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)

The substrate specificity of monomeric and dimeric forms of alpha-sarcin was investigated by membrane blotting procedures. Dimeric alpha-sarcin fails to inactivate ribosomes as well as to hydrolyze mini-stem-loop RNA, whereas monomeric alpha-sarcin catalyzes both substrates. Both monomeric and dimeric alpha-sarcin are effective ribonucleases that are displayed by in situ RNA-impregnated gel electrophoresis. The same purine base specificity was detected for both dimeric and monomeric forms. alpha-Sarcin is also an effective deoxyribonuclease to supercoiled DNA. The action of alpha-sarcin as deoxyribonuclease and ribonuclease is inhibited by the presence of SDS (3.5 x 10(-6) M); the inhibition on ribonuclease, but not on deoxyribonuclease, is reversible if the proteins are renatured.
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PMID:Substrate specificity of monomeric and dimeric alpha-sarcin. 863 5

Mammalian pancreatic ribonuclease (RNase) was conjugated chemically via a disulfide bond to human or murine epidermal growth factor (EGF). The conjugation between EGF and RNase was ascertained by SDS-PAGE using reduced and nonreduced conjugates. The EGF-RNase conjugate retained potent RNase activity and competed with 125I-EGF for binding to EGFR to the same extent as unconjugated EGF. Both the human and murine EGF-RNase conjugates showed dose-dependent cytotoxicity against EGFR-overexpressing A431 human squamous carcinoma cells with IC50 values of 3 x 10(-7) M and 6 x 10(-7) M, respectively, whereas free RNase had an IC50 of 10(-4) M. Against the EGFR-deficient small-cell lung cancer cell line H69, the EGF-RNase conjugate had no cytotoxic effect. The Human EGF-RNase conjugate showed dose-dependent cytotoxicity against other squamous carcinoma cell lines (TE-5, TE-1) and breast cancer cell lines (BT-20, SK-BR-3, MCF-7) and the cytotoxicity of the conjugate correlated positively with the level of expression of EGFR by each cell line. An unconjugated mixture of EGF and RNase had no greater effect than RNase alone on any cell line. Excess free EGF blocked EGF-RNase conjugate cytotoxicity against A431 cells. These results suggest that the EGF-RNase conjugate may be a more effective anticancer agent with less immunogenicity than coventional chimeric toxins.
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PMID:Epidermal growth factor receptor-dependent cytotoxic effect by an EGF-ribonuclease conjugate on human cancer cell lines--a trial for less immunogenic chimeric toxin. 867 51

Some cysteine-containing proteins upon sulfitolysis have been found to show anomalously retarded SDS-PAGE mobilities in non-reducing gels. These proteins include bovine serum albumin, ovalbumin, aldolase, ribonuclease and a recombinant fusion protein (XA) consisting of a portion of gamma-interferon linked to the A chain of human insulin. This mobility shift has been employed to determine the stability of the sulfonated products and to study the kinetics of the sulfitolysis reaction. Partially sulfonated products of intermediate shifts were observed at 0.01% beta-ME, while 0.05% beta-ME gave a shift characteristic of the completely reduced protein. The undiluted sulfitolysis reagent reacted with XA to give within 1 min a gel shift characteristic of the fully sulfitolysed protein. Its transition stages could be visualized at 15, 30 and 60 min when the reagent was diluted four-fold. In the presence of 8 M urea, the sulfitolysis of BSA was nearly complete at 30 min when the sulfitolysis reagent was used at a dilution of 1:5. However, under the same conditions BSA was predominantly unsulfitolysed in the absence of urea. In order to elucidate the mechanism of sulfonation shift, several derivatives of XA, e.g. performic acid oxidized, alkylated with (a) iodoacetamide and (b) iodoacetate, have been prepared. While the mobility of XASSO3- was sensitive to the presence of beta-ME, all other derivatives moved in a beta-ME-insensitive fashion. Furthermore, while the nonreducing mobilities of the acidic derivatives (-SSO3-, -SO3- and -SCH2CO2-) were anomalously retarded and identical, the mobility of the iodoacetamide derivative was intermediate between the retarded acidic derivatives above and XA below. These studies have suggested a role of the extended conformation of the A chain of insulin in causing a mobility shift of the acidic derivatives in this series. Similar results were observed in an analogous series of derivatives prepared from BSA. Non-denaturing gel filtration analyses of native vs. sulfitolysed samples of serum albumin, ovalbumin and ribonuclease have indicated that the sulfitolysed proteins elute earlier than their native counterparts and appear to be significantly larger than their true molecular weights. Circular dichroism analysis has indicated significant loss in helicity of sulfitolysed BSA. This suggests that the retarded mobility of sulfitolysed proteins seen on SDS-PAGE is likely to be due to an expansion in the hydrodynamic volumes of these proteins, a phenomenon triggered by cleavage of disulfide bonds and further accentuated by the introduction of strongly negatively charged sulfonates.
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PMID:Anomalous mobility of sulfitolysed proteins in SDS-PAGE. Analysis and applications. 889 91

