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)

A previously studied species of ubiquitin-protein ligase contains specific sites for the binding of basic (Type I) and bulky hydrophobic (Type II) NH2-terminal amino acid residues of protein substrates. We now describe another enzyme that ligates ubiquitin specifically to proteins that have NH2-terminal residues other than the above two categories (Type III substrates). The new species of ligase, that we call E3 beta, is separable from the formerly described ligase (termed E3 alpha) by affinity chromatography on protein substrate columns. E3 beta was partially purified from extracts of rabbit reticulocytes and was shown to be required for the breakdown of Type III proteins. Apart from its different substrate specificity, it resembles E3 alpha in some physical properties, in a requirement for ubiquitin carrier protein (E2) for conjugate formation, and in its action to ligate multiple ubiquitin units to the substrate protein. The denatured derivative of bovine pancreatic ribonuclease is a specific substrate for E3 alpha, while that of ribonuclease S-protein is a good substrate for E3 beta. Since S-protein is formed by the removal from ribonuclease of NH2-terminal S-peptide, it is suggested that E3 beta interacts with an NH2-terminal determinant exposed in ribonuclease S-protein.
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PMID:A ubiquitin-protein ligase specific for type III protein substrates. 232 89

The complete amino acid sequence of ribonuclease U1 (RNase U1), a guanine-specific ribonuclease from a fungus, Ustilago sphaerogena, was determined by conventional protein sequencing, using peptide fragments obtained by several enzymatic cleavages of the performic acid-oxidized protein. The oxidized protein was first cleaved by trypsin and the resulting peptides were purified and their amino acid sequences were determined. These tryptic peptides were aligned with the aid of overlapping peptides isolated from a chymotryptic digest of the oxidized protein. The amino acid sequence thus deduced was further confirmed by isolation and analysis of peptides obtained by digestion of the oxidized protein with lysyl endopeptidase. The location of the disulfide bonds was deduced by isolation and analysis of cystine-containing peptides from a chymotryptic digest of heat-denatured RNase U1. These results showed that the protein is composed of a single polypeptide chain of 105 amino acid residues cross-linked by two disulfide bonds, having a molecular weight of 11,235, and that the NH2-terminus is blocked by a pyroglutamate residue. It has an overall homology with other guanine-specific or related ribonucleases, and shows 48% identity with RNase T1 and 38% identity with RNase U2.
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PMID:The amino acid sequence of ribonuclease U1, a guanine-specific ribonuclease from the fungus Ustilago sphaerogena. 316 89

Two glycopeptide hydrolases, an endo-beta-N-acetylglucosaminidase and peptide:N-glycanase (amidase), have been isolated from defatted jack bean meal by standard procedures involving differential solubility and column chromatography. The purified products appear to be free of contaminating proteases and exoglycosidases, and their substrate specificity has been explored with regard to both glycan and peptide structure of the substrates. The endoglycosidase appears to be specific for high mannose glycans; no hydrolysis of either hybrid or complex glycans has been observed. It shows limited activity with two intact glycoproteins, ribonuclease B and yeast invertase, and gives optimal rate with glycopeptides. Free glycan-Asn derivatives are poor substrates in comparison with glycopeptides or glycan-Asn derivatives where the alpha-amino group has been dansylated. The amidase will liberate both high mannose, hybrid, and asialo-complex glycans from both proteins and peptides, but many glycans in intact proteins or in long peptides are resistant to the amidase and become active as substrates only after further proteolytic cleavage. The best substrates appear to be those with the glycosylated asparagine no more than 4-5 residues in from either the NH2- or COOH-terminal end of the peptide. Sialylated glycans do not appear to be released by the amidase.
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PMID:Purification and characterization of two glycopeptide hydrolases from jack beans. 333 94

Previous studies have indicated that at least part of the selection of proteins for degradation takes place at a binding site on ubiquitin-protein ligase, to which the protein substrate is bound prior to ligation to ubiquitin. It was also shown that proteins with free NH2-terminal alpha-NH2 groups bind better to this site than proteins with blocked NH2 termini (Hershko, A., Heller, H., Eytan, E., and Reiss, Y. (1986) J. Biol. Chem. 261, 11992-11999). In the present study, we used simple derivatives of amino acids, such as methyl esters, hydroxamates, or dipeptides, to examine the question of whether the protein binding site of the ligase is able to distinguish between different NH2-terminal residues of proteins. Based on specific patterns of inhibition of the binding to ligase by these derivatives, three types of protein substrates could be distinguished. Type I substrates are proteins that have a basic NH2-terminal residue (such as ribonuclease and lysozyme); these are specifically inhibited by derivatives of the 3 basic amino acids (His, Arg, and Lys) with respect to degradation, ligation to ubiquitin, and binding to ligase. Type II substrates (such as beta-lactoglobulin or pepsinogen, that have a Leu residue at the NH2 terminus) are not affected by the above compounds, but are specifically inhibited by derivatives of bulky hydrophobic amino acids (Leu, Trp, Phe, and Tyr). In these cases, the amino acid derivatives apparently act as specific inhibitors of the binding of the NH2-terminal residue of proteins, as indicated by the following observations: (a) derivatives in which the alpha-NH2 group is blocked were inactive and (b) in dipeptides, the inhibitory amino acid residue had to be at the NH2-terminal position. An additional class (Type III) of substrates comprises proteins that have neither basic nor bulky hydrophobic NH2-terminal amino acid residues; the binding of these proteins is not inhibited by homologous amino acid derivatives that have NH2-terminal residues similar to that of the protein. It is concluded that Type I and Type II proteins bind to distinct and separate subsites of the ligase, specific for basic or bulky hydrophobic NH2-terminal residues, respectively. On the other hand, Type III proteins apparently predominantly interact with the ligase at regions of the protein molecule other than the NH2-terminal residue.
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PMID:Specificity of binding of NH2-terminal residue of proteins to ubiquitin-protein ligase. Use of amino acid derivatives to characterize specific binding sites. 334 27

