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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

2,5-Hexanedione (2,5-HD) is the neurotoxic gamma-diketone metabolite of the industrial solvent n-hexane. Substantial evidence indicates that 2,5-HD reacts with neurofilament protein lysine epsilon-amines to yield 2,5-dimethylpyrrole adducts and that this reaction is critical to the mechanism of toxicity. Alkylpyrroles are susceptible to autoxidative dimerization, a process that has also been suggested as an obligatory step in 2,5-HD neuropathy. In the present study, we characterized pyrrole autoxidation products of a 2,5-HD-treated lysine analogue and of a model, lysine-containing dipeptide and examined mechanistic aspects of pyrrole-mediated protein cross-linking. Incubation of 2,5-HD with N alpha-acetyllysine or the dipeptide N alpha-acetylglycyllysine methyl ester in physiological buffer (pH 7.4) under oxidative conditions resulted in time-dependent formation of the N epsilon-pyrrole derivative and two major pyrrole autoxidation products, as demonstrated by HPLC, on-line thermospray MS, and UV photodiode array detection. An autoxidative pyrrole dimer containing a methylene bridge between C-2 of one pyrrole ring and C-3 of a second ring was characterized by thermospray MS and 1H-NMR spectroscopy. 13C-NMR spectroscopy provided evidence for an identical pyrrole-to-pyrrole bridge in autoxidized, pyrrolylated ribonuclease (RNase). MS analysis also revealed a second major product--a stable, oxygen-containing monomeric pyrrole derivative. This product exhibited a UV absorbance maximum (lambda max = 355 nm) consistent with extended conjugation. Polymerization of pyrrolylated acetyllysine was accelerated by persulfate, a free-radical initiator, and inhibited by ascorbate, an antioxidant.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Formation and structure of cross-linking and monomeric pyrrole autoxidation products in 2,5-hexanedione-treated amino acids, peptides, and protein. 798 20

Changes in biological properties of serum albumin, egg white lysozyme, human serum alpha-1 antiproteinase and human leukocyte ribonuclease in effect of interaction with the enzyme system composed of myeloperoxidase from human neutrophilic polymorphonuclear leukocytes, Cl- and H2O2 were investigated. All the studied proteins lost their biological functions and were denaturated, but the amounts of hydrogen peroxide necessary to produce these effects differed remarkably for each individual protein. The alpha-1 antiproteinase ability of binding to trypsin was abolished upon employing 1.2 mols of H2O2 per mol of alpha-1 antiproteinase. The lysozyme enzymatic activity was abolished when 1.4 mols of H2O2 per mol of lysozyme were employed. Albumin decreased its binding to specific antialbumin antibodies and entirely lost the binding properties when 2 mols and about 10 mols of H2O2 per mol of albumin were employed, respectively. On the other hand 18 mols of H2O2 per mol of human leukocyte ribonuclease were necessary to inactivate this enzyme. All the mentioned proteins were protected from losing their biological functions by excess of specific amino acids with affinity to hypochlorite: Alpha-1 antiproteinase by excess of N-acetylmethionine, lysozyme by N-acetylmethionine and N-acetyl glycyltryptophane, albumin by N-acetyl derivatives of methionine, cysteine, tryptophane and lysine, whereas ribonuclease was protected from denaturation by all above mentioned amino acid derivatives. None of the studied proteins was protected from denaturation by N-acetyl tyrosine, or phenylalanine.
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PMID:Inactivation and denaturation of some proteins by enzyme system: myeloperoxidase, chloride and hydrogen peroxide. 840 71

