Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Purified Escherichia coli recA protein catalyzed ATP-dependent pairing of superhelical DNA and homologous single-stranded fragments. The product of the reaction: (i) was retained by nitrocellulose filters in 1.5 M NaCl/0.15 M Na citrate at pH 7, (ii) was dissociated at pH 12.3 but was not dissociated by heating at 55 degrees C for 4 min or by treatment with 0.2% sodium dodecyl sulfate and proteinase K, (iii) contained covalently closed circular double-stranded DNA (form I DNA), (iv) contained single-stranded fragments associated with replicative form (RF) DNA, and (v) contained a significant fraction of D-loops as judged by electron microscopy. Linear and nicked circular double-stranded DNA did not substitute well for superhelical DNA; intact circular single-stranded DNA did not substitute well for single-stranded fragments. Homologous combinations of single-stranded fragments and superhelical DNA from phages phiX174 and fd reacted, whereas heterologous combinations did not. The reaction required high concentrations of protein and MgCl2. The ATPase activity of purified recA protein was more than 98% dependent on the addition of single-stranded DNA. In 1 mM MgCl2, the ability of superhelical DNA to support the ATPase activity was two-thirds as good as that of single-stranded DNA.
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PMID:Purified Escherichia coli recA protein catalyzes homologous pairing of superhelical DNA and single-stranded fragments. 15 61

A comparative study of some commonly employed laboratory procedures for studying DNA synthesis in isolated nuclei was carried out. Nuclei isolated from baby-hamster kidney (BHK-21/C13) cells synthesize DNA for 30-60min at 37 degrees C in a reaction requiring uni- and bi-valent cations, ATP and all four deoxyribonucleoside 5'-triphosphates. The addition of either ribonucleotides or cytosol from S-phase cells had no effect, but DNA synthesis was stimulated by some dextrans (mol.wt. 5x10(6)). The extent of synthesis was influenced by apparently minor variations in experimental conditions. For example, DNA synthesis by nuclei in Tris/HCl, pH7.5, was only 50% of that observed in Hepes/NaOH, pH7.5; the presence of detergents Triton X-100, Triton N-101, Nonidet P-40, Brij 58 and Tween 80 in the incubation medium altered the amount of synthesis to different extents. Although most detergents inhibited synthesis, a stimulation occurred with Tween 80 (150% of controls). These effects were reversed on washing the nuclei, except that of Brij 58, which inhibited DNA synthesis by 90-95% irreversibly. Anomalous sucrose-density-gradient sedimentation behaviour of the DNA, and of precursor [(3)H]-dTTP, was observed when nuclei were lysed with solutions of sodium dodecyl sulphate/Mg(2+) or with Sarkosyl/Mg(2+), but consistent results, showing that the DNA synthesized in vitro sedimented exclusively at about 4S, were obtained when nuclei were lysed with sodium dodecyl sulphate (without Mg(2+))/EDTA, digested with proteinase K and heated at 100 degrees C with 11% (v/v) formaldehyde to prevent macromolecular association. These results, coupled with density-labelling studies with bromodeoxyuridine and CsCl-density-gradient analysis, showed that DNA synthesis in these nuclei was replicative and was restricted to a covalent extension of Okazaki pieces previously initiated in vivo. No new initiations were observed, and the DNA was not ligated into larger molecules. The cessation of DNA synthesis after about 60 min was due to the complete utilization of available primer/template DNA.
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PMID:Deoxyribonucleic acid synthesis in isolated nuclei from baby-hamster kidney cells (BHK-21/C13). Characterization of the system. 45 70

