Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

N-(p-Azido[3,5-3H]benzoyl)daunorubicin ([3H]NABD), a radioactive photoactive anthracycline analogue, was used to photoaffinity label anthracycline binding polypeptides in P388 murine leukemic cell lines. Whole cell homogenates were mixed with 6 X 10(-8) M [3H]NABD, exposed to ultraviolet light, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for radiolabel incorporation. Autoradiofluorography showed incorporation of radioactivity into a Mr 18,000 component independent of polypeptides prominently stained with Coomassie blue. Photolabeling of subcellular fractions showed predominant mitochondrial localization of the Mr 18,000 radiolabel. The protein composition of the photolabeled constituents was confirmed by treatment with proteinase K, DNase and RNase, or by lipid extraction with organic solvent. [3H]NABD photolabeling of homogenates from anthracycline sensitive and resistant cells resulted in Mr 18,000 radiolabel incorporation of 3,966 +/- 355 and 6,487 +/- 533 dpm per 50 micrograms cellular protein for anthracycline sensitive and resistant cells, respectively (P less than 0.005). These studies characterize the photoaffinity labeling of a low molecular weight mitochondrial polypeptide using a photoactive anthracycline analogue. The role for this polypeptide as a mediator of anthracycline activity remains to be determined.
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PMID:Anthracycline photoaffinity labeling of a mitochondrial polypeptide in P388 murine leukemic cell lines. 346 35

Proteinase K (EC 3.4.21.14) from the fungus Tritirachium album Limber is the most active known serine endopeptidase. The sequence of its 275-residue long polypeptide chain and its three-dimensional folding show a high degree of homology with the bacterial subtilisin proteases. Using difference Fourier methods, the binding mode of the synthetic carbobenzoxy-Ala-Ala-chloromethyl ketone inhibitor to the active site of proteinase K was determined. In several cycles of restrained least-squares, the enzyme-inhibitor complex was refined to a current R = 22% for 9400 X-ray diffraction data between 2.2 and 5.0 A resolution. The inhibitor is attached to proteinase K by two covalent bonds: one between the methylene carbon of the inhibitor and N epsilon 2 of the catalytic His 68, the other between the ketone carbon atom of the inhibitor and O gamma of the catalytic Ser 221. In addition, two hydrogen bonds donated by the peptide NH of Ser 221 and by the side chain NH2 of Asn 160 hold the hemiketal O- in the oxyanion hole. The peptide inhibitor is further hydrogen bonded to the proteinase polypeptide chain in a three-stranded antiparallel pleated sheet.
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PMID:Active-site geometry of proteinase K. Crystallographic study of its complex with a dipeptide chloromethyl ketone inhibitor. 351 98

Considerable evidence suggests that the scrapie prion protein (PrP) is a component of the infectious particle. We studied the biogenesis and transmembrane orientation of an integral-membrane form of PrP in a cell-free transcription-linked translation-coupled translocation system programmed with a full-length PrP cDNA cloned behind the SP6 promoter. Translation of SP6 transcripts of the cDNA or of native mRNA from either normal or infected hamster brain in the absence of dog pancreas membranes resulted in the synthesis of a single PrP immunoreactive polypeptide (each polypeptide was the same size; Mr, 28,000), as predicted from the known sequence of the coding region. In the cotranslational presence of membranes, two additional forms were observed. Using peptide antisera specific to sequences from the amino- or the carboxy-terminal domain of PrP together with proteinase K or endoglycosidase H digestion or both, we showed that one of these forms included an integrated and glycosylated form of PrP (Mr = 33,000) which spans the bilayer twice, with domains of both the amino and carboxy termini in the extracytoplasmic space. By these criteria, the other form appeared to be an unglycosylated intermediate of similar transmembrane orientation. The PrP cell-free translation products did not display resistance to proteinase K digestion in the presence of nondenaturing detergents. These results suggest that the PrP cell-free translation products most closely resemble the normal cellular isoform of the protein, since its homolog from infected brain was proteinase K resistant. The implications of these findings for PrP structure and function are discussed.
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PMID:Biogenesis and transmembrane orientation of the cellular isoform of the scrapie prion protein [published errratum appears in Mol Cell Biol 1987 May;7(5):2035]. 354 85