The binding efficiency of high affinity monoclonal antifluorescyl antibody 4.4-20 with the homologous ligand situated in different protein environments has been investigated to quantitate the effect of non-active site secondary factors. To synthesize monofluoresceinated proteins, fluorescein 5-isothiocyanate was reacted with a 100-fold molar excess of ribonuclease, lysozyme, lactalbumin and bovine serum albumin. Absorption and emission spectra, as well as fluorescence life-time measurements which yielded discrete components and proteolytic studies suggested that fluorescein was conjugated to a specific lysine residue consistent with a non-random distribution of lysines within each protein population. The derivatized residue was probably a surface moiety based on accessibility analyses with iodide as a dynamic quencher. Dissociation rate analyses indicated that the relative release time of 4.4-20 with each monofluoresceinated protein was Fl-RNAse > or = Fl-lyso > or = FDS > Fl-lact > or = Fl-BSA which correlated with changes in free energy of binding. Relative fluorescence quenching measurements of the fluorescein moiety indicated that 4.4-20 showed decreasing quenching in the order FDS > Fl-RNAse > Fl-lyso > or = Fl-lact > Fl-BSA. Because spectral data indicated that fluorescein was conjugated to a specific residue or a non-random distribution of residues in each protein population, the results represented the effect of a single distinct environment or a weighted average of different microenvironments. Results have been interpreted within the theoretical framework of a dynamic antibody model involving conformer selection and the relative effects of primary and secondary interactions.
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PMID:Effects of secondary forces on the primary antibody-ligand interaction. 855 47

1 The role of nitric oxide (NO) derived from constitutive and inducible nitric oxide synthase (cNOS and iNOS) and its relationship to oxygen-derived free radicals and prostaglandins (PG) was investigated in a carrageenan-induced model of acute hindpaw inflammation. 2 The intraplantar injection of carrageenan elicited an inflammatory response that was characterized by a time-dependent increase in paw oedema, neutrophil infiltration, and increased levels of nitrite/nitrate (NO2-/NO3-) and prostaglandin E2(PGE2) in the paw exudate. 3 Paw oedema was maximal by 6 h and remained elevated for 10 h following carrageenan administration. The non-selective cNOS/iNOS inhibitors, NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME) given intravenously (30-300 mg kg-1) 1 h before or after carrageenan administration, inhibited paw oedema at all time points. 4 The selective iNOS inhibitors, N-iminoethyl-L-lysine (L-NIL) or aminoguanidine (AG), failed to inhibit carrageenan-induced paw oedema during the first 4 h following carrageenan administration, but inhibited paw oedema at subsequent time points (from 5-10 h). iNOS mRNA was detected between 3 to 10 h following carrageenan administration using ribonuclease protection assays. iNOS protein was first detected 6 h and was maximal 10 h following carrageenan administration as shown by Western blot analysis. Administration of the iNOS inhibitors 5 h after carrageenan (a time point where iNOS was expressed) inhibited paw oedema at all subsequent time points. Infiltrating neutrophils were not the source of iNOS since pretreatment with colchicine (2 mg kg-1) suppressed neutrophil infiltration, but did not inhibit the iNOS mRNA expression or the elevated NO2-/NO3- levels in the paw exudate. 5 Inhibition of paw oedema by the NOS inhibitors was associated with attenuation of both the NO2-/NO3- and PGE2 levels in the paw exudate. These inhibitors also reduced the neutrophil infiltration at the site of inflammation. 6 Recombinant human Cu/Zn superoxide dismutase coupled to polyethyleneglycol (PEGrhSOD; 12 x 10(3) u kg-1), administered intravenously either 30 min prior to or 1 h after carrageenan injection, inhibited paw oedema and neutrophil infiltration, but had no effect on NO2-/NO3- or PGE2 production in the paw exudate. The administration of catalase (40 x 10(3) u kg-1), given intraperitoneally 30 min before carrageenan administration, had no effect on paw oedema. Treatment with desferrioxamine (300 mg kg-1), given subcutaneously 1 h before carrageenan, inhibited paw oedema during the first 2 h after carrageenan administration, but not at later times. 7 These results suggest that the NO produced by cNOS is involved in the development of inflammation at early time points following carrageenan administration and that NO produced by iNOS is involved in the maintenance of the inflammatory response at later time points. The potential interactions of NO with superoxide anion and PG is discussed.
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PMID:Nitric oxide: a key mediator in the early and late phase of carrageenan-induced rat paw inflammation. 879 51

