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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Affinity-purified human placental ribonuclease inhibitor (PRI) was digested by trypsin. Subsequent fractionation of the hydrolysate by HPLC yielded 44 fractions, 3 of which retained the ability to inhibit ribonuclease. One of these, the most active, was a 15 amino acid peptide which had an amino acid composition corresponding to a tryptic fragment of PRI. This peptide was synthesised, and preliminary experiments were carried out on its interactions with ribonuclease. These experiments suggested that the behaviour of the peptide in terms of effect of pH, and effect of salt concentration were similar to the protein from which it was derived. These studies together with the strategic positioning of the peptide in the sequence of the ribonuclease inhibitor, suggest that this segment of PRI has an important role in the inhibitory activity of the intact protein.
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PMID:Characterisation of a tryptic peptide from human placental ribonuclease inhibitor which inhibits ribonuclease activity. 163 92

The region of human angiogenin containing residues 8-21 is highly conserved in angiogenins from four mammalian species but differs substantially from the corresponding region of the homologous protein ribonuclease A (RNase A). Regional mutagenesis has been employed to replace this segment of angiogenin with the corresponding RNase A sequence, and the activities of the resulting covalent angiogenin/RNase hybrid, designated ARH-III, have been examined. The ribonucleolytic activity of ARH-III is unchanged toward most substrates, including tRNA, naked 18S and 28S rRNA, CpA, CpG, UpA, and UpG. In contrast, the capacity of ARH-III to inhibit cell-free protein synthesis is decreased 20-30-fold compared to that of angiogenin. The angiogenic activity of ARH-III is also different; it is actually more potent. It induces a maximal response in the chick chorioallantoic membrane assay at 0.1 ng per egg, a 10-fold lower dose than required for angiogenin. In addition, binding of ARH-III to the placental ribonuclease inhibitor is increased by at least 1 order of magnitude (Ki less than or equal to 7 x 10(-17) M) compared to angiogenin. Thus, mutation of a highly conserved region of angiogenin markedly affects those properties likely involved in its biological function(s); it does not, however, alter ribonucleolytic activity toward most substrates.
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PMID:Replacement of residues 8-22 of angiogenin with 7-21 of RNase A selectively affects protein synthesis inhibition and angiogenesis. 169 38

Ribonuclease inhibitor (RI) is a protein that forms a very tight complex with ribonucleases (RNases) of the pancreatic type. RI contains 30 thiol groups, some of which are important for the enzyme-inhibitor interaction. To examine which thiols are affected by the binding of RNase, differential labeling experiments were performed. Reaction of porcine RI with the cysteine-specific labeling reagent 4-N,N-dimethylaminoazobenzene-4'-iodoacetamido-2'-sulfonic acid resulted in labeling of an average of 7.4 of the 30 cysteinyl residues. Binding of bovine pancreatic RNase A caused a 3.2-fold reduction in the extent of modification. Peptide mapping showed that in free RI, Cys-57, -371, and -404 were labeled to the greatest extent (yield, 0.4-0.6 mol/mol). RNase A did not protect Cys-57 against modification, whereas the labeling of Cys-371 and -404 was reduced by more than 90%. A second group of residues was labeled to a lesser extent in free RI (yield, 0.04-0.2 mol/mol). Within this group 11 residues were protected by RNase A by more than 90%, 2 were not affected at all, and 7 were protected between 10 and 90%. Seven cysteinyl residues in RI that were protected in the RI.RNase A complex were no longer protected in the RI.S-protein complex. These residues were mainly present in the N-terminal region of RI. However, when the S-peptide was included to yield the RI.RNase S complex, the same pattern of labeling was obtained as with the RI.RNase A complex. Addition of the S-peptide alone had no effect on the labeling. The implications of these observations with respect to RNase binding areas of RI are discussed in relation to the results obtained from the analysis of active RI molecules that contain deletions.
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PMID:Studies on the interaction of ribonuclease inhibitor with pancreatic ribonuclease involving differential labeling of cysteinyl residues. 174 89

