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:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two general methods for the isolation of DNA from various sources based on the use of cetyltrimethylammonium bromide (cetavlon, CTA-Br) are described. Cetavlon is a strong cationic detergent precipitating DNA from diluted salt solutions. Cells are lysed and cellular components are dissolved in the presence of cetavlon, 5 M urea, 0.1 M EDTA and 2 M NaCl (KCl). In the first method pure DNA is precipitated in the form of CTA-salt by direct dilution of the lysate to bring the concentration of NaCl (KCl) down to 0.5 M after the removal of the main part of proteins by deproteinization with chloroform. In the first method pure DNA is precipitated in the form of CTA-salt by direct dilution of the lysate to bring the concentration of NaCl (KCl) down to 0.5 M after the removal of the main part of proteins by deproteinization with chloroform. In the second method DNA is purified on the hydroxyapatite column after cell lysis and the removal of cell debris by centrifugation. Both methods are suitable for rapid isolation of pure DNA from various sources with recovery about 80% and average molecular weight 20-10(6) and higher without use of ribonuclease, pronase and amylase.
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PMID:[Two simple methods for isolation of DNA from various sources using cetavlon]. 92 66

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.
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PMID:The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells. 98 39

A general strategy and a specific tactic for affinity purification of polypeptides synthesized on solid supports are desbribed and demonstrated. The desired peptide chains were distinguished from terminated peptide chains before removal from the support by attachment of an affinity reagnet (cysteinyl-methionine) bearing an affinity group (thiol) and a binding group (carboxylic acid). After cleavage from the synthetic support, the affinity-labeled peptides (Cys-Met-peptides) were bound to an affinity receptor (organomercurial-agarose) and thus separated from terminated peptides and all other peptides lacking the affinity group. The desired synthetic peptide was obtained by separation of the affinity reagent (loss of Cys-Met by cyanogen bromide cleavage). This general affinity purification strategy is independent of the length or amino acid sequence of the desired peptide. After assembly of ribonuclease-(111-124)-tetradecapeptide, using radiolabeled acetic anhudride for termination of uncoupled in termediates, essentially all (greater than 98.5%) of the acetylated delection peptides were removed by employing the organomercurial Cys-Met tactic. Similarly, the purity of crude synthetic histone H4-(1-37)-heptatriacontapeptide was increased six-fold by using this tactic to remove terminated peptides. A related dimeric Cys-Met tactic is outlined for affinity purification of peptides containing internal cysteine and methionine residues.
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PMID:Affinity purification of synthetic peptides. 106 9

A zymogram method for detection of in situ ribonuclease (RNase) activity, combined with isoelectric focusing in a thin layer of polyacrylamide gel (IEF-PAGE), has been developed. After incubation with a dried agarose film containing substrate RNA, ethidium bromide, and an appropriate reaction buffer, which was placed tightly on the top of the focused gel, sharp and distinct dark bands corresponding to RNase isoenzymes on a fluorescent background appeared under uv light. Addition of urea to the IEF-PAGE gel at a final concentration of 4.8 M permitted optimal focusing of the RNases. This method had not only a high sensitivity of less than 0.1 ng purified RNase A, but also a high band resolution compared with the immunostaining method. It was also useful for analysis of purified enzymes, including bovine pancreatic RNases and two types of human urine RNase as mammalian enzymes, and RNases T1 and T2 as microbial enzymes, as well as for detection of RNases present in crude tissue extracts, resulting in more detailed elucidation of the multiplicity of these enzymes.
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PMID:The zymogram method for detection of ribonucleases after isoelectric focusing: analysis of multiple forms of human, bovine, and microbial enzymes. 128 Sep 19

The effects of hCG, 8-bromo-cAMP, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, and forskolin on insulin-like growth factor-I (IGF-I) receptor gene expression of Leydig cells were studied. The treatment of purified Leydig cells with hCG caused a dose-dependent increase in [125I]IGF-I binding to Leydig cells without changes in binding affinity, indicating that the increased binding was due to increased receptor numbers and not to increased affinity. The minimal time required for hCG to induce IGF-I binding was 6 h, and it had reached a plateau at 16 h. 8-Bromo-cAMP (1 mM) increased IGF-I binding about 2-fold, and forskolin (10 microM) increased binding about 51%. Using the ribonuclease protection assay, we found that hCG and 8-bromo-cAMP could increase IGF-I receptor mRNA expression as early as 2 h before the increase in IGF-I binding. The induction by hCG was over 3.5-fold at 4 h and decreased to about 2-fold at 6 h. 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate had a very small effect on IGF-I receptor mRNA levels (1.5-fold increase at 2 h and no changes at 4 and 6 h). In conclusion, IGF-I receptors can be up-regulated by hCG, 8-bromo-cAMP, and forskolin. The up-regulation of IGF-I receptor number is associated with transient increases in IGF-I receptor mRNA levels. This could be a mechanism by which hCG and IGF-I interact to enhance Leydig cell steroidogenesis.
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PMID:Human chorionic gonadotropin up-regulates insulin-like growth factor-I receptor gene expression of Leydig cells. 165 15

