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)

Numerous studies have indicated that a modified proteinase K-resistant form of an endogenous brain protein, prion protein (PrP), is associated with scrapie infection in animals. This scrapie-associated PrP modification appears to occur posttranslationally in brain, but its molecular nature is not known. To learn about the normal PrP biosynthesis and whether it is altered by scrapie infection in vitro, we did metabolic labeling experiments with uninfected and scrapie-infected mouse neuroblastoma tissue culture cells. Pulse-chase labeling experiments indicated that, in both cell types, two major PrP precursors of 28 and 33 kilodaltons (kDa) were processed to mature 30- and 35- to 41-kDa forms. Endoglycosidase H, tunicamycin, and phospholipase treatments revealed that the 28- and 33-kDa precursors resulted from the addition of high-mannose glycans to a 25-kDa polypeptide containing a phosphatidylinositol moiety and that maturation of the precursors involved the conversion of the high-mannose glycans to hybrid or complex glycans. Treatments of the live cells with trypsin and phosphatidylinositol-specific phospholipase C indicated that the mature PrP species were expressed solely on the cell surface, where they were anchored by covalent linkage to phosphatidylinositol. Once on the cell surface, the major PrP forms had half-lives of 3 to 6 h. No differences in PrP biosynthesis were observed between the scrapie-infected versus uninfected neuroblastoma cells.
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PMID:Prion protein biosynthesis in scrapie-infected and uninfected neuroblastoma cells. 256 14

The mosquitocidal crystal of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2 was purified, bioassayed against third-instar Aedes aegypti larvae (50% lethal concentration, 7.5 micrograms/ml), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealing polypeptides of 125, 50, 47, and 28 kilodaltons (kDa). When solubilized and proteolytically activated by insect gut proteases or proteinase K, the crystal was cytotoxic to insect and mammalian cells in vitro and was hemolytic. By using nondenaturing polyacrylamide gel electrophoresis, a polypeptide of 23 kDa, derived from the 28-kDa protoxin, was identified which was hemolytic and cytotoxic to Aedes albopictus, A. aegypti, and Choristoneura fumiferana CF1 insect cell lines. The 23-kDa polypeptide was purified by ion-exchange chromatography and gave 50% lethal dose values of 3.8, 3.3, and 6.9 micrograms/ml against A. albopictus, A. aegypti, and C. fumiferana CF1 cells lines, respectively. Cytotoxicity in vitro was both dose and temperature dependent, with a sigmoidal dose-response curve. The cytotoxicity of the 23-kDa toxin and the solubilized and proteolytically activated delta-endotoxin was inhibited by a range of phospholipids containing unsaturated fatty acids and by triglyceride and diglyceride dispersions. An interaction with membrane phospholipids appears important for toxicity. Polyclonal antisera prepared against the 23-kDa polypeptide did not cross-react with polypeptides in the native crystals of four other mosquitocidal strains.
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PMID:Purification and properties of a 28-kilodalton hemolytic and mosquitocidal protein toxin of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2. 256 94

Polypeptide and Western immunoblot profiles of subcellular fractions of Treponema denticola ATCC 33520 have been determined by SDS-PAGE of Triton X-100-soluble and -insoluble fractions, a lipopolysaccharide-enriched fraction and purified flagella. Major Triton X-100-soluble polypeptides of 72, 68, 54 and 52 kDa were detected. The 54 kDa polypeptide appeared to be a breakdown product of a larger, heat-modifiable polypeptide. Based on the results of SDS-PAGE analysis and immunoblotting of proteinase K digests of T. denticola, a 'rough' lipopolysaccharide appeared to be present. Electron microscopy has been used to monitor the effect of detergent treatment on the morphology of the organism and to examine the detailed structure of the flagella. Treatment with Triton removed the T. denticola outer membrane, resulting in exposure of the flagella. The flagella were shown to have a complex sheath and core structure and polypeptide composition characteristic of that observed for other treponemes. Polypeptides of 38, 35, 32 and 28 kDa were present in purified flagella preparations. Immunoelectron microscopy, iodine-labelling and Western blotting were used to demonstrate the exposure of antigens on the T. denticola surface. Surface iodination located polypeptides of 72, 68 and 54 kDa. Antiserum raised against whole cells of T. denticola recognized these polypeptides and an additional polypeptide of 52 kDa. These data provide a basis for future detailed molecular analysis of the ultrastructure and antigenicity of T. denticola.
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PMID:Antigenic and structural analysis of Treponema denticola. 263 57

