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)

Using a rapid phenol extraction assay, an enzyme was purified from uninfected HeLa cells that can cleave the 5'-terminal protein (VPg) from poliovirus RNA. Both cytoplasmic and nuclear extracts had enzymes with similar behavior. A polypeptide of molecular weight 27,000 was the major one present in the purified preparation. Assuming that this protein is the enzyme, a very low turnover number was calculated for it. The purified enzyme would cleave the tyrosine-phosphate bond linking VPg to poliovirus RNA with minimal degradation of the RNA or of VPg. If the RNA was first treated with proteinase K to degrade VPg, leaving a small peptide on the RNA, this peptide could also be removed by the enzyme. If the RNA was degraded with T1 RNase, leaving VPg attached to a nonanucleotide, the enzyme still would cleave off VPg, although incompletely. If the RNA was degraded completely, leaving either pUp or pU attached to VPg, the enzyme would not remove the nucleotides from the protein. Thus, for the enzyme to be active requires some length of polynucleotide attached to the protein but only a short peptide need be present for the enzyme to act.
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PMID:Purification and properties of a HeLa cell enzyme able to remove the 5'-terminal protein from poliovirus RNA. 624 32

Membrane-bound acetylcholinesterase (AChE) from the electric organ of Torpedo marmorata was labeled with the hydrophobic photoactivatable reagent 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ( [125I]TID). Labeling with [125I]TID was restricted to the membranous polypeptide segment of AChE as shown upon conversion of the amphiphilic form to the hydrophilic one by limited digestion with proteinase K. The labeled membranous segment, which has an Mr of approx. 3000 was isolated by gel filtration on Sephadex LH-60 in ethanol/formic acid.
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PMID:Hydrophobic labeling of the membrane binding domain of acetylcholinesterase from Torpedo marmorata. 637 63

The exposure of the three polypeptide subunits H, M, and L of the photochemical reaction center (RC) on both surfaces of the membrane of Rhodopseudomonas capsulata was studied by partial proteolysis with proteinase K and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of of degradation products. The possible association of RC subunits with bacteriochlorophyll a and bacteriopheophytin was investigated by spectroscopical measurements. Chromatophores (inside-out oriented) and spheroplasts (right-side-out oriented), as well as purified, detergent-solubilized RCs and RCs reconstituted into phosphatidyl choline liposomes, were used. Subunit H of the RC was degraded to fragments with apparent MrS of 15,000 and 12,500, which were possibly derived from cleavage of a loop exposed on the cytoplasmic surface. Polypeptide M was digested at a comparable rate. The apparent Mr of M decreased by roughly 4,000 upon proteolytic cleavage. Subunit L was relatively insensitive to protease attack, except that a small peptide was clipped off. The primary donor P870 was also found to be only slightly affected proteinase K. All three RC subunits appear to be exposed on the chromatophore surface.
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PMID:Transverse topography of the photochemical reaction center polypeptides in the Rhodopseudomonas capsulata membrane. 637 44

Accessibility of nascent chains of periplasmic proteins to externally added proteinase K was used as the criterion for translocation of polypeptides across the cytoplasmic membrane of E. coli during the process of export. It is concluded for maltose-binding protein and ribose-binding protein that nascent chains carrying the signal sequence are not accessible to the proteinase while chains that have been matured span the membrane and are degraded. Translocation of polypeptides is a late event relative to extent of elongation, occurring only after maltose-binding protein has reached molecular weight 33,000 (80% of its entire length) and after ribose-binding protein has been fully elongated (molecular weight 29,000). The data presented here are inconsistent with postulated mechanisms of export requiring a strict coupling of translocation to elongation of nascent polypeptide chains. In contrast, the data support the idea that entire domains of polypeptides are transferred after their synthesis. This is the case whether the translocation of a protein is initiated post-translationally or begins before synthesis of the entire protein is completed.
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PMID:Translocation of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation. 638 Jul 53