The complete amino acid sequence of ribonuclease (RNase MC) from the seeds of bitter gourd (Momordica charantia) has been determined. This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with trypsin, lysylendopeptidase, and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The protein contains 191 amino acid residues and has a calculated molecular mass of 21,259 Da. Comparison of this sequence with sequences of the fungal RNases, RNase T2, and RNase Rh, revealed that there are highly conserved residues at positions 32-38 (TXHGLWP) and 81-92 (FWXHEWXKHGTC). Furthermore, the sequence of RNase MC was found to be homologous to those of Nicotiana alata S-glycoproteins involved in self-incompatibility sharing 41% identical residues.
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PMID:The complete amino acid sequence of ribonuclease from the seeds of bitter gourd (Momordica charantia). 189 1

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

Brome mosaic virus (BMV) is an icosahedral virus with a tripartite RNA genome which infects monocotyledonous plants, while the cowpea or legume strain of tobacco mosaic virus (CcTMV) is a rod-shaped virus with a single component RNA genome which infects dicotyledonous plants. To examine the potential for exchanging entire genes between RNA viruses, biologically active cDNA clones were used to replace the natural coat gene of BMV RNA3 with the coat gene and encapsidation origin of CcTMV. In protoplasts coinoculated with BMV RNAs 1 and 2, the resulting hybrid RNA3 was replicated by BMV trans-acting factors but was packaged in TMV coat protein to give rod-shaped particles rather than the usual BMV icosahedra. When the CcTMV encapsidation origin was suitably inserted in derivatives of BMV RNAs 1 and 2, these RNAs were also packaged in a ribonuclease-resistant form in protoplasts coinoculated with the hybrid RNA3 expressing TMV rather than BMV coat protein. Thus, despite the markedly divergent nature of BMV and TMV, replicating hybrids bearing characters derived from both parent viruses were produced. Such hybrid viruses could be of considerable value for studying specific steps in infection and for assigning functions to particular virus genes.
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PMID:Hybrid brome mosaic virus RNAs express and are packaged in tobacco mosaic virus coat protein in vivo. 284 11

The complete amino acid sequence of ribonuclease N1 (RNase N1), a guanine-specific ribonuclease from a fungus, Neurospora crassa, was determined by conventional protein sequencing, using peptide fragments obtained by tryptic digestion of cyanogen bromide-treated RNase N1 and by Staphylococcus aureus V8 protease digestion of heat-denatured RNase N1. The results showed that the protein is composed of a single polypeptide chain of 104 amino acid residues cross-linked by two disulfide bonds and has a molecular weight of 11,174: (sequence; see text) (Disulfide bonds: C2-C10, C6-C103) The amino acid sequence was homologous with those of RNase T1 (65% identity) and related microbial RNases.
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PMID:The amino acid sequence of ribonuclease N1, a guanine-specific ribonuclease from the fungus Neurospora crassa. 297 30

The preparation and analysis of a mutant ribonuclease (RNase) T1 which possesses higher nucleolytic activity than the wild-type enzyme are described. The gene for the mutant RNase T1 (Tyr45----Trp45), in which a single amino acid at the binding site of the guanine base has been changed, was constructed by the cassette mutangenesis method using a chemically synthesized gene [Ikehara, M. et al. (1986) Proc. Natl Acad. Sci. USA 83, 4695-4699]. In order to reduce the nucleolytic activity of the enzyme in vivo, this gene was expressed in Escherichia coli as a fused protein connected through methionine residues to other proteins at both the N- and C-termini. After liberation from the fused protein by cleavage with cyanogen bromide at the methionine junctions, the mutant RNase T1 was purified by column chromatography. The nucleolytic activity toward pGpC increased to 120% of that of wild-type RNase T1. The kinetic parameters of the mutant enzyme demonstrate that this higher nucleolytic activity is due to a higher affinity for the substrate, probably because of an increased stacking effect in the binding pocket for the guanine base. This mutant enzyme also possessed a higher nucleolytic activity against pApC than wild-type RNase T1.
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PMID:Increase in nucleolytic activity of ribonuclease T1 by substitution of tryptophan 45 for tyrosine 45. 312 93


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