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)

The pyrE gene, encoding the pyrimidine biosynthetic enzyme orotate phosphoribosyltransferase, is the promoter distal gene of the dicistronic orfE-pyrE operon. The promoter proximal orfE gene, whose transcription and translation is important for regulation of the pyrE attenuator, encodes a 238-amino acid residue protein which was recently identified as the phosphorolytic ribonuclease, RNase PH, that removes nucleotides from the 3' ends of tRNA precursors. In this paper we report the construction of a plasmid, which overexpresses the orfE and pyrE gene products substantially, as well as the purification of the OrfE protein by ammonium sulfate precipitation and chromatography on phosphocellulose. The highly purified protein catalyzes the phosphorolytic cleavage of poly(A) at a rate of 1.6 mumol/min/mg and the formation of CDP from tRNA-CCA-Cn and orthophosphate at a rate equal to 0.14 mumol/min/mg, as characteristic for RNase PH. OrfE/RNase PH contains helix-turn-helix motifs resembling those in DNA-binding proteins, and it binds nonspecifically to DNA. On SDS gels, OrfE/RNase PH migrates as two distinct protein bands. This heterogeneity might be caused by post-translational modification other than proteolysis, or may be an electrophoretic artifact. The native protein is composed of two or more subunits.
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PMID:Overexpression and rapid purification of the orfE/rph gene product, RNase PH of Escherichia coli. 151 52

A ribonuclease activity in a 100,000 x g supernatant of a Triton lysate of a mitochondrial-kinetoplast fraction from Leishmania tarentolae is activated by incubation with heparin or by predigestion of the lysate with proteinase k or pronase. In vitro-transcribed pre-edited cytochrome b mRNA is cleaved at several sites. With time, complete degradation of the RNA occurs. All cleavages occurred within putative single-stranded regions of the RNA. No cleavage was observed with 9 S rRNA. The presence of a nonspecific nucleotide or nucleoside slows the rate of cleavage. The cleavage activity is inhibited by sodium dodecyl sulfate or phenol/chloroform extraction, is retained by a 10-kDa cutoff filter, and passes through a 30-kDa filter. Micrococcal nuclease inhibits the proteinase-induced activity but not the heparin-induced activity.
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PMID:A ribonuclease activity is activated by heparin or by digestion with proteinase K in mitochondrial extracts of Leishmania tarentolae. 155 86

We have reported previously [Sakakibara, et al. (1991) Chem. Pharm. Bull. 39, 146-149] that a protein purified from a partially purified pharmaceutical preparation of human chorionic gonadotropin (a urinary protein preparation from pregnant women) is a unique nonsecretory ribonuclease (RNase)-like protein on the basis of its amino terminal sequence homology. We purified the protein further from the same materials by gel filtration and reversed-phase column chromatographies with RNase activity as an index. The purified protein was designated RNase UpI-2. The catalytic activity and its sensitivity to inhibition by divalent cations suggest that the protein is related to nonsecretory RNase. The estimated molecular weight of RNase UpI-2 (38 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was significantly higher than that of urinary nonsecretory RNases (13 to 19 kDa) reported so far. After trifluoromethanesulfonic acid treatment, the molecular weight of RNase UpI-2 was reduced and approached that of nonsecretory RNase, which indicated that the protein contains a significant amount of carbohydrate (approximately 50%). RNase UpI-2 was immunoreactive with antibodies to a nonsecretory RNase, RNAase 1 [Yasuda et al. (1988) Biochim. Biophys. Acta 965, 185-194]. By immunoblot analysis of the protein freshly prepared from various urine samples, it was shown that a considerable amount of RNase UpI-2 is present in urine of pregnant women, but only a trace of RNase UpI-2, if any, was detected in urine of nonpregnant women and men. These results suggest the possibility that RNase UpI-2 may have been formed via a specific protein modification in pregnant women.
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PMID:Characterization of a unique nonsecretory ribonuclease from urine of pregnant women. 158 93