Glucose dehydrogenase from B. megaterium is subjected to proteolysis with proteinase K. Upon proteolysis the enzyme is inactivated and the polypeptide chain is cleaved into two distinct fragments. These components designated as K-protein and K-peptide have molecular masses of 26 000 and 3 000 Da, respectively. Under native conditions the K-protein and K-peptide remain associated and the tetrameric structure of the proteolytically modified enzyme is preserved. The K-protein and K-peptide were isolated and characterised. The cleavage occurs in the C-terminal region of the polypeptide chain. -Leu Ala decreases Ser-Ser-Glu is proposed as the cleavage site.
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PMID:Limited proteolysis of glucose dehydrogenase from Bacillus megaterium by proteinase K. 641 54

The unspecific proteinase K and the specific proteases alpha-chymotrypsin, trypsin and S. aureus V 8 protease were used in order to determine the orientation of the polypeptides B 870-alpha and B 870-beta from the major antenna complex B 870 of Rs. rubrum G-9+ within the chromatophore membrane (inside-out vesicle). Although B 870-alpha exhibits cleavable peptide bonds, treatment with specific proteases yielded splitting only in B 870-beta within the N-terminal region. In the case of proteinase K, which was most effective, mainly 6 (B 870-alpha) and 16 (B 870-beta) amino acid residues were removed from their N-terminal parts as proved by means of Edman degradation of cleavage products. The major peptide bonds cleaved were identified as Gln6-Leu7 in B 870-alpha and as Lys16-Glu17 in B 870-beta. The central hydrophobic stretch regions and the relatively hydrophilic C-terminal parts of both light-harvesting polypeptides were not affected by proteinase K. On the basis of these degradation experiments a transmembrane orientation of B 870-alpha and B 870-beta is postulated, with their N-terminal towards the cytoplasm and their C-termini towards periplasm with regard to the photosynthetic membrane. This hypothesis is supported by the transmembrane model proposed by Brunisholz et al. (Hoppe-Seyler's Z., Physiol. Chem., (1984) 365, 675-688) in which the hydrophobic stretch of B 870-alpha and of B 870-beta forming an alpha-helix would span the membrane once. Organic solvent extraction of chromatophores treated with proteinase K yielded a fairly pure polypeptide fragment with an apparent molecular mass of 14000 Da. Its N-terminal amino-acid sequence is identical with the sequence within the N-terminal region of the reaction centre subunit L of Rs. rubrum G-9+. Thus it is most likely that as in the case of B 870-beta, proteinase K removed 16 amino acid residues from the N-terminal part of subunit L. This subunit therefore also seems to be exposed at the surface of the cytoplasmic side of the chromatophore membrane.
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PMID:The light-harvesting polypeptides of Rhodospirillum rubrum. II. Localisation of the amino-terminal regions of the light-harvesting polypeptides B 870-alpha and B 870-beta and the reaction-centre subunit L at the cytoplasmic side of the photosynthetic membrane of Rhodospirillum rubrum G-9+. 643 97

A procedure is described for purification of the primary bactericidal component of normal rabbit serum active in vitro against Bacillus subtilis. A 65 000-fold increase in specific bactericidal activity per milligram of serum protein was obtained, yielding a low molecular weight, heat-stable polypeptide fraction (PC-III) exhibiting biological activity at protein concentrations below 10 ng/mL. This preparation appeared homogeneous as judged by column chromatography and analytical NaDodSO4-polyacrylamide gel eletrophoresis; recovery of serum bactericidal activity was routinely greater than 80%. Analysis of dansylated or 125I-labeled samples in peptide-resolving polyacrylamide gels revealed a single band with an Mr of 1800. Optimal antibacterial activity of PC-III against B. subtilis occurred at an ionic strength of 0.24 and was absolutely dependent upon divalent cations; calcium was the most effective. Under optimum conditions, 4 ng/mL of PC-III reduced the viability of B. subtilis test innocula by 90% within 10 min at 37 degrees C. Listeria monocytogenes, Escherichia coli, and Salmonella typhimurium were all sensitive to the action of PC-III, but higher bactericide concentrations were required to produce similar reductions in viability as observed with B. subtilis. All strains were killed by PC-III concentrations well below 1 microgram/mL, roughly that found in normal serum. The activity of PC-III preparations was significantly reduced by pretreatment with trypsin or proteinase K but not by neuraminidase or periodate.
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PMID:Antibacterial peptide from normal rat serum. 1. Isolation from whole serum, activity, and microbicidal spectrum. 679 8