A highly selective affinity labeling procedure has been applied to map the active center of DNA primase from the yeast Saccharomyces cerevisiae. Enzyme molecules that have been modified by covalent attachment of benzaldehyde derivatives of adenine nucleotides are autocatalytically labeled by incubation with a radioactive ribonucleoside triphosphate. The affinity labeling of primase requires a template DNA, is not affected by DNase and RNase treatments, but is sensitive to proteinase K. Both the p58 and p48 subunits of yeast DNA primase appear to participate in the formation of the catalytic site of the enzyme, although UV-photocross-linking with [alpha-32P]ATP locates the ribonucleoside triphosphate binding site exclusively on the p48 polypeptide. The fixation of the radioactive product has been carried out also after the enzymatic reaction. Under this condition the RNA primers synthesized by the DNA polymerase-primase complex under uncoupled DNA synthesis conditions are linked to both DNA primase and DNA polymerase. When DNA synthesis is allowed to proceed first, the labeled RNA chains are fixed exclusively to the DNA polymerase polypeptide. These results, in accord with previous data, have been used to propose a model illustrating the interactions and the putative roles of the polypeptides of the DNA polymerase-primase complex.
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PMID:Affinity labeling of the active center and ribonucleoside triphosphate binding site of yeast DNA primase. 264 56

The effects of a hydrophobic peptide segment inserted into the amino-terminal region of the mature domain of OmpC, an outer membrane protein, on its translocation across the cytoplasmic membrane was studied. Both the intact OmpC and central domain-deleted OmpC were examined. The hydrophobic segment was derived from the signal peptide of OmpF. Secretory translocation across the cytoplasmic membrane was examined by means of proteinase K treatment. Four monoclonal antibodies that recognize different regions of OmpC were used to characterize proteinase K-resistant fragments. Insertion of the hydrophobic segment did not appreciably prevent the translocation of these proteins across the cytoplasmic membrane, larger parts of them being found as mature forms, which were mostly localized outside the cytoplasmic membrane. Circumstantial evidence supports the view, on the other hand, that the inserted hydrophobic domain was retained in the cytoplasmic membrane. It is concluded, therefore, that the hydrophobic segment, although it is not exported across the cytoplasmic membrane, does not prevent the secretion of the following polypeptide chain. The secretion was dependent on the amino-terminal signal peptide. Insertion of positive charges immediately after the hydrophobic segment resulted in suppression of the translocation. Based on these results possible mechanisms by which the secretion of the polypeptide chain after the hydrophobic segment are discussed.
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PMID:Insertion of a signal peptide-derived hydrophobic segment into the mature domain of OmpC, an outer membrane protein, does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E. coli. 268 Nov 78

Limited proteolysis of a recombinant, soluble form of the Tac protein, a human interleukin-2 receptor (rIL-2R), was performed using trypsin, Staphylococcus aureus V8 protease and proteinase K to study the structural requirements of interleukin-2 receptor (IL-2R) for interleukin-2 (IL-2) binding. Sensitive proteolytic sites were found to be clustered in the regions of the polypeptide encoded by exons 3, 5, and 6, with a few semi-sensitive sites located within the two homologous domains encoded by exons 2 and 4. A number of nicked and truncated rIL-2R species generated by proteolysis were assayed for IL-2 binding using recombinant IL-2 (rIL-2) affinity gel and then structurally characterized. The results demonstrated that only the species that consist of the regions encoded by exons 2 and 4, joined by five disulfide bonds, are capable of binding IL-2 and that the presence of semi-sensitive cleavage sites within the two homologous domains had no apparent effect on IL-2 binding. These results suggest that the pattern of the sensitive cleavage sites in rIL-2R is closely related to the structural requirements for IL-2 binding. Based on the experimental results, a highly symmetrical core structure of IL-2R with a total of 135 amino acid residues was identified. This is the smallest protein moiety so far known to be capable of binding IL-2.
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PMID:Limited proteolysis of recombinant human soluble interleukin-2 receptor. Identification of an interleukin-2 binding core. 268 72

Exposure of bovine pulmonary artery smooth muscle cells (SMC) to anoxia for 24 h resulted in an approximate twofold stimulation of serotonin (5-HT) uptake compared with SMC exposed to 20% O2. The stimulation of 5-HT uptake by exposure to anoxia was eliminated when bovine pulmonary artery endothelial cells (EC) were cocultured with SMC. Incubation with EC-conditioned medium produced similar inhibitory effects on 5-HT uptake of SMC exposed to anoxia, a result not seen with SMC-conditioned medium. The inhibitory effect of EC-conditioned medium on the anoxia-stimulated 5-HT uptake was concentration dependent and absent at a dilution of 1:16. The production of the inhibitor was time-dependent. The EC-derived inhibitory factor was heat-stable at 100 degrees C for as long as 10 min and was stable in a pH range from 5.0 to 10.0. Significant losses of inhibitory activity of EC-conditioned medium were observed after treatment with trypsin, pronase E, and proteinase K. The molecular weight on the inhibitory factor from EC-conditioned medium was estimated to be approximately 66,000 by size-exclusion chromatography. The data show that stimulated uptake of 5-HT by SMC under anoxic conditions is under regulation by a protein (or polypeptide) produced by EC.
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PMID:Endothelial cell inhibition of hypoxia-induced stimulation of serotonin uptake by vascular smooth muscle cells. 271 40