A procedure for the purification of Neisseria meningitidis lipopolysaccharide (LPS) from outer membrane vesicles (OMV) in spent growth media was developed. Five different LPS strains of group A N. meningitidis were grown in tryptic soy broth with vigorous aeration for 36-48 h, and centrifuged to collect both cells and supernatants. The amount of LPS in the OMV in the supernatants was higher or at least equal to that in the cells. The OMV in each supernatant were concentrated, pelleted by ultracentrifugation, and treated with 2% sodium deoxycholate to dissociate LPS from OMV. The LPS was then separated from capsular polysaccharides, proteins and phospholipids by gel filtration on Sephacryl S-300 column in 1% sodium deoxycholate, and precipitated from the column fractions in 70% ethanol. In addition, LPS was also extracted from cells with hot phenol-water, ultracentrifuged once after treatment with ribonuclease, and purified on Sephacryl S-300. When compared with an improved phenol-water extraction method, the LPS obtained from either OMV or cells by the above methods gave a 40-180% increase in yield. The LPS also had much higher activities in limulus amebocyte lysate assay, rabbit pyrogenic test, and enzyme-linked immunosorbent assay. The LPS purified from cells and from OMV were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.
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PMID:Purification of rough-type lipopolysaccharides of Neisseria meningitidis from cells and outer membrane vesicles in spent media. 177 80

Two neutral ribonucleases have been purified from developing tomato fruit. Their activity is maximal 5 days after anthesis, declines during maturation, and then increases slightly in the mature green through breaker stages. The ribonucleases Tf1 and Tf2 have molecular weights of 59 and 29 K, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and are glycoproteins. The reduced and denatured Tf1 is composed of two subunits, 30 and 29 K, of which only the 30-K subunit displays ribonuclease activity after renaturation. Reduced and denatured Tf2 is a single 29-K polypeptide that is renaturable to an active ribonuclease. Only the 30-K, active subunit of Tf1 is immunologically cross-reactive with Tf2. Both ribonucleases are cyclyzing endoribonucleases with a strong preference for cleavage at pyrimidine residues, thus generating oligonucleotide products ending with pyrimidine 2',3'-cyclic phosphate. These tomato fruit ribonucleases share a number of properties in common with the S-glycoprotein ribonucleases that are involved in self-incompatibility reactions in some solanaceous plants.
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PMID:Purification and characterization of two ribonucleases from developing tomato fruit. 192 99

The ribonuclease excreted by Bacillus amyloliquefaciens, Barnase, was co-crystallized with the deoxy-dinucleotide d(GpC). The crystal structure was determined by molecular replacement from a model of free Barnase previously derived by Mauguen et al. Refinement was carried out using data to 1.9 A resolution. The final model, which has a crystallographic R factor of 22%, includes 869 protein atoms, 38 atoms from d(GpC), a sulfate ion and 73 water molecules. Only minor differences from free Barnase are seen in the protein moiety, the root-mean-square C alpha movement being 0.45 A. The dinucleotide has a folded conformation. It is located near the active site of the enzyme, but outside the protein molecule and making crystal packing contacts with neighboring molecules. The guanine base is stacked on the imidazole ring of active site His102, rather than binding to the so-called recognition loop as it does in other complexes of guanine nucleotides with microbial nucleases. The deoxyguanosine is syn, with the sugar ring in C-2'-endo conformation; the deoxycytidine is anti and C-4'-exo. In addition to the stacking interaction, His102 hydrogen bonds to the free 5' hydroxyl, which is located near the position where the 3' phosphate group is found in other inhibitors of microbial ribonucleases. While the mode of binding observed with d(GpC) and Barnase would be non-productive for a dinucleotide substrate, it may define a site for the nucleotide product on the 3' side of the hydrolyzed bond.
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PMID:Crystal structure of a barnase-d(GpC) complex at 1.9 A resolution. 202 57