Rats were treated with clofibrate, a hypolipidemic drug, and with thyroxine. Both drugs which are known to cause peroxisome proliferation, and a concomitant increase in peroxisomal fatty acid beta-oxidation activity in liver increased one of the major integral peroxisomal membrane polypeptides (PMPs), with apparent molecular mass of 69-kDa, six- and twofold, respectively. On the other hand hypothyroidism caused a decrease in peroxisomal fatty acid beta-oxidation activity and considerably lowered the concentration of PMP 69 in the peroxisomal membrane. Two other PMPs with apparent molecular masses of 36 and 22 kDa were not influenced by these treatments. The PMPs with apparent molecular masses of 42, 28, and 26 kDa were shown to be derived from the 69-kDa polypeptide by the activity of a yet uncharacterized endogenous protease during isolation of peroxisomes. Limited proteolysis of intact peroxisomes using proteinase K and subtilisin further substantiated that some portion of the 69-kDa polypeptide extends into the cytoplasm. The 36- and the 22-kDa polypeptides were accessible to proteolytic attack to a much lower extent and, therefore, are supposed to be rather deeply embedded within the peroxisomal membrane. It is demonstrated that peroxisomal acyl-CoA synthetase, an integral PMP extending partially into the cytoplasm, and PMP 69 are not identical polypeptides. Comparison of the peroxisomal membrane with that of mitochondria and microsomes revealed that the 69- and 22-kDa polypeptides as well as the bifunctional protein of the peroxisomal fatty acid beta-oxidation pathway were specifically located only in peroxisomes. Considerable amounts of a polypeptide cross-reacting with the antiserum against the 36-kDa polypeptide were found in mitochondria.
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PMID:Integral membrane polypeptides of rat liver peroxisomes: topology and response to different metabolic states. 359 54

The cell surface antigen defined by monoclonal antibody CO17-1A is sensitive to proteinase K but not to neuraminidase digestion. Immunoprecipitation of two polypeptide chains of 30 and 40 kDa using large amounts of CO17-1A antibody confirmed that the CO17-1A antigen is a protein. The requirement for large quantities of CO17-1A antibody may relate to the binding properties of this antibody, as bivalent but not monovalent (Fab) forms of the antibody bind effectively to carcinoma cells. The 30 kDa form of the CO17-1A antigen was purified by immunoaffinity chromatography using GA733, another monoclonal antibody that recognizes the CO17-1A antigen. Treatment of purified antigen with endoglycosidase F revealed a 25 kDa and a 28 kDa species, demonstrating that the antigen has at least two N-linked oligosaccharide chains. Protease treatment of the purified antigen revealed a 26 kDa protease-resistant polypeptide.
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PMID:Identification and characterization of the CO17-1A carcinoma-associated antigen. 374 82

Pertussigen (Ptx), referred to by many different names, including pertussis toxin, was separated into five polypeptide subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a discontinuous Tris-glycine buffer system. Under non-reducing conditions, the apparent molecular weights of the polypeptides (mean 10(-3)) were: S1 (26.3), S2 (24.4), S3 (22.7), S4 (12.2), and S5 (11.3). Under reducing conditions, the apparent molecular weights (mean 10(-3)) were: S1 (28.2), S2 (24.8), S3 (24.3), S4 (12.2) and S5 (13.9). The identity of the individual polypeptide subunits was further confirmed by their unique two-dimensional peptide maps. The polypeptides which showed an apparent increase in molecular weight under reducing conditions were those previously found to contain at least two cysteine residues. Reducing conditions also altered the reactivity of S3 and S2 to polyclonal rabbit antibody in electrophoretic transfer (Western) blot analysis. When Ptx was stored in solution at 4 degrees C, S1 and S5 underwent a gradual decrease in apparent molecular weight, as judged by SDS-PAGE. This decrease occurred in three different buffer systems, and was similar to a decrease in apparent molecular weight of S1 and S5 after treatment with the proteolytic enzymes subtilisin or proteinase K. Neither the changes due to storage nor proteolysis affected the activity of Ptx in regard to hemagglutination, lymphocytosis promotion or histamine sensitization. These changes did, however appear to modify the reactivity of S5 in the Western blot. Both the "endogenous" and enzyme-induced changes in S1 and S5 could be stopped by phenylmethanesulfonyl fluoride. These data suggest that S1 and S5 have exposed determinants in the intact Ptx molecule which are readily cleaved by proteases, but have little bearing on the biological activity of the intact molecule. Resistance to inactivation by proteolytic cleavage may help explain the long duration of Ptx activity within in vivo biological systems.
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PMID:Effect of proteolytic enzymes, storage and reduction on the structure and biological activity of pertussigen, a toxin from Bordetella pertussis. 391 65

