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)

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

We have synthesized and analyzed the functional properties of a novel DNA capture reagent containing a methidium moiety attached to a sepharose bead by a spermine linker. DNA present in a biological fluid or other complex sample binds to the reagent. The DNA-capture reagent complex is then separated from the sample by centrifugation and the DNA is released from the reagent by brief incubation in 0.1 to 0.5 N NaOH or KOH. Capture of DNA from complex samples is independent of the salt concentration of the sample, and occurs in the presence of high concentrations of EDTA, proteinase K and detergents. Many samples can be processed simultaneously. The following specific applications, in which denatured DNA is quantitated or characterized, are demonstrated: 1). Isolation of hepatitis B virus DNA from serum and quantitation by dot-blot hybridization, 2). Isolation and quantitation of DNA from urine, 3). Isolation of human genomic DNA from one microliter of blood or 100 HeLa cells followed by amplification of a specific gene sequence using the Polymerase Chain Reaction, 4). Isolation of single stranded phage M13 sequencing templates from bacterial cultures. These investigations suggest that a capture reagent containing an intercalating moiety bound to a solid support may be useful for many applications in molecular biology and molecular diagnostics.
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PMID:Rapid isolation of DNA from complex biological samples using a novel capture reagent--methidium-spermine-sepharose. 278 Mar 16

The specific recognition by mitochondria of the precursor of porin and the insertion into the outer membrane were studied with a radiolabeled water-soluble form of porin derived from the mature protein. High-affinity binding sites had a number of 5-10 pmol/mg mitochondrial protein and a ka of 1-5 X 10(8) M-1. Binding was abolished after trypsin pretreatment of mitochondria indicating that binding sites were of protein-aceous nature. Specifically bound porin could be extracted at alkaline pH but not by high salt and was protected against low concentrations of proteinase K. It could be chased to a highly protease resistant form corresponding to mature porin. High-affinity binding sites could be extracted from mitochondria with detergent and reconstituted in asolectin-ergosterol liposomes. Water-soluble porin competed for the specific binding and import of the precursor of the ADP/ATP carrier, an inner membrane protein. We suggest that (i) binding of precursors to proteinaceous receptors serves as an initial step for recognition, (ii) the receptor for porin may also be involved in the import of precursors of inner membrane proteins, and (iii) interaction with the receptor triggers partial insertion of the precursor into the outer membrane.
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PMID:High-affinity binding sites involved in the import of porin into mitochondria. 296 May 20

The interactions of 16 alpha-hydroxyestrone (16 alpha-OHE1), a metabolite of estradiol (E2), with estrogen receptors (ERs) were compared in this study to the classic E2-receptor mechanism in human breast cancer cells MCF-7 in culture. When MCF-7 cells were incubated with radioinert 16 alpha-OHE1 or its 3H-labeled form for 4 weeks, the estrogen bound extensively and irreversibly in a time-dependent fashion to nuclear protein species that correspond to the ER. Here we show that the interactions of 16 alpha-OHE1 with the ER are different from those of E2 with the receptor. Dissociation of tritiated E2-ER or 16 alpha-OHE1-ER complexes, salt extraction, DNase and proteinase K digestion, and ethanol treatment demonstrated that the binding of 16 alpha-OHE1 to the ER corresponds to two different forms: a classical noncovalent interaction similar to that of E2, and a covalent adduct formation between the metabolite and the ER. These complexes localized preferentially in nuclear matrix components as revealed by cell fractionation and probing with a monoclonal anti-ER antibody. [3H]16 alpha-OHE1-ER complexes analyzed by polyacrylamide gel electrophoresis demonstrated a radiolabeled band at approximately 66 kDa that was absent when the exposure of cells was done in the presence of E2 in competition and that was also absent in [3H]E2 incubations. The present results when considered together with our previous findings of elevated activities of estrogen 16 alpha-hydroxylase, the enzyme responsible for the formation of 16 alpha-OHE1, in breast cancer patients and in women at enhanced risk for the disease, suggest that covalent modification of the ER may be one mechanism of malignant transformation in estrogen target tissues.
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PMID:Covalent binding of the endogenous estrogen 16 alpha-hydroxyestrone to estradiol receptor in human breast cancer cells: characterization and intranuclear localization. 318 93