Estrogen administration to male Xenopus causes the cytoplasmic destabilization of the hepatic serum protein coding mRNAs, most notably, albumin, yet has little effect on mRNAs encoding intracellular proteins such as ferritin. This report describes an estrogen-inducible ribonuclease activity found in liver polysomes that degrades albumin mRNA 4 times faster in vitro than it degrades ferritin mRNA. This differential rate of degradation was observed upon incubation of polysome extract with free liver RNA, isolated liver mRNPs, or transcripts from plasmid vectors. A cleavage fragment consisting of a doublet of approximately 194 nucleotides in length was consistently observed upon digestion of transcripts for the full length or 5' half of albumin mRNA. The generation of this cleavage fragment was used as an assay to study properties of the polysome nuclease activity. The 194 doublet is produced by the action of a Mg(2+)-independent endonuclease. This distinguishes the Xenopus liver enzyme from the enzymes that degrade histone or c-myc mRNA in vitro. It is inactivated by 400 mM NaCl or heating at 90 degrees C, but not by placental ribonuclease inhibitor or N-ethylmaleimide. Finally, the polysomal nuclease activity does not degrade double-stranded RNA. We believe the estrogen-induced nuclease activity contains an enzyme(s) that may mediate hormone-regulated changes in mRNA stability in this tissue.
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PMID:Estrogen-induced ribonuclease activity in Xenopus liver. 193 72

The effect of aluminum on alkaline ribonuclease (RNase) and RNase inhibitor, purified from bovine brain, was investigated. Incubation of alkaline RNase with aluminum interrupted binding of RNase inhibitor to alkaline RNase. A stoichiometry of 1:1 for the binding of aluminum to brain alkaline RNase was estimated, whereas no aluminum was found to be bound to the RNase inhibitor. Aluminum-bound alkaline RNase, however, retained a full alkaline RNase activity. None of the enzyme-bound aluminum was dissociated by dialysis against 50 mM Hepes, pH 7.0, at 4 degrees C for 24 h. Citrate, EDTA, NaF and apotransferrin protected the alkaline RNase against aluminum binding. Aluminum did not bind to the incubated alkaline RNase-inhibitor complex, suggesting that aluminum might compete with RNase inhibitor for the binding site. However, the data from chemical modification and spectroscopic studies indicate that it is also highly possible that aluminum binding to the enzyme induces conformational changes at or near the inhibitor-binding site, which subsequently interrupt binding of RNase inhibitor to alkaline RNase. These results suggest that accumulation of aluminum in brain might affect the regulation of RNA metabolism.
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PMID:Aluminum interrupts the formation of alkaline-ribonuclease-inhibitor complex from bovine brain. 193 68

The interactions of human placental ribonuclease inhibitor (PRI) with bovine pancreatic ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed previously, nor have any placental RNases been identified. In the present work, the major human placental RNase (PR) was purified to homogeneity by a five-step procedure and was obtained in a yield of 110 micrograms/kg of tissue. The placental content of angiogenin was also examined and was found to be at least 10-fold lower than that of PR. On the basis of its amino acid composition, amino-terminal sequence, and catalytic properties, PR appears to be identical with an RNase previously isolated from eosinophils (eosinophil-derived neurotoxin), liver, and urine. The apparent second-order rate constant of association for the PR.PRI complex, measured by examining the competition between PR and angiogenin for PRI, is 1.9 X 10(8) M-1 s-1. The rate constant for dissociation of the complex, determined by HPLC measurement of the rate of release of PR from its complex with PRI in the presence of a scavenger for free PRI, is 1.8 X 10(-7) s-1. Thus the Ki value for the PR.PRI complex is 9 X 10(-16) M, similar to that obtained with angiogenin, and 40-fold lower than that measured with RNase A. Complex formation causes a small red shift in the protein fluorescence emission spectrum, with no significant change in overall intensity. The fluorescence quantum yield of PR and the Stern-Volmer constant for fluorescence quenching by acrylamide are both high, possibly due to the presence of an unusual posttranslationally modified tryptophan residue at position 7 in the primary sequence.
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PMID:Interaction of human placental ribonuclease with placental ribonuclease inhibitor. 199 83