Synthesis of the vinyl sulfone and chloroethyl sulfone derivatives of poly(ethylene glycol) (PEG) is described. The chloroethyl sulfone (CES-PEG) is rapidly converted to the vinyl sulfone (VS-PEG) in the presence of base but is stable in water at neutral pH. Reactions with small molecules such as beta-mercaptoethanol and N alpha-acetyllysine show that the vinyl sulfone derivative is highly selective for reaction with sulfhydryl groups relative to reaction with amino groups. Also, VS-PEG is stable in water. These properties indicate that VS-PEG should be useful for selective attachment of PEG to protein cysteine groups. This hypothesis was verified by reacting VS-PEG with cysteine groups of reduced ribonuclease (RNase); the reaction is rapid and selective at pH 7-9. Reaction at lysine sites of unreduced RNase occurs slowly at pH 9.3 and is essentially complete after 100 h. Amino acid residues other than lysine and cysteine are not reactive toward VS-PEG. The covalent linkage between VS-PEG and lysine or cysteine groups is shown to be stable.
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PMID:Preparation of characterization of poly(ethylene glycol) vinyl sulfone. 881 61

The discovery of Ribonuclease k6 (RNase k6) was an unexpected result of our ongoing efforts to trace the evolutionary history of the ribonuclease gene family. The open reading frame of RNase k6, amplified from human genomic DNA, encodes a 150 amino acid polypeptide with eight cysteines and histidine and lysine residues corresponding to those found in the active site of the prototype, ribonuclease A. The single-copy gene encoding RNase k6 maps to human chromosome 14 and orthologous sequences were detected in both primate and non-primate mammalian species. A single mRNA transcript (1.5 kb) was detected in all human tissues tested, with lung representing the most abundant source. At the cellular level, transcripts encoding RNase k6 were detected in normal human monocytes and neutrophils (but not in eosinophils) suggesting a role for this ribonuclease in host defense. Of the five previously identified human ribonucleases of this group, RNase k6 is most closely related to eosinophil-derived neurotoxin (EDN), with 47% amino acid sequence identity; slight cross-reactivity between RNase k6 and EDN was observed on Western blots probed with polyclonal anti-EDN antiserum. The catalytic constants determined, Km = 5.0 microM and Kcat = 0.13 s-1, indicate that recombinant RNase k6 has approximately 40-fold less ribonuclease activity than recombinant EDN. The identification and characterization of RNase k6 has extended the ribonuclease gene family and suggests the possibility that there are others awaiting discovery.
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PMID:Molecular cloning and characterization of a novel human ribonuclease (RNase k6): increasing diversity in the enlarging ribonuclease gene family. 883 75

In search of synthetic high affinity ligands for the mannose receptor, we synthesized a series of lysine-based oligomannosides containing two (M2L) to six (M6L5) terminal alpha-D-mannose groups that are connected with the backbone by flexible elongated spacers (16 A). The synthesized cluster mannosides were all able to displace binding of biotinylated ribonuclease B and tissue-type plasminogen activator to isolated human mannose receptor. The affinity of these cluster mannosides for the mannose receptor was continuously enhanced from 18-23 microM to 0.5-2.6 nM, with mannose valencies increasing from two to six. On average, expansion of the cluster mannoside with an additional alpha-D-mannose group resulted in a 10-fold increase in its affinity for the mannose receptor. M3L2 to M6L5 displayed negative cooperative inhibition of ligand binding to the mannose receptor, suggesting that binding of these mannosides involves multiple binding sites. The nanomolar affinity of the most potent ligand, the hexamannoside M6L5 makes it the most potent synthetic cluster mannoside for the mannose receptor yet developed. As a result of its high affinity and accessible synthesis, M6L5 not only is a powerful tool to study the mechanism of ligand binding by the mannose receptor, but it is also a promising targeting device to accomplish cell-specific delivery of genes and drugs to liver endothelial cells or macrophages in bone marrow, lungs, spleen, and atherosclerotic plaques.
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PMID:Lysine-based cluster mannosides that inhibit ligand binding to the human mannose receptor at nanomolar concentration. 891 Apr 12