We have previously shown that membranes from the retinal pigment epithelium can transform added all-trans-retinol into a mixture of 11-cis-retinoids, demonstrating the "missing reaction" in the visual cycle for the first time (Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853). In this article, this isomerase activity is further characterized. Double-label experiments with [15-3H]- and [15-14C]all-trans-retinol as the substrate show that the tritium label is retained in the 11-cis-retinol and 11-cis-retinyl palmitate products. This requires that isomerization occur at the alcohol level of oxidation. All-trans-retinyl esters, such as the palmitate, acetate, butyrate, and hexanoate esters, are not directly transformed into their 11-cis counterparts by the membranes. The data are consistent with the presence of an all-trans-retinol isomerase enzyme system or enzyme complex, which produces 11-cis-retinol. Other isomeric retinols were tested for substrate activity. Neither 9-cis-retinol(al) nor 13-cis-retinol were processed by the isomerase. Since the membranes containing the isomerase possess other retinol metabolizing activities, such as retinyl ester synthetase and dehydrogenase activities, further purification was attempted. Appreciable quantities of all detergents tested led to the disappearance of isomerase activity, and high salt or EDTA did not dissociate isomerase activity from the membranes. However, extensive sonication of the membranes did produce a 100,000 x g supernatant fraction of light membranes depleted of other all-trans-retinol processing activities. The isomerase activity in these membranes was saturable with all-trans-retinol, as required for a biologically significant process, and showed a Vmax of 5 pmol/h/mg of protein, a KM of 0.8 microM, and a pH optimum of 8. The isomerase was destroyed by proteinase K, by phospholipase C, by heating, or by ethanol at concentrations greater than 1%. The addition of high energy compounds, such as MgATP, MgGTP, or palmitoyl-CoA, did not appear to stimulate isomerase activity in the 100,000 x g supernatant.
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PMID:Biochemical characterization of the retinoid isomerase system of the eye. 350 Jan 73

We examined Escherichia coli K-12 lipopolysaccharide (LPS), which is known to be an R-form LPS, for its ability to form a hexagonal lattice structure in vitro. The LPS from E. coli K-12 strain JE1011 did not form a hexagonal lattice structure when it was precipitated by addition of two volumes of 10 mM MgCl2-ethanol, but it did form such a structure when it was electrodialyzed and then converted to the magnesium or calcium salt form. The lattice constant of the magnesium salt form was 15.2 +/- 0.3 nm and that of the calcium salt form 18.5 +/- 0.3 nm. Since prior treatment of the LPS with proteinase K in the presence of sodium dodecyl sulfate did not affect its capability of hexagonal assembly, the lattice formation by the LPS does not require the presence of proteins.
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PMID:In vitro hexagonal assembly of lipopolysaccharide of Escherichia coli K-12. 354 25

A protein with an apparent molecular weight of 14500 (14.5K) was extractable from homogenates of Borna disease virus-infected brains and tissue cultures using high concentrations of detergent and salt and by differential centrifugation procedures. The protein, present in an aggregated form, was remarkably resistant to proteinase K. Specific antibodies prepared in the homologous system (rat) recognized the 14.5K protein from various sources (infected brain of rat, mouse or chicken, and tissue cultures), but did not neutralize infectivity nor stain Borna disease virus-specific antigens from in vitro or in vivo preparations. Post-infection immune sera from different animal species did not detect the protein. This 14.5K protein was infection-specific but not disease-specific, and is inferred to be part of an internal virion component.
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PMID:Isolation and characterization of a 14500 molecular weight protein from brains and tissue cultures persistently infected with borna disease virus. 393 95