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

Human monoamine oxidase A that had been synthesized in a reticulocyte lysate translation system was capable of binding to and inserting into either rat liver mitochondria or isolated mitochondrial outer membranes. The inserted form was as resistant to proteinase K as endogenous mitochondrial monoamine oxidase A. The insertion, but not the binding, of monoamine oxidase A was prevented by depleting the reaction mixture of either ATP (with apyrase) or ubiquitin (with purified antibodies against this polypeptide). Addition of ATP or ubiquitin, respectively, to these depleted mixtures restored the insertion of the enzyme. In the absence of mitochondria, in vitro synthesized monoamine oxidase A did not catalyze its own alkylation by the mechanism-based inhibitor, [3H]clorgyline. However, both monoamine oxidase A that had been membrane-inserted in vitro and monoamine oxidase A that had been bound to the mitochondria under conditions of ATP depletion catalyzed adduct formation. Furthermore, reaction of either clorgyline or another mechanism-based inhibitor, pargyline, with the membrane-bound enzyme during ATP depletion inhibited the insertion of monoamine oxidase A when ATP was restored. These observations indicate that monoamine oxidase A acquired a catalytically active conformation on interaction with the mitochondrial outer membranes prior to its ATP and ubiquitin-dependent insertion into the membrane.
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PMID:The insertion of monoamine oxidase A into the outer membrane of rat liver mitochondria. 130 56

This paper extends our recent report that renal Na+,K(+)-ATPase is digested by trypsin in the absence of Ca2+ and presence of Rb+ ions to a stable 19-kDa fragment and smaller membrane-embedded fragments of the alpha chain and essentially intact beta chain. These are referred to as "19-kDa membranes." Occlusion of both Rb+ (K+) or Na+ ions is preserved, but ATP-dependent functions are lost (Karlish, S. J. D., Goldshleger, R., and Stein, W. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4566-4570). We now show that extensive digestion with nonselective fungal proteases (Pronase and proteinase K) alone, in combination, or after tryptic digestion can remove up to 70% of membrane protein without destroying Rb+ occlusion. In the most heavily digested membranes, the 19-kDa fragment or a slightly shorter 18.5-kDa fragment and smaller fragments of the alpha chain remain, whereas the beta chain is largely digested, leaving smaller membrane-embedded fragments (13-15 kDa). For either trypsin or Pronase digestion, preservation of Rb+ occlusion and the specific fragmentation pattern is observed only in the absence of divalent metal ions (Mg2+ or Ca2+) and presence of either Rb+ or Na+ or congener ions. Tryptic digestion at pH 7.0 can split the beta chain into two fragments of approximately 50 and 16 kDa joined by an S-S bridge. The 16-kDa fragment is protected against further digestion by the presence of Rb+ ions, but probably is not directly involved in occluding cations. Tryptic 19-kDa membranes show a clear and reproducible fragmentation pattern in which all predicted membrane segments are identifiable. Families of fragments from 19-kDa membranes, including seven peptides of 7.6-11.7 kDa, have been separated by size-exclusion high performance liquid chromatography, concentrated, and resolved on 16.5% Tricine gels. N-terminal sequences of the different fragments have been determined after transfer to polyvinylidene difluoride paper. The most interesting findings are as follows. (a) Whereas the 19-kDa tryptic fragment begins at Asn831 as reported previously, the 18.5-kDa Pronase fragment begins at Thr834. (b) Fragments in tryptic 19-kDa membranes of 7.6-11.7 kDa begin at Asp68, Ile263, and Gln737, respectively. These include all putative transmembrane segments other than those in the 19-kDa fragment. (c) A Pronase fragment of 7.8 kDa begins at Thr834, i.e. apparently the 19-kDa fragment has been partially cut, without loss of Rb+ occlusion. (d) Tryptic 16- and approximately 50-kDa fragments of the beta chain begin at Ala5 and Gly143, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Extensive digestion of Na+,K(+)-ATPase by specific and nonspecific proteases with preservation of cation occlusion sites. 130 64