The Saccharomyces cerevisiae mutant rpo B1 produces a DNA-dependent RNA polymerase B defective in RNA synthesis in vitro. RNA polymerase B purified from the mutant is altered both structurally and functionally. The enzyme is defective in the RNA chain initiation and elongation reactions. Enzyme-DNA binding is comparatively much less affected. These enzymological defects in the mutant enzyme are enhanced at elevated ionic strengths. Purified rpo B1 RNA polymerase B is lacking B32 and B16.5 subunits. However, the low activity of the mutant enzyme cannot be accounted for only by the loss of these two polypeptides. Wild type enzyme devoid of B32 and B16.5 subunits can be obtained after a mild urea treatment. This enzyme variant, called RNA polymerase B, does not share the enzymological properties of the mutant RNA polymerase. Immunoprecipitation of the enzyme from crude extracts shows that, in the rpo B1 mutant, a normal amount of RNA polymerase B is synthesized which contains the full complement of subunits. The polypeptide chain altered by the rpo B1 mutation was identified by partial proteolysis with proteinase K in the presence of sodium dodecyl sulfate. The 35S-labeled peptide pattern generated from the B220 subunit of the mutant enzyme differs markedly from the peptide pattern of the wild type subunit. The rpo B1 mutation therefore alters the B220 subunit, suggesting a role for this subunit in RNA chain elongation and in the association of the B32 and B16.5 subunits to the RNA polymerase molecule.
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PMID:A mutation of the B220 subunit gene affects the structural and functional properties of yeast RNA polymerase B in vitro. 699 72

Cell walls from Streptococcus mutans were prepared by conventional technique and subjected to a series of extraction procedures involving classical protein solvents. The extracted walls contained several non-peptidoglycan amino acids and were also amenable to radiolabeling with [125I]sodium iodide and chloramine T. The cell walls could be chemically modified with tetranitromethane and diazo-1H-tetrazole, suggesting the presence of tyrosine or histidine or both. Flourescence spectra of the walls revealed the presence of either tyrosine or tryptophan. Several proteases, including pronase, trypsin, subtilisin, and proteinase K, removed some of the label from the walls. In contrast, treatment of the walls with salts or denaturants did not result in the solubilization of label. When the walls were solubilized with mutanolysin and subjected to chromatography, three peaks of radioactivity with apparent molecular weights of 73,000, 39,000, and 9,600 were observed. Wall digests subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band of radioactivity corresponding to an apparent molecular weight of 79,000. Isoelectric focusing of labeled wall digest gave rise to two major bands of radioactivity with isoelectric points of approximately 2.4 and 5.6. The results suggest that the cell wall of S. mutans contains tightly and possibley covalently bound polypeptide molecules. We propose that the cell wall polypeptides of S. mutans serve as factors in the attachment of the bacteria to smooth surfaces.
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PMID:Association of protein with the cell wall of Streptococcus mutans. 738 May 60

Hepatitis B virus DNA contains a tightly bound protein which was not removed by healing to 60 degrees C with 2% SDS, 2% mercaptoethanol. The protein was indirectly demonstrated by the extraction of the DNA-protein complex with phenol before but not after its digestion with proteinase K. The DNA-protein complex had a lower buoyant density than protease-treated or free DNA; it was bound to glass fiber filters; it migrated at a slower rate in gel electrophoresis; and it could be radiolabeled by oxidative iodination. The binding site of the protein was mapped by extraction of restriction endonuclease digests with phenol and analysis of the digests for missing DNA fragments. The protein was localized to a site near the 5' end of the complete viral DNA strand. It remained attached to this strand after heating with SDS to 90 degrees C or treatment with 0.1 N NaOH, suggesting a covalent linkage. The 5' end of neither viral DNA strand could be phosphorylated in a reaction with polynucleotide kinase, consistent with attachment of protein to the 5' ends. The incomplete DNA strand, however, which is the strand elongated by the virion DNA polymerase reaction, did not contain a detectable amount of polypeptide as did the complete strand. The reasons for the apparent block of the 5' end of the incomplete DNA strand is thus not known. The protein bound covalently to HBV DNA could be involved in the replication of the complete viral DNA strand and/or endonucleolytic generation of linear unit-length DNA pieces from replicative intermediates, although its function and origin are not yet known.
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PMID:Hepatitis B virus contains protein attached to the 5' terminus of its complete DNA strand. 743 7


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