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)

The scrapie agent causes a degenerative neurologic disease and can be transmitted to laboratory rodents. The unusual properties of the scrapie agent prompted introduction of the term prion in order to distinguish this class of novel pathogens from viruses and viroids. The scrapie prion protein (PrPSc) is the only component of the infectious scrapie prion identified, to date. Although many biochemical and genetic lines of evidence argue that PrPSc is a major component of the infectious particle, the most convincing data is derived from immunoaffinity purification studies. Limited proteinase K digestion of hamster brain PrPSc produced PrP 27-30. After dispersion of brain microsomes isolated from scrapie-infected hamsters into detergent-lipid-protein complexes (DLPC), copurification of PrPSc and scrapie infectivity was obtained with PrP 27-30 monoclonal antibody affinity columns. PrPSc was enriched approximately 5700-fold with respect to total brain protein while scrapie prion infectivity was enriched approximately -4000-fold. The ratio of prion titer to PrPSc remained constant throughout purification. Heterologous monoclonal antibody columns failed to bind either PrpSc or scrapie infectivity. Polyclonal rabbit PrP antiserum raised against sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-purified PrP 27-30 reduced scrapie infectivity dispersed into DLPC by a factor of 100. Our findings represent the first direct immunologic and chromatographic demonstrations of a relationship between PrPSc and prion infectivity as well as providing additional support for the contention that PrPSc is a major component of the infectious scrapie particle. While these results and those of other studies establish that PrPSc is a component of the infectious prion, the possibility of a second component such as a small nucleic acid which might be required for infection must still be considered. PrPSc is encoded by a single copy cellular gene and not by a hypothetical-nucleic acid within purified prion preparations. Normal, uninfected cells express the cellular prion protein (PrPC). Both PrPSc and PrPC appear to be translated from the same 2.1-kb mRNA. The N-terminal amino acid sequences of hamster PrPC and PrPSc are identical; both correspond to that predicted by the translated prion protein (PrP) gene sequence. While the chemical difference between PrPC and PrPSc remains unknown, the organization of the PrP gene argues that it results from a posttranslational event. The mouse PrP gene is on chromosome 2 and is linked to a gene controlling the scrapie incubation time (Prn-i).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Immunoaffinity purification and neutralization of scrapie prions. 257 71

An extracellular inhibitory substance produced by the marine Alteromonas strain P-31 (NCMB 2144) was isolated and purified. The inhibitor was a macromolecule with a molecular weight of 90,000 estimated by Sephadex G-100 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitory activity was antagonized by proteinase K and beta-amylase and inactivated by heating at 80 degrees C for 30 min. The purified substance exhibited two typical absorption bands in the infrared spectrum at 1,650 and 1,075 cm-1, corresponding to peptide linkages and carbohydrate residues, respectively. These findings allowed us to characterize the antimicrobial compound as a thermolabile glycoprotein. The substance exhibited a broad inhibitory spectrum, being active against clinical and environmental isolates from related and nonrelated taxonomical bacterial groups as well as against the producer strain and other similar marine bacterial strains. The inhibitory glycoprotein did not display cytotoxicity toward mammalian and fish cell lines.
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PMID:Purification and characterization of an antibacterial substance produced by a marine Alteromonas species. 258 40

A procedure is described for assaying antibodies based on the application of antigen to nitrocellulose as a line with an ink pen point. The method requires no expensive apparatus, is easy to perform, and requires less than 0.25 micrograms of antigen per assay. More than 45 antigens can be assayed simultaneously with a single antibody. Antigens can be applied as purified proteins, extracts, or sodium dodecyl sulfate solubilized extracts. The application of the line blot assay for the detection of monoclonal antibodies which recognize heat-sensitive and insensitive epitopes on the typhus rickettsia surface protein antigen is described. A new serotyping assay for Gram-negative bacteria is also described in which sodium dodecyl sulfate solubilized antigens are applied as lines with and without prior proteinase K digestion. The value of the line blot serotyping assay is demonstrated with Proteus. Rickettsia, Rochalimaea, and Legionella antigens. The line blot immunoassay is a simple, but powerful and flexible, alternative to dot and cross-dot immunoassays.
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PMID:The line blot: an immunoassay for monoclonal and other antibodies. Its application to the serotyping of gram-negative bacteria. 260 66

T lymphocyte-mediated immunity is important for resistance to Francisella tularensis. To characterize the specificity of this immunity, we used membrane proteins and two lipopolysaccharide (LPS) preparations. Both membrane proteins were heat-modifiable, as indicated by their migration in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). One had an apparent molecular mass (Mm) of 120 kilodaltons (kDa) when solubilized in the SDS buffer at room temperature, but 17 kDa after heating. The respective values for the other protein were 35 kDa before and 40 kDa after heating. Both proteins were purified by a preparative SDS-PAGE. The LPS-containing preparations were isolated by aqueous phenol (WP) or PCP (phenol-chloroform-petroleum ether) extraction (LPS-R), and rendered protein-free by treatment with proteinase K. Lymphocytes from nine subjects immunized with a live tularemia vaccine from one to three years earlier responded specifically to both an F. tularensis whole cell antigen and the 17 kDa protein in the lymphocyte blast transformation test. By contrast, the 40 kDa protein and the two LPS preparations did not stimulate any detectable lymphocyte proliferation.
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PMID:Membrane proteins of Francisella tularensis LVS differ in ability to induce proliferation of lymphocytes from tularemia-vaccinated individuals. 262 30