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 orientation of the enzyme Mg(2+)-ATPase (EC 3.6.1.3) in the transverse tubule (TT) membranes of skeletal muscle was investigated using highly purified chicken and rabbit TT vesicles. The percentage of sealed vesicles present in these preparations averaged 88 and 78%, respectively, as calculated from the detergent-induced increase in ouabain-sensitive (Na+, K+)-ATPase activity, ATP-dependent ouabain binding, and lactate dehydrogenase activity (sarcoplasmic enzyme trapped in the TT vesicles). Sidedness of the sealed vesicles, estimated from latency of 5'-nucleotidase, acetylcholinesterase, and adenylate cyclase, was predominantly right-side out (69-76%, chicken TT and 62-70%, rabbit TT). In both chicken and rabbit native vesicles, high Mg(2+)-ATPase activity was detected by addition of ATP to the extravesicular medium; this activity was increased 14-12% by alamethicin pointing to the external localization of the active site. Furthermore, the enzymatic activity resulted partially inhibited by treatment of the chicken TT vesicles with proteinase K or p-hydroxymercuribenzoate. Concanavalin A stimulated 4-fold the chicken TT Mg(2+)-ATPase activity, an effect not potentiated by detergent permeabilization of the intact vesicles, indicating that lectin-binding sites were also solvent accessible. This stimulatory effect was not observed in native or permeabilized rabbit TT vesicles. From these results we conclude that the TT Mg(2+)-ATPase is an ectoenzyme with its nucleotide-hydrolyzing site and glycosylated regions facing the extracellular space. Inhibitors of ion-motive ATPases did not modify the enzyme activity, suggesting a different physiological role for the TT Mg(2+)-ATPase which may be involved in the regulation of muscle fiber functions affected by extracellular ATP levels.
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PMID:Transverse tubule Mg(2+)-ATPase of skeletal muscle. Evidence for extracellular orientation of the chicken and rabbit enzymes. 166 Apr 76

The beta-2 adrenergic receptor has been proposed to have seven membrane-spanning domains. Expression of functional beta-2 adrenergic receptor was achieved in a heterologous cell-free system composed of rabbit reticulocyte lysate and microsomal membranes from Xenopus laevis oocytes. The functional state of the receptor protein can be determined by ligand-binding assays and by the ability of ligands to alter the susceptibility of the receptor to proteinase K digestion. The process by which functional receptor is made was studied. The receptor protein remains nonfunctional immediately following translocation and glycosylation, and additional processing steps are needed before the receptor is able to interact with ligands. These processing steps require intact microsomal membranes as well as several cytosolic factors including ATP and one or more high molecular mass (greater than 30 kDa) factors but do not require receptor glycosylation and are not inhibited by nonhydrolyzable GTP analogues.
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PMID:The role of cytosolic and membrane factors in processing of the human beta-2 adrenergic receptor following translocation and glycosylation in a cell-free system. 169 24

We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastid DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and alpha-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thio UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensitivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA-protein complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubation of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast transcriptional complex.
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PMID:Photoaffinity labelling of the pea chloroplast transcriptional complex by nascent RNA in vitro. 171 36

We have studied the import of the precursor to yeast cytochrome c oxidase subunit Va, a protein of the mitochondrial inner membrane. Like the majority of mitochondrial precursor proteins studied thus far, import of presubunit Va was dependent upon both a membrane potential (delta psi) and the hydrolysis of ATP. However, the levels of ATP necessary for the import of presubunit Va were significantly lower than those required for the import of a different mitochondrial precursor protein, the beta subunit of the F1-ATPase. The rate of import of presubunit Va was found to be unaffected by temperature over the range 0 to 30 degrees C, and was not facilitated by prior denaturation of the protein. These results, in conjunction with those of an earlier study demonstrating that presubunit Va could be efficiently targeted to mitochondria with minimal presequences, suggest that the subunit Va precursor normally exists in a loosely folded conformation. Presubunit Va could also be imported into mitochondria that had been pretreated with high concentrations of trypsin or proteinase K (1 mg/ml and 200 micrograms/ml, respectively). Furthermore, the rate of import into trypsin-treated mitochondria, at both 0 and 30 degrees C, was identical to that observed with the untreated organelles. Thus, import of presubunit Va is not dependent upon the function of a protease-sensitive surface receptor. When taken together, the results of this study suggest that presubunit Va follows an unusual import pathway. While this pathway uses several well-established translocation steps, in its entirety it is distinct from either the receptor-independent pathway used by apocytochrome c, or the more general pathway used by a majority of mitochondrial precursor proteins.
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PMID:An unusual mitochondrial import pathway for the precursor to yeast cytochrome c oxidase subunit Va. 184 27


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