Group V major allergen Phl p 5b of timothy grass pollen induces allergic rhinitis and bronchial asthma in 90% of grass pollen-allergic patients. In addition to its allergenicity ribonuclease activity has recently been attributed to this 29-kDa protein. The allergen was expressed in Escherichia coli and subsequently purified. Spontaneous conversion of these preparations to a mixture of various forms with molecular sizes between 10 and 29 kDa was consistently observed. Surprisingly, crystals could be grown from this heterogenous preparation. Single crystals, redissolved and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot, yielded one distinct low molecular weight protein, which was identified by amino acid sequencing as the C-terminal 13-kDa portion of the allergen. Histamine release assays with single crystal solutions using basophils of an allergic patient demonstrated allergenicity comparable with that of the holo-allergen. By contrast, RNase activity of the crystallized C-terminal form was 23 times higher than that of the full-length parent allergen. Crystals were used to collect preliminary diffraction data; the space group was evaluated to I4122 with cell dimensions of a = 87.7 A, b = 87.7 A, and c = 59.6 A. We conclude that preferential crystal growth of the 13-kDa form is indicative of a compact conformation of this particular C-terminal portion of the allergen. Thus, we show here that protein crystallization is not only a prerequisite for structural analyses, but it also can provide a unique separation technique to localize the functional domain of a major allergen.
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PMID:Crystallization and preliminary diffraction data of a major pollen allergen. Crystal growth separates a low molecular weight form with elevated biological activity. 891 Feb 84

A generally applicable, rapid, and sensitive method for profiling and sequencing of glycoprotein-associated N-linked oligosaccharides from protein gels was developed. The method employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation and purification and in-gel deglycosylation using PNGase F for glycan release. Profiles of the neutral glycans from bovine ribonuclease B, chicken ovalbumin, and human immunoglobulin G (IgG), as well as sialic acid-containing sugars (following esterification of the acidic groups) of bovine fetuin and bovine alpha1-acid glycoprotein, were obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and by normal-phase high-performance liquid chromatography following fluorescent labeling. Oligosaccharides were sequenced using specific exoglycosidases, and digestion products were analyzed by MALDI MS. Between 50 and 100 pmol (1.5 to 15 microg) of glycoprotein applied to the gel was sufficient to characterize its oligosaccharide contents. The identity of all glycoproteins investigated could be confirmed after deglycosylation by in-gel trypsin treatment followed by MALDI MS mass mapping and matching the measured molecular weights to a sequence database. The technique was used for the characterization of the glycan moieties of human immunodeficiency virus recombinant gp120 (Chinese hamster ovary cells) and to monitor changes in the glycosylation of this glycoprotein when produced in the presence of a glucosidase I inhibitor. Furthermore, since heavy and light chains of IgG became separated by SDS-PAGE, it could be established that most glycans were associated with the heavy chains.
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PMID:Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography. 923 2

The ability of intact protein antigens to bind to purified class II histocompatibility molecules was investigated. Intact bovine ribonuclease (RNase) inhibited peptide binding to DR1 with a potency similar to that of a high affinity peptide or irreversibly denatured RNase. Similarly, horse myoglobin (Mb) was a potent inhibitor of peptide binding to I-E(k). I-E(k)-Mb complexes were directly visualized as a distinct band with reduced mobility on SDS PAGE. Direct binding experiments with biotin-labeled proteins demonstrated that Mb and RNase bind to class II molecules through the peptide-binding groove with high affinity, and that binding occurs in the absence of detergent. The possibility that HLA-DM can catalyse the binding of intact protein antigens was supported by the observation that DM enhances the binding of biotin-RNase to DR1. Our results provide further support for the hypothesis that intact, partially unfolded protein antigens can act as ligands for initial interaction with class II molecules.
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PMID:Intact proteins can bind to class II histocompatibility molecules with high affinity. 930 63