A cDNA encoding porcine ribonuclease inhibitor was used to express this protein in yeast under control of the PHO5 promoter. The recombinant protein was purified to homogeneity with a yield of 0.2 mg/g of yeast cells (wet weight) and was found to be indistinguishable from the inhibitor isolated from porcine liver on the basis of the following criteria: the amino acid composition, the number of free sulfhydryl groups, the molecular weight of the native and the denatured protein, peptide mapping, and amino acid sequence analysis of the N- and C-terminal regions of the protein. A simple method was developed for measuring accurately the slow, tight-biding kinetics of the inhibition of ribonuclease by ribonuclease inhibitor. From the dependence of the observed inhibition constant on the substrate concentration, it could be concluded that RI was competitive with the substrate UpA. The dependence of the observed association rate constant on the substrate concentration was consistent with a two-step mechanism in which the substrate only competed in the second (isomerization) step. The values for the inhibition constant for the inhibition of RNase by the recombinant inhibitor, 67 fM, the association rate constant, 1.5 x 10(8) M-1.s-1, and the dissociation rate constant, 8.3 x 10(-6) s-1, were in good agreement with those obtained for the porcine liver RNase inhibitor.
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PMID:Protein chemical and kinetic characterization of recombinant porcine ribonuclease inhibitor expressed in Saccharomyces cerevisiae. 227 59

Human placental ribonuclease inhibitor (PRI), a 50-kDa tight-binding inhibitor of angiogenin and pancreatic ribonuclease, consists predominantly of 7 internal repeats, each 57 residues long. Repeats 3 plus 4 (residues 144-257) or repeat 6 (residues 315-371) can be deleted to give mutant proteins, PRI delta 3-4 and PRI delta 6, respectively, that retain inhibitory activity [Lee, F. S., & Vallee, B. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1879-1883]. We describe here the isolation and characterization of these two active mutant proteins. Both inhibit the enzymatic activities of either angiogenin or bovine pancreatic ribonuclease A (RNase A) with a 1:1 stoichiometry, and the mode of inhibition of RNase A by either is competitive. PRI delta 3-4 binds to angiogenin and RNase A with Ki values of 0.72 and 170 pM, respectively The corresponding values for PRI delta 6 are 22 and 43 pM, respectively. Since recombinant PRI to angiogenin and RNase A with Ki values of 0.29 and 68 fM, respectively, deletion of repeats 3 plus 4 weakens both interactions 2500-fold while deletion of repeat 6 weakens them 76,000- and 630-fold, respectively. Therefore, either the deletion of these repeats has altered the conformation of the angiogenin/RNase binding site in PRI or the deleted repeats contribute directly to the binding site, or both. In addition, the tighter binding to angiogenin versus RNase A seen with native PRI has been preserved in PRI delta 3-4 but has been almost completely abolished in PRI delta 6.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic characterization of two active mutants of placental ribonuclease inhibitor that lack internal repeats. 239 4

Human placental ribonuclease inhibitor (PRI) is a potent protein inhibitor of pancreatic ribonucleases and the homologous blood vessel-inducing protein angiogenin. Although inhibition by PRI occurs with a 1:1 stoichiometry, its primary structure is composed predominantly of seven internal leucine-rich repeats. These internal repeats were systematically deleted either singly or in combination by "modular" mutagenesis. Deletion of repeat units 3 plus 4 or repeat unit 6 results in mutants that both bind to and inhibit ribonuclease A. Therefore, the angiogenin/ribonuclease binding site in PRI must reside primarily or entirely in repeats 1, 2, 5, or 7, the short N- or C-terminal segments, or a combination of these. Deletion of repeat units 3-5, 5-6, or 5 alone results in mutants that exhibit only binding activity. Hence, the binding site cannot reside exclusively in repeat 5. Other internal deletions or N- or C-terminal deletions of 6-86% of the protein all abolish activity. These results suggest that PRI has a modular structure, with one primary structural repeat constituting one module. The approach taken may be applicable to other proteins with repeat structures.
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PMID:Modular mutagenesis of human placental ribonuclease inhibitor, a protein with leucine-rich repeats. 240 43

A small (Mr less than 500) anionic, heat-stable molecule has been identified in rat liver cytosol which prevents the RNase-induced decrease in the glucocorticoid receptor sedimentation properties which we described previously. This factor, which can be removed by dialysis, molecular exclusion chromatography, or ultrafiltration, functions as a true stabilizer of the RNA-glucocorticoid receptor association, and not as a RNase inhibitor. Preliminary characterization shows that the factor is not a protein, nucleic acid, or nucleotide, is not absorbed by activated dextran-charcoal, and is unaffected by extraction with organic solvents. This factor prevents activation of the glucocorticoid receptor by dilution. The relationship of this stabilization factor to a low molecular weight activation inhibitor described by others is discussed.
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PMID:Stabilization of glucocorticoid receptor association with RNA by a low molecular weight factor from rat liver cytosol. 241 81


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