages include speed, quantitative recovery, low background, and elimination of the usual baking step. Essential elements of the procedure for selectively immobilizing mRNA include dissolving cells in Brij-35 and desoxycholate, proteinase K treatment, solubilizing nucleic acids in room temperature 12.2 molal NaI, filtration through nitrocellulose, and acetylation of residual protein. Advantages include selective immobilization of mRNA but not tRNA, rRNA, or DNA, and the maintenance of biological activity of the immobilized mRNA. Control experiments to optimize the procedures and examples of their application are shown.
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PMID:Quick-blot: selective mRNA or DNA immobilization from whole cells. 664 75

The remarkable resistance of isolated ribosomes to gelonin is overcome by cofactors present in post-ribosomal supernatants. In rat liver post-ribosomal supernatant RNA is the cofactor responsible of the sensitization of ribosomes. Isolated RNA, which consists mostly of deacylated tRNA, accounts for less than 10 per cent of the activity of the original supernatant. The activity of the supernatant is completely destroyed by micrococcal nuclease and RNAase A and also by proteinase K, suggesting that some protein enhances the effect of RNA. RNA has a role also in the sensitization of ribosomes to alpha-sarcin, an RNAase which inactivates ribosomes by hydrolyzing a single phosphodiester bond in the same region of 28S rRNA which is the target of the N-glycosidase activity of gelonin.
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PMID:RNA present in post-ribosomal supernatants makes ribosomes susceptible to inactivation by gelonin and alpha-sarcin. 751 79

Ribonuclease P (RNase P) from Dictyostelium discoideum has been purified 470-fold. D. discoideum RNase P cleaves the precursor to Schizosaccharomyces pombe suppressor tRNA(Ser) at the same site as S. pombe RNase P, producing the mature 5' end of tRNA(Ser). pH and temperature optima for enzyme activity are 7.6 and 37 degrees C, respectively. The enzyme shows optimal activity in the presence of 5 mM MgCl2 and 10 mM NH4Cl or 5 mM KCl. The apparent Km for the S. pombe tRNA precursor derived from the supS1 tRNA(Ser) gene is 240 nM, and the apparent Vmax is 3.6 pmol/min. Inhibition of D. discoideum RNase P by proteinase K and micrococcal nuclease strongly indicates that the activity requires both protein and RNA components. In cesium sulfate density gradients, the enzyme has a buoyant density of 1.23 g/ml, indicating a low RNA/protein ratio for the holoenzyme.
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PMID:Partial purification and characterization of RNase P from Dictyostelium discoideum. 773 3

Two distinct RNase P-like activities which cleave leader sequences from pre-tRNA molecules to give mature 5' ends have been identified in carrot suspension-culture cells. An Escherichia coli pre-tRNA(Phe) and a tobacco pre-tRNA(Tyr) were transcribed in vitro then used as substrates for processing reactions in a cell-free extract. The pre-tRNA(Tyr) transcript was used to establish optimal salt and divalent cation requirements for processing. Kinetic experiments were then carried out on both substrates to determine if 5' and 3' processing were ordered. Primer extension analysis of processing intermediates and stable products verified that an ammonium sulfate fraction of the extract was indeed capable of accurately processing the 5' ends of both pre-tRNAs. Subsequent fractionation of the 5' end-processing activity by chromatography on phosphocellulose revealed two distinct activities, eluting at 0.1 and 0.5 M KCI, when assayed with the tobacco pre-tRNA(Tyr) substrate. When the same fractions were assayed with the E. coli pre-tRNA(Phe), only the 0.1 M KCI fraction exhibited activity. Both of the active fraction display sensitivity to micrococcal nuclease (MN) and proteinase K indicating each is a ribonucleoprotein, a result not seen with other plant RNase Ps. Subsequent FPLC fractionation of the two activities using Mono Q and Mono S columns demonstrated that the two activities could be further distinguished on the basis of their chromatographic behavior.
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PMID:Characterization and partial purification of two pre-tRNA 5'-processing activities from Daucus carrota (carrot) suspension cells. 774 55

We have identified and partially characterized a novel class III transcription factor fraction (TFIIIE) from yeast nuclear extracts. TFIIIE is functionally distinct from the standard yeast transcription factor fractions, TFIIIB and TFIIIC. It is also different from either of the TFIIIB subfractions, B' and B". TFIIIE is essential for specific transcription of both tRNA and 5 S RNA genes, its activity is sensitive to proteinase K, and it exhibits an apparent sedimentation coefficient of 4.0 S when analyzed on glycerol gradients. In the case of a tRNA gene, TFIIIE does not play a role in the formation of stable preinitiation complexes containing TFIIIB and TFIIIC. It is required for single as well as multiple rounds of transcription, however. Thus, TFIIIE is involved in the utilization of stable transcription complexes, but its action is not restricted to reinitiation events.
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PMID:A novel RNA polymerase III transcription factor fraction that is not required for template commitment. 849 77