A new ribonuclease from Saccharomyces cerevisiae, specific for poly(U) and poly(C) substrate, was purified near to homogeneity by successive fractionation with DEAE-Sepharose, Heparin-Sepharose and CM-Sepharose chromatography. The purified molecule detected by SDS/polyacrylimide gel electrophoresis has a molecular mass of 29 kDa. The optimum pH for the enzyme activity is 5.5-7 and its isoelectric point is 7.5. The purified enzyme was able to degrade 26S, 18S and 5S rRNAs as well as mRNA obtained from in vitro transcription. No catalytic activity was observed when the RNase was incubated with tRNA and double stranded substrate. Our findings suggest that this novel RNase may play an important role in the processing of RNA in Saccharomyces cerevisiae.
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PMID:Purification and characterization of a novel poly(U), poly(C) ribonuclease from Saccharomyces cerevisiae. 936 71

An extracellular ribonuclease from Rhizopus stolonifer (designated as RNase Rs) was purified to homogeneity by chromatography on DEAE-cellulose followed by CM-cellulose. The Mr of the purified enzyme determined by gel filtration and SDS-PAGE is 25,000 and 28,200, respectively. RNase Rs is a glycoprotein and contains 10.5% neutral sugar. It is an acidic protein with a pI of 5.0 and has a blocked N-terminus. The optimum pH and temperature are 5.5 and 45 degrees C, respectively. RNase Rs shows high stability between pH 6.0-10.0. Divalent cations like Zn2+, Hg2+ and Cu2+ inhibit the enzyme activity whereas, mononucleotides does not have any significant effect. The enzyme cleaves RNA to 3'-mononucleotides via 2',3'-cyclic nucleotides, with preferential liberation of 2',3'-cyclic GMP, suggesting that RNase Rs is a guanylic acid preferential cyclizing RNase. Moreover, cyclic nucleotides generated are highly resistant to further hydrolysis.
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PMID:Extracellular ribonuclease from Rhizopus stolonifer: characteristics of an atypical--guanylic acid preferential--enzyme from ribonuclease T2 family. 952 62

We generated a recombinant immunotoxin, named scFv(MGR6)-Cla, composed of the Fv region of an anti ErbB2 monoclonal antibody (MGR6) fused to clavin, a type 1 ribosome-inactivating protein (RIP) from Aspergillus clavatus. ErbB2 is a tyrosine kinase receptor which is overexpressed in most adenocarcinomas; clavin is a 17 kDa ribonuclease which inhibits protein synthesis by inactivating ribosomes. A recombinant DNA construct containing the cDNA of the single chain Fv fragment (scFv) of the MGR6 antibody fused to the clavin cDNA, was expressed at high levels in Escherichia coli as an insoluble fusion protein containing an N-terminal affinity tag of six consecutive histidine residues. Inclusion bodies were denatured and the recombinant fusion protein was purified under denaturing conditions by single-step purification using immobilised metal ion affinity chromatography (IMAC). The purified immunotoxin was renatured at high yield and histidine tag removed by digestion with enterokinase. The purity of the immunotoxin obtained after refolding was confirmed by SDS-PAGE, RP-HPLC, GPC-HPLC and N-terminal sequence analysis. Cell-free protein synthesis inhibition and binding assays showed that both clavin and scFv(MGR6) maintained their properties after refolding.
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PMID:Production and characterisation of a recombinant single-chain anti ErbB2-clavin immunotoxin. 985 10

An antifungal protein designated sativin was isolated from the legumes of the sugar snap (also known as honey pea) Pisum sativum var. macrocarpon. The procedure entailed extraction, affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The protein exhibited a molecular weight of 38 kDa in SDS-polyacrylamide gel electrophoresis. It possessed an N-terminal amino acid sequence which showed similarity to those of miraculin (a sweet protein) and pisavin (a ribosome-inactivating protein from Pisum sativum var arvense Poir manifesting similarity to miraculin). Unlike pisavin, however, sativin demonstrated negligible ribonuclease activity and inhibited translation in a rabbit reticulocyte lysate system with a very low potency (IC50= 14 microM). Sativin exerted antifungal activity against Fusarium oxysporum, Coprinus comatus and Pleurotus ostreatus but not against Rhizoctonia solani.
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PMID:Sativin: a novel antifungal miraculin-like protein isolated from legumes of the sugar snap Pisum sativum var. macrocarpon. 1096 7


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