In order to determine the actual distance between the active site and the substrate binding site, termed the basic protrusion, of Escherichia coli ribonuclease HI, synthetic oligonucleotide duplexes with gradually extended overhangs were used, in which the enzymatic cleavage was restricted to a single site with 2'-O-methylnucleosides. The affinity of the enzyme for each substrate was determined by kinetic analysis. It was found that the affinity increased markedly when one nucleotide was attached to the 3' end of the DNA strand of the nine-base-pair hybrid duplex and then increased slightly as the DNA strand was extended further, whereas elongation of the strand in the other direction caused no change. When a mutant enzyme, in which three lysine residues in the basic protrusion were altered to alanine, was used, no increase in the kcat/K(m) value was observed. The results indicate that, for the productive binding, the axis from the 3' to the 5' end of the RNA strand of the substrate duplex must be oriented in agreement with the direction from the active site to the basic protrusion of the enzyme. The distance between the active site and the basic protrusion in the enzyme-substrate complex was shorter than that anticipated in modeling studies. A dynamic structure refinement, referred to as the normal mode analysis, was carried out in order to simulate the fluctuations of the basic protrusion.
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PMID:Interaction of the basic protrusion of Escherichia coli ribonuclease HI with its substrate. 894 69

We have characterized four novel murine ribonuclease genes that, together with the murine eosinophil-associated ribonucleases 1 and 2, form a distinct and unusual cluster within the RNase A gene superfamily. Three of these genes (mR-3, mR-4, mR-5) include complete open reading frames, encoding ribonucleases with eight cysteines and appropriately spaced histidines (His11 and His124) and lysine (Lys35) that are characteristic of this enlarging protein family; the fourth sequence encodes a non-functional pseudogene (mR-6P). Although the amino acid sequence similarities among these murine ribonucleases varies from 60 to 94%, they form a unique cluster, as each sequence is found to be more closely related to another of this group than to either murine angiogenin or to murine pancreatic ribonuclease. Interestingly, the relationship between the six genes in this 'mR cluster' and the defined lineages of the RNase A gene family could not be determined by amino acid sequence homology, suggesting the possibility that there are one or more additional ribonuclease lineages that have yet to be defined. Although the nature of the evolutionary constraints promoting this unusual expansion and diversification remain unclear, the implications with respect to function are intriguing.
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PMID:Molecular cloning of four novel murine ribonuclease genes: unusual expansion within the ribonuclease A gene family. 933 52

We have localized the gene encoding human RNase k6 to within approximately 120 kb on the long (q) arm of chromosome 14 by HAPPY mapping. With this information, the relative positions of the six human RNase A ribonucleases that have been mapped to this locus can be inferred. To further our understanding of the individual lineages comprising the RNase A superfamily, we have isolated and characterized 10 novel genes orthologous to that encoding human RNase k6 from Great Ape, Old World, and New World monkey genomes. Each gene encodes a complete ORF with no less than 86% amino acid sequence identity to human RNase k6 with the eight cysteines and catalytic histidines (H15 and H123) and lysine (K38) typically observed among members of the RNase A superfamily. Interesting trends include an unusually low number of synonymous substitutions (Ks) observed among the New World monkey RNase k6 genes. When considering nonsilent mutations, RNase k6 is a relatively stable lineage, with a nonsynonymous substitution rate of 0.40 x 10(-9) nonsynonymous substitutions/nonsynonymous site/year (ns/ns/yr). These results stand in contrast to those determined for the primate orthologs of the two closely related ribonucleases, the eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), which have incorporated nonsilent mutations at very rapid rates (1.9 x 10(-9) and 2.0 x 10(-9) ns/ns/yr, respectively). The uneventful trends observed for RNase k6 serve to spotlight the unique nature of EDN and ECP and the unusual evolutionary constraints to which these two ribonuclease genes must be responding. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF037081-AF037090.]
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PMID:Ribonuclease k6: chromosomal mapping and divergent rates of evolution within the RNase A gene superfamily. 964 35


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