The topology of several of the cytoplasmically made subunits of beef heart cytochrome c oxidase has been determined by protease digestion of oriented membrane preparations, using subunit-specific antibodies to identify cleavage products. Reconstituted vesicles of cytochrome c oxidase and asolectin were used as a vesicle preparation with the C domain of the enzyme available for protease digestion. Submitochondrial particles were used as vesicles with the M domain outermost. Trypsin and/or proteinase K cleaved polypeptides CIV, ASA, AED, STA, and IHQ. Cleavage of CIV, STA, and IHQ was from the M domains only and involved the removal of a fragment from the N-terminus in each case. Polypeptide AED was cleaved from the C side in the N-terminal part, while ASA was cleaved from both the C and M domains. Polypeptide fragments were electroblotted from polyacrylamide gels onto derivatized glass paper and sites of proteolytic cleavage determined by N-terminal sequence analysis.
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PMID:Orientation of the cytoplasmically made subunits of beef heart cytochrome c oxidase determined by protease digestion and antibody binding experiments. 283 91

The simian rotavirus SA11 genome segment 10 codes for a nonstructural glycoprotein, NS28, that has been hypothesized to be involved in budding of viral particles into the endoplasmic reticulum (ER) membrane. Previous studies had suggested that NS28 is an integral membrane protein of the ER, possibly a transmembrane protein. We have examined the topography of NS28 inserted in microsomal membranes following cell-free translation of genome segment 10 transcripts. These transcripts were obtained either by hybrid selection of mRNA synthesized by the endogenous viral RNA polymerase or by in vitro transcription of genome segment 10 cDNA using SP6 polymerase. Full-length and truncated gene 10 transcripts were translated in a cell-free system supplemented with dog pancreatic microsomes. The existence of a cytoplasmic domain of the translation product was demonstrated by protease protection experiments. An 18,000 (18K) mol wt glycosylated polypeptide was protected from digestion with proteinase K and trypsin, whereas chymotrypsin digestion yielded a 23K mol wt glycosylated polypeptide. Correlation of these biochemical data with the known sequence of NS28 suggests that a 10K mol wt hydrophilic, carboxy-terminal fragment (from amino acid number 86 to amino acid number 175) of this glycoprotein is exposed on the cytoplasmic side of the ER membrane. A model of how NS28 folds in the ER membrane is proposed.
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PMID:Topography of the simian rotavirus nonstructural glycoprotein (NS28) in the endoplasmic reticulum membrane. 283 61

Pig small intestinal mucosal explants, labelled with [35S]-methionine, were fractionated into Mg2+-precipitated (intracellular and basolateral) and microvillar membranes, and the orientation of newly synthesized aminopeptidase N (EC 3.4.11.2) in vesicles from the two fractions was studied by its accessibility to proteolytic cleavage. The mature polypeptide of Mr 166 000 from the latter fraction was cleaved by trypsin, proteinase K and papain, consistent with an extracellular location of the enzyme at its site of function. In contrast, both the mature form and the transient form of Mr 140 000 from the Mg2+-precipitated fraction were equally well protected from proteolytic cleavage (in the absence of Triton X-100). This indicates that the basolateral plasma membrane is unlikely to be involved in the post-Golgi transport of newly synthesized aminopeptidase N and suggests instead a direct delivery of the enzyme to the apical plasma membrane. A crude membrane preparation from labelled explants was used in immunoelectrophoretic purification of membranes to determine at what stage during intracellular transport newly synthesized microvillar enzymes are sorted, i.e., accumulated in areas of the membrane from where other proteins are excluded. The transient form of aminopeptidase N was only moderately enriched by immunopurification, using antibodies against different microvillar enzymes, but the mature form was enriched approximately 30-fold from explants, labelled for 30 min. This suggests that for microvillar enzymes, the aspects of sorting studied take place in, or shortly after exit from, the Golgi complex.
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PMID:Biosynthesis of intestinal microvillar proteins. Evidence for an intracellular sorting taking place in, or shortly after, exit from the Golgi complex. 286 38


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