Penicillin-binding proteins (PBPs) of Treponema pallidum subsp. pallidum (T. pallidum) were characterized by using [3H]penicillin G and a conjugate consisting of ampicillin and 125I-labeled Bolton-Hunter reagent. Both antibiotics specifically radiolabeled proteins with molecular masses of 94, 80, 63, and 58 kilodaltons (kDa); 125I-labeled Bolton-Hunter reagent-ampicillin also radiolabeled several polypeptides with lower molecular masses. The 94- and 58-kDa proteins demonstrated the highest binding affinities for [3H]penicillin G and were radiolabeled at concentrations of 8 and 40 nM, respectively. Radiolabeling of PBPs was detectable after 1 min of incubation in 1 microM [3H]penicillin G and was nearly maximal within 10 min. The rapidity of penicillin binding contrasted with the observation that only 40% of virulent treponemes became immobilized during prolonged incubation in vitro with a much higher concentration (1 mM) of unlabeled penicillin. Two lines of evidence indicated that most, if not all, of the PBPs are integral cytoplasmic membrane proteins: (i) preincubation of organisms in 0.1% Triton X-100 solubilized nearly all of the outer membranes but did not affect radiolabeling of PBPs, and (ii) except for the 80-kDa protein, the PBPs partitioned into the detergent phase following extraction with the nonionic detergent Triton X-114. The presence of peptidoglycan in T. pallidum was confirmed by the detection of muramic acid in the sodium dodecyl sulfate-insoluble, proteinase K-resistant residue obtained from Triton X-114-extracted organisms.
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PMID:Penicillin-binding proteins and peptidoglycan of Treponema pallidum subsp. pallidum. 264 34

A novel replicating agent (IFDO) was isolated from ileal fluid. Growth occurred in vitro under aerobic and anaerobic conditions, and was faster at 37 degrees C than at room temperature. The doubling time was 15.8 min. Colonies were dark brown in colour and occurred beneath the surface of agar after conventional surface inoculation. Provisional data indicate that the agent may be a normal intestinal commensal. The agent was remarkably resistant to inactivation by steam at 134 degrees C, formaldehyde and glutaraldehyde; it was relatively resistant to ionising radiation, and it was filterable through membranes with a nominal pore diameter of 10 nm. Such properties, with the exception of growth in cell-free medium, are shared by "unconventional agents" such as those of Creutzfeldt-Jakob disease and scrapie. Further comparison of the properties of the intestinal agent and of slow viruses revealed additional shared characteristics, including resistance to proteinase K and trypsin, and inactivation by guanidine thiocyanate, diethyl pyrocarbonate, phenol and sodium hydroxide. The agent differs from that of scrapie in being inactivated by ethidium bromide, zinc nitrate, EDTA, hydroxylamine in the presence Sarkosyl, and, under certain circumstances, by ribonuclease. Broth cultures of the agent contained particles possessing considerable size heterogeneity. The smaller filterable particles were generally more susceptible to inactivation, did not survive autoclaving, and were inactivated by papaya protease and lipase. It is possible that the replicating agent may be formed by crystallisation from constituents of the medium, and not by a biological process. This does not exclude the postulated relationship to slow viruses.
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PMID:A novel replicating agent isolated from the human intestinal tract having characteristics shared with Creutzfeldt-Jakob and related agents. 265 97

Various extracellular signals (i.e. transmitters, hormones, growth factors, etc.), together with their respective second-messenger pathways, regulate transmitter biosynthesis and neuronal function by altering gene expression. In this study we validated a protocol for isolating rat striatum and adrenal medullary nuclei for the purpose of extracting, identifying, and characterizing, nuclear regulatory factors which may serve a functional role in signal-transduction processes. Through gel retardation studies using a 299 base pair (bp) XmnI-SacI 32P-labeled probe (derived from the 5' untranslated region of the rat preproenkephalin gene), we show that different patterns of retained bands result from nuclear extracts derived from rat adrenal medulla and striatum (as well as from other tissue). These tissue differences may have biological significance since rat adrenal medullae have low basal enkephalin levels while the striatum has high levels of this peptide and its respective mRNA. Additionally, certain retained bands were common to both cytosolic and nuclear compartments, suggesting binding factors may be located in either cell space. An initial biochemical characterization of these factors was also undertaken. Generally, salt levels of 100 mM or more reduced factor binding while 10-50 mM sodium ion levels showed preferentially enhanced bands. Binding activity appeared optimal at pH 6.8. As all retained bands were abrogated by proteinase K treatment, these factors appear to have a significant protein component. Finally, of particular interest is that this 299 bp region contains many sequences showing over 80% sequence identity with several previously characterized transcriptional control elements (i.e. cAMP and phorbol ester inducible enhancers, GCN4, AP1, Sp1, CCAAT binding factor, ATF, and AP2). If binding is confirmed (footprint analysis) and function validated (transfection studies), the evolutionary significance of the apparent presence of gene regulatory sequences and functional element divergence of the DNA region between different species can be evaluated.
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PMID:Preproenkephalin DNA-binding proteins in the rat: 5' flanking region. 271 96