The mitochondrial DNA-binding protein P16 was isolated from rat liver mitochondrial lysates by affinity chromatography on single strand DNA agarose and separated from DNA in the preparation by alkaline CsCl isopycnic gradients. The top fraction of the gradients contained a single polypeptide species (Mr approximately equal to 15,200) based upon SDS PAGE. Digestion of single strand DNA-bound P16 with proteinase K produced a protease-insensitive, DNA-binding fragment (Mr approximately equal to 6,000) that has been purified by essentially the same procedures used for intact P16. The partial amino acid compositions for P16 and the DNA-binding fragment were obtained by conventional methods. Analysis of subcellular fractions revealed that nearly all of the cellular P16 was located in the mitochondria and that only trace amounts of protein of comparable electrophoretic mobility could be isolated from the nuclear or cytoplasmic fractions. The labeling of P16 with [35S]methionine in primary rat hepatocyte cultures was inhibited by more than 90% by the cytoplasmic translation inhibitor cycloheximide, but unaffected by the mitochondrial-specific agent chloramphenicol. These results indicate that P16 is synthesized on cytoplasmic ribosomes and imported into the mitochondria. The addition of purified P16 to deproteinized mitochondrial DNA resulted in the complete protection of the labeled nascent strands of displacement loops against branch migrational loss during cleavage of parental DNA with SstI, thus providing strong evidence that P16 is the single entity required for this in vitro function. Incubation of P16 with single strand phi X174 DNA, double strand (RF) phi X174 DNA, or Escherichia coli ribosomal RNA and subsequent analysis of the nucleic acid species for bound protein indicated a strong preference of P16 for single strand DNA and no detectable affinity for RNA or double strand DNA. Examination of P16-single strand phi X174 DNA complexes by direct electron microscopy revealed thickened, irregular fibers characteristic of protein-associated single strand DNA.
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PMID:Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria. 403

Total polysomal RNA of rat liver was translated in vitro in a rabbit reticulocyte lysate system. The translation products were mixed with a postnuclear supernatant fraction of rat liver and incubated post-translationally at 26 degrees C for 15-60 min. The import assay mixture was separated into a particulate fraction and supernatant by centrifugation, both of which were analyzed by immunoprecipitation with a goat antibody against rat liver peroxisomal proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. One peroxisomal translation product (Mr 72,000) appeared in the particulate fraction, was partly proteinase K-resistant, and addition of detergents prior to proteolysis abolished this resistance. In isopycnic centrifugation of the uptake assay mixture, the protease-resistant 35S-polypeptide of Mr 72,000 cosedimented with the peroxisomes. This translation product was identified immunochemically as fatty acyl-CoA oxidase; both before and after import it was indistinguishable in size from subunit A of the purified enzyme by prolonged sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the cell-free translation products were incubated with highly purified peroxisomes, 35S-catalase entered peroxisomes (by the criterion of protease resistance), and its entry was stimulated by the addition of a high speed supernatant (cytosolic) fraction of rat liver. These results demonstrate the post-translational import into peroxisomes in vitro of at least two cell-free translation products.
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PMID:Post-translational import of fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro. 403 22

We disrupted bacteriophage Mu particles by freeze-thaw treatment and recovered the DNA by CsCl density gradient centrifugation. This CsCl-purified DNA had a buoyant density which was indistinguishable from that of phenol-extracted Mu DNA. It was, however, 10(3) times more infective than phenol-extracted DNA for spheroplasts of exoV endI Escherichia coli. Infectivity was destroyed by proteinase K as well as by pancreatic DNase, indicating that the infective form was a DNA-protein complex. The infective properties of the complex demonstrated that the protein protects. Mu DNA against degradation by exonuclease V and that it serves at least one other function in bacteriophage Mu infection. The infectivity of the CsCl-purified DNA was due to a small class of highly infective molecules which sedimented 1.2. times faster than phenol-extracted Mu DNA on neutral sucrose gradients. This change in sedimentation rate is best explained by the formation of protein-linked circular monomers or linear dimers of Mu DNA. In vitro labeling of the DNA-protein complex, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that the CsCl-purified DNA contained a noncovalently associated 65,000-dalton polypeptide. A 65,000-dalton protein was also found to be a minor component of the bacteriophage Mu particle. No protein was found in phenol-extracted Mu DNA. These results suggest that the 65,000-dalton protein is necessary for successful phage infection and is normally injected into the host cell with the Mu genome.
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PMID:Transfection of Escherichia coli spheroplasts with a bacteriophage Mu DNA-protein complex. 621 51

Cytochrome c oxidase from rat liver was incubated with various proteinases of different specificities and the enzymic activity was measured after various incubation times. A loss of catalytic activity was found after digestion with proteinase K, aminopeptidase M and a mitochondrial proteinase from rat liver. In each case the decrease in enzymic activity was compared with the changes in intensities of the polypeptide pattern obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibilities of the subunit polypeptides of the soluble cytochrome c oxidase to proteinases were very different. Whereas subunit I was most susceptible, subunits V--VII were rather resistant to degradation. From the relative inaccessibility of subunits V--VII to proteinases it is likely that these polypeptides are buried in the interior of the enzyme complex.
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PMID:Catalytic activity and arrangement of subunit polypeptides in rat liver cytochrome c oxidase as studied by proteolysis. 624 69


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