Extraction of human caudate nucleus under high-ionic-strength conditions solubilized 20-30% of total acetylcholinesterase (AChE) activity. Density gradient centrifugation revealed monomeric (5.0 S) and tetrameric (11.0 S) enzyme species. The purified, tetrameric salt-soluble (SS) AChE sedimented at 10.6 S and did not bind detergents. It showed an immunochemical reaction of identity with the detergent-soluble (DS) AChE species from human caudate nucleus and human erythrocytes, but did not cross-react with antibodies raised against human serum cholinesterase. The remaining activity was solubilized under low-ionic-strength conditions in the presence of 1.0% Triton X-100. The purified tetrameric, DS-AChE sedimented at 10.0 S as detergent-protein mixed micelle and on extensive removal of the detergent this enzyme formed defined aggregates by self-micellarization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed that the salt-soluble and detergent-soluble tetrameric enzyme species both contained a heavy and a light dimer; under reducing conditions mainly one band corresponding to the light subunit was seen. Molecular weights of 300,000 dalton and 280,000 dalton were calculated for SS-AChE and DS-AChE, respectively. Limited digestion of DS-AChE with proteinase K led to isolation of an enzyme that no longer bound detergents and lacked the intersubunit disulfide bridges.
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PMID:Molecular forms of acetylcholinesterase from human caudate nucleus: comparison of salt-soluble and detergent-soluble tetrameric enzyme species. 397 87

The nature of the association of U1 RNA with rapidly sedimenting RNP structures in rat hepatoma nuclei was investigated. The effects of salt and proteinase K treatment on the stability of this 'bound' form of U1 RNA were studied using sucrose density gradient analyses. Quantitation of the amount of U1 RNA remaining associated with large structures after treatment was used to assess the relative contribution of protein-protein (and protein-RNA) versus RNA-RNA interactions. Forty-eight percent of the total nuclear U1 RNA released by sonication was found in a 'bound' form when the sonicate was centrifuged through gradients containing 50 mM NaCl. Fifty percent of this 'bound' U1 RNA remained associated with rapidly sedimenting RNPs when the NaCl concentration was raised to 500 mM. To assess the contribution of protein independent interactions, large RNPs were completely deproteinized and their RNA moieties were then recentrifuged on gradients. By this analysis, 27% of the U1 RNA originally 'bound' to hnRNPs was associated with rapidly sedimenting (greater than 30 S) RNA (at 50 mM NaCl) suggesting their association by RNA-RNA hydrogen bonds. When the concentration of NaCl was 500 mM, 31% of the U1 RNA was associated with large RNA. Hence, approximately 30% of the U1 RNA molecules originally 'bound' (or about 15% of the total nuclear U1 RNA) were found to be associated by RNA-RNA hydrogen bonds while the remainder of the binding of U1 RNP to hnRNP was by protein-protein and/or protein-RNA interactions.
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PMID:Interactions of U1 RNP with heterogeneous nuclear RNA in rat Novikoff hepatoma nuclei. 608 66

hnRNP are made of two classes of unit, monoparticles and heterogeneous complexes. The monoparticles are much more easily dissociated by salt than the heterogeneous complexes. We made use of this differential salt sensitivity to determine the localization of snRNA in hnRNP. 1, About 50% of the snRNA were released by NaCl under the conditions of dissociation of monoparticles, U1 RNA which was enriched in monoparticles was preferentially released. 2, When the proteins resistant to salt dissociation were digested with proteinase K, an additional small proportion of snRNA was released, in particular a species designated 5 Sa RNA. Therefore, 5 Sa RNA seems to be preferentially associated with the proteins of heterogeneous complexes. 3, 40% of the snRNA remained associated with the hnRNA in the absence of any detectable protein. U1 and U2 RNA were the major RNAs in this fraction. The same RNA pattern was obtained for phenol-extracted RNA. The results indicate that all snRNA species are associated with the proteins of monoparticles, with those of heterogeneous complexes and with hnRNA. The existence of these pools of snRNA may reflect different functional states.
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PMID:The status of small nuclear RNA in the ribonucleoprotein fibrils containing heterogeneous nuclear RNA. 616 62


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