Methods are described that allow DNA to be prepared from widely different yeasts (Candida utilis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe). The methods are reliably reproducible, and the DNA obtained is of appropriate quality for the construction of gene libraries (upper limit of size range consistently 50-150 kbp). In method A, yeast cells are converted into spheroplasts by treatment with a highly purified mixture of enzymes from Trichoderma harzianum, the spheroplasts are lysed in a lauroylsarcosinate/EDTA buffer, and the lysate is incubated with proteinase K and then directly centrifuged through a cesium trifluoroacetate gradient. DNA is recovered from the appropriate fractions by ethanol precipitation, and the redissolved precipitate is incubated with ribonuclease. For the rest of the isolation, two protocols are given, one avoiding and one including phenol/chloroform extraction. In this way, DNA up to about 150 kbp in size can be obtained. In method B, spheroplasts are not made. Yeast cells are broken by grinding under liquid nitrogen and are then worked up in a manner similar to method A, protocol 2. Subsequent steps depend on the purpose for which the DNA is required. Traditional methods of sucrose or salt density gradient centrifugation or agarose gel electrophoresis are applicable for size selection. A sodium iodide/silica matrix technique allows fast and effective DNA recovery from agarose gels.
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PMID:Isolation of DNA from yeasts. 272 83

Hen oviduct N alpha-acetyltransferase was clarified to have a nucleic acid as an existing constituent by the following three results: (i) an ultraviolet absorption spectrum of the purified N alpha-acetyltransferase free of S-acetyl coenzyme A (Ac-CoA) had an absorption maximum at 260 nm. (ii) A nucleic acid band stained with ethidium bromide was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (iii) An ethidium bromide band co-migrated with a fluorescent band of the protein treated with N-(7-dimethylamino-4-methylcoumarinyl)maleimide, a reagent specific for thiol groups, on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate. N alpha-Acetyltransferase lost its activity partially or completely by digestion with bovine pancreatic RNase A, Staphylococcus aureus nuclease, or proteinase K, showing that both the nucleic acid and the protein subunit were necessary for the enzyme activity. The nucleic acid component was identified as an RNA but not a DNA because the RNase T2 digest of the nucleic acid was composed of four 3'-ribomononucleotides and completely separated from 3'- and 5'-deoxyribomononucleotides on TLC. The chain length of the nucleic acid of 260 nucleotides estimated by formamide-polyacrylamide gel electrophoresis was calculated to be about 83,000 of the molecular weight. The contents of RNA (35.0%) and protein (65.0%) in N alpha-acetyltransferase determined on weight basis corresponded reasonably well to the contents of RNA (34.4%) and protein (65.6%) calculated based on the assumption that N alpha-acetyltransferase consisted of one molecule of 7 S RNA (Mr 83,000) and two identical Mr 79,000 protein subunits. The total molecular weight (241,000) of the holoenzyme calculated based on the above result was identical to the molecular weight (240,000) of N alpha-acetyltransferase estimated by Sepharose 6B gel filtration.
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PMID:Hen oviduct N alpha-acetyltransferase is a ribonucleoprotein having 7 S RNA. 275 10

Isolates of Haemophilus influenzae type b (Hib) can be divided into three antigenic groups based on their reactivities with a set of two monoclonal antibodies (MAbs) directed against epitopes in the oligosaccharide region of Hib lipooligosaccharide (LOS) (P. A. Gulig, C. C. Patrick, L. Hermanstorfer, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 55:513-520, 1987). Approximately 24% of Hib strains react with both of these LOS-specific MAbs. Immunoprecipitation experiments involving several of these strains indicated that the epitopes recognized by these MAbs resided in two different LOS molecules, both of which were synthesized by these particular Hib strains. In addition, Western blot (immunoblot) analysis of proteinase K-treated cell extracts of these strains that had been subjected to sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis revealed two different LOS staining patterns when they were probed independently with the two MAbs. Colony blot radioimmunoassay of hundreds of colonies of one of these Hib strains showed that each colony bound both MAbs. Immune electron microscopy confirmed that individual cells of this same Hib strain expressed both types of LOS molecule at the same time. An antibody accessibility radioimmunoassay was used to show that different Hib strains of this type varied in the relative amounts of each of the two MAbs that they could bind to their cell surfaces. These findings indicate that some Hib strains can synthesize two antigenically distinct LOS molecules simultaneously.
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PMID:Antigenic evidence for simultaneous expression of two different lipooligosaccharides by some strains of Haemophilus influenzae type b. 278 4


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