Ribonuclease P (RNase P) is responsible for the generation of mature 5' termini of tRNA. The RNA component of this complex encodes the enzymatic activity in bacteria and is itself catalytically active under appropriate conditions in vitro. The role of the subunits in eucaryotes has not yet been established. We have partially purified RNase P activity from the ciliate protozoan Tetrahymena thermophila to learn more about the biochemical characteristics of RNase P from a lower eucaryote. The Tetrahymena RNase P displays a pH optimum and temperature optimum characteristic of RNase P enzymes isolated from other organisms. The Km of the T. thermophila enzyme for pre-tRNAGln is 1.6 x 10(-7)M, which is comparable to the values reported for other examples of RNase P. The Tetrahymena RNase P is a ribonucleoprotein complex, as supported by its sensitivity to micrococcal nuclease and proteinase K. The buoyant density of the enzyme in Cs2SO4 is 1.42 g/ml, which suggests that the RNA component of the Tetrahymena enzyme comprises a significantly greater percentage of the holoenzyme than that determined for RNase P of other Eucarya or Archaea. The holoenzyme has a requirement for divalent cations displaying characteristics that are unique for RNase P but closely resemble preferences reported for the Tetrahymena group I intron RNA. Puromycin inhibits pre-tRNA processing by the Tetrahymena complex, and implications of the similarities between recognition of tRNA by ribosomal components and RNase P are discussed.
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PMID:Ribonuclease P of Tetrahymena thermophila. 866 80

The intervening sequences of nuclear tRNA precursors are known to be excised by tRNA splicing endonuclease. We show here that a T7 transcript corresponding to a pre-tRNA(Tyr) from Arabidopsis thaliana has a highly specific activity for autolytic intron excision. Self-cleavage occurs precisely at the authentic 3'-splice site and at the phosphodiester bond one nucleotide downstream of the authentic 5'-splice site. The reaction results in fragments with 2',3'-cyclic phosphate and 5'-OH termini. It is resistant to proteinase K and/or SDS treatment and is not inhibited by added tRNA. The self-cleavage depends on Mg2+ and is stimulated by spermine and Triton X-100. A set of sequence variants at the cleavage sites has been analysed for autolytic intron excision and, in parallel, for enzymatic in vitro splicing in wheat germ S23 extract. Single-stranded loops are a prerequisite for both reactions. Self-cleavage not only occurs at pyrimidine-A but also at U-U bonds. Since intron self-excision is only about five times slower than the enzymatic intron excision in a wheat germ S23 extract, we propose that the splicing endonuclease may function by improving the preciseness and efficiency of an inherent pre-tRNA self-cleavage activity.
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PMID:Another heritage from the RNA world: self-excision of intron sequence from nuclear pre-tRNAs. 871 Apr 88

Ribosomes prepared from somatic tissue of Xenopus laevis inhibit transcription by RNA polymerase III. This observation parallels an earlier report that a high speed fraction from activated egg extract, which is enrichedin ribosomes, inhibits RNA polymerase III activityand destabilizes putative transcription complexes assembled on oocyte 5S rRNA genes. Transcription of somatic- and oocyte-type 5S rRNA genes and a tRNA gene are all repressed in the present experiments. We find that 5S rRNA genes incubated in S150 extract prepared from immature oocytes exhibit an extensive DNase I protection pattern that is nearly identical to that of the ternary complex of TFIIIA and TFIIIC bound to a somatic 5S rRNA gene. The complexes formed in this extract are stable at concentrations of ribosomes that completely repress transcription, indicating that formation of the TFIII(A+C) complex is not the target of inhibition. Ribosomes taken through a high salt treatment no longer repress transcription of class III genes, establishing that the inhibition is due to an associated factor and not the particle itself. The inhibitory activity released from ribosomes is inactivated by treatment with proteinase K, but not micrococcal nuclease. Preincubation of ribosomes with a general protein kinase inhibitor, 6-dimethylaminopurine, eliminates repression of transcription. Western blot analysis demonstrates that p34(cdc2), which is known to mediate repression of transcription by RNA polymerase III, is present in these preparations of ribosomes and can be released from the particles upon extraction with high salt. These results establish that a kinase activity, possibly p34(cdc2), is the actual agent responsible for the observed inhibition of transcription by ribosomes.
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PMID:Inhibition of RNA polymerase III transcription by a ribosome-associated kinase activity. 975 46


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