Unfolded ribonuclease (RNase) from porcine pancreas consists of a mixture of fast and slow-refolding species. The equilibrium distribution of these species differs strongly from other homologous RNases, because an additional proline residue is present at position 115 of the porcine protein. The major slow-folding species of porcine RNase contains incorrect proline isomers at Pro93 and at Pro114-Pro115. Both positions are presumably part of beta-turn structures in the native protein, as deduced from the structure of the homologous bovine RNase A. The folding kinetics of these molecules depend strongly on the conditions used. Under unfavorable conditions (near the unfolding transition), refolding is virtually blocked by the presence of the incorrect proline peptide bonds and partially folded intermediates with incorrect isomers could not be detected. As a consequence, folding is very slow under such conditions and the re-isomerization of Pro114-Pro115 is the first and rate-limiting step of folding. Under strongly native conditions (such as in the presence of ammonium sulfate), refolding is much faster. A largely folded intermediate accumulates with the turns around Pro93 and Pro114-Pro115 still in the non-native conformation. These results suggest that incorrect proline isomers strongly influence protein folding and that, under favorable conditions, the polypeptide chain can fold with two beta-turns locked into a non-native conformation. We conclude, therefore, that early formation of correct turn structure is not necessarily required for protein folding. However, the presence of incorrect turns, locked-in by non-native proline isomers, strongly decreases the rate of refolding. Alternative pathways of folding exist. The choice of pathway depends on the number and distribution of incorrect proline isomers and on the folding conditions.
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PMID:Role of two proline-containing turns in the folding of porcine ribonuclease. 231 96

A ribonuclease (RNase) was isolated from the urine of a 35-year-old male and purified to electrophoretic homogeneity. The enzyme was tentatively designated RNase 2. A rabbit antibody produced by injection of the purified RNase 2 was able to distinguish RNase 2 from another type of RNase coexisting in body fluids. With this antibody it was possible to detect RNase 2 isozymes in human serum and urine without difficulty using isoelectric focusing or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting. Both RNase 2 in serum and urine seemed to exist in multiple forms with regard to their molecular masses and pI values. This technique may prove to be useful in genetic and forensic studies of RNase polymorphism.
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PMID:New detection method for ribonuclease 2 (RNase 2) using immunoblotting with specific antibody. 235 60

The eosinophil granule major basic protein, the eosinophil cationic protein, and the eosinophil-derived neurotoxin were found to be lytic for Trypanosoma cruzi trypomastigotes from blood, cell cultures, or insect vectors and for cultured amastigotes. The toxic effects of the major basic and cationic proteins were inhibited by the polyanions heparin and dextran sulfate, in keeping with the cationic nature of these proteins, or by heat denaturation, suggesting that molecular conformation was also relevant. The lytic activity of the neurotoxin was not inhibited by heating at 56 degrees C for 4 hr, establishing an additional difference with the eosinophil cationic protein. Heparin had only a slight inhibitory effect on the toxicity of the neurotoxin, and dextran sulfate was inactive even at 25 mg/ml. Although both the eosinophil cationic protein and the neurotoxin possess ribonuclease activity, only the toxicity of the latter was abolished by the ribonuclease inhibitor RNasin (Promega, Madison, Wisconsin) or by a competitive substrate, yeast ribonucleic acid. Eosinophil peroxidase significantly increased the extent of trypomastigote or amastigote killing by hydrogen peroxide in the presence of iodide. This effect was abrogated by sodium azide, bovine serum albumin, or gelatin, known inhibitors of the eosinophil peroxidase + halide + hydrogen peroxide system. These results suggest that the destruction of T. cruzi trypomastigotes and amastigotes by eosinophils may result from toxic mechanisms involving several granule proteins.
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PMID:Toxic effects produced or mediated by human eosinophil granule components on Trypanosoma cruzi. 245 44

The ribonuclease inhibitor from pig brain has been purified 1,500-fold by a combination of ammonium sulfate fractionation, ion-exchange chromatography, hydroxylapatite chromatography, and gel filtration. The inhibitor has a Mr 50,000. It is a noncompetitive inhibitor for pancreatic ribonuclease A with a Ki of 1 nM, forming a 1:1 complex. Both ribonuclease A and B, but not ribonuclease U1 and T1, are inactivated by the inhibitor. The inhibition capacity was abolished by sulfhydryl reagents such as p-chloromercuribenzoate. Incubation of the enzyme-inhibitor complex with the sulfhydryl reagent caused dissociation into active ribonuclease and inactive inhibitor. Dithiothreitol was required during purification to retain the activity of the inhibitor.
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PMID:Ribonuclease inhibitor from pig brain: purification, characterization, and direct spectrophotometric assay. 254 Jun 74


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