Gabonase, an enzyme which acts on fibrinogen and factor XIII in uniquely thrombin-like ways, was purified to electrophoretic homogeneity from the venom of Bitis gabonica. On sodium dodecyl sulfate-polyacrylamide electrophoresis, the reduced protein behaved as a single chain with Mr = 30,600. The enzyme contains 20.6% carbohydrate, no free sulfhydryl groups and hence, from amino acid analysis, five disulfide bonds. Its extinction coefficient (E1%1cm) at 280 nm is 9.6. Its pI is 5.3. Gabonase has an active serine residue, is inactivated by phenylmethanesulfonyl fluoride, and has an active histidine which reacts with the chloromethyl ketone of tosyl-L-lysine. Its NH2-terminal amino acid sequence (Val-Val-Gly-Gly-Ala-Glu-Cys-Lys-Ile-Asp-Gly-His-Arg-Cys-Leu-Ala-Leu-Leu -Tyr-) is homologous to the B chain of thrombin. The activity of the enzyme is stabilized by calcium ion. It exhibits strong N alpha-p-tosyl-L-arginine methyl esterase activity, hydrolyzes tripeptide nitroanilide derivatives weakly or not at all, and cleaves no peptide bonds in insulin, glucagon, or the S peptide of ribonuclease. Gabonase clots fibrinogen with a specific activity of 45 NIH thrombin-equivalent units/mg, releasing both fibrinopeptides A and B and showing substrate inhibition at fibrinogen concentrations of 3 mg/ml or greater. The enzyme also activates factor XIII. It is not inactivated by either heparin or hirudin.
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PMID:Thrombin-like enzyme from the venom of Bitis gabonica. Purification, properties, and coagulant actions. 352 80

The molecular basis of the high reactivity toward reducing agents of intersubunit disulfides at positions 31 and 32 of dimeric bovine seminal ribonuclease was investigated by studying in the monomeric enzyme the fast reaction kinetics with disulfides of the adjacent cysteine-31 and -32, exposed by selective reduction of the intersubunit disulfides. Negatively charged and neutral disulfide reagents were used for measuring the thiol reaction rates at neutral pH. The kinetics studied as a function of pH permitted us to define pK values for the thiols of interest and indicated the possibility of determining pK values of SH groups in proteins indirectly by measuring the kinetics of reactivity of the SH groups with a disulfide reagent. The results were compared with those obtained under identical conditions with synthetic thiol peptides and model compounds. The data indicate that the superreactivity of intersubunit disulfides of seminal ribonuclease is matched by the high reactivity at neutral pH of adjacent cysteine residues 31 and 32, as compared to all small thiol compounds tested. The synthetic hexapeptide segment of seminal ribonuclease Ac-Met-Cys-Cys-Arg-Lys-Met-OH, which includes the two cysteine residues of interest, was even more reactive. These data, and the other results reported in this paper, led to the conclusion that the superreactivity at neutral pH of cysteine residues at positions 31 and 32 of bovine seminal ribonuclease is primarily dependent on the nearby presence of positively charged groups, particularly the epsilon-NH2 of lysine-34, and is influenced by the adjacency of the two thiols and by the protein tertiary structure.
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PMID:Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease. 409 91

1. The kinetics of the reaction of 2,4,6-trinitrobenzenesulphonic acid with various amino acids, peptides and proteins were studied by spectrophotometry. 2. The reaction of the alpha- and in-amino groups in simple amino acids was found to be second-order, and the unprotonated amino group was shown to be the reactive species. 3. By allowing for the concentration of unreactive -NH(3) (+) group, intrinsic reactivities for the free amino groups were derived and shown to be correlated with the basicities. 4. The SH group of N-acetylcysteine was found to be more reactive to 2,4,6-trinitrobenzenesulphonic acid than most amino groups. 5. The reactions of insulin, chymotrypsinogen and ribonuclease with 2,4,6-trinitrobenzenesulphonic acid were analysed in terms of three exponential rate curves, each referring to one or more amino groups of the proteins. 6. The reaction of lysozyme with 2,4,6-trinitrobenzenesulphonic acid was found to display an acceleration effect. 7. From the reaction of 2,4,6-trinitrobenzenesulphonic acid with glutamate dehydrogenase at several enzyme concentrations, it was possible to discern two sets of amino groups of different reactivity, and to show that the number of groups in each set was decreased by aggregation of the enzyme.
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PMID:The reaction of 2,4,6-trinitrobenzenesulphonic acid with amino acids, Peptides and proteins. 566 53

We have designed and synthesized a model pentadecapeptide predicted to have the essential sequence information needed to form a stable and enzymatically active noncovalent complex with bovine pancreatic ribonuclease S-protein. The model peptide sequence, based on the conformational approach of simplifying the native sequence in a manner consistent with retention of essential noncovalent contacts and of secondary structure features, contained alanine at all positions except for Glu 2, Lys 7, Phe 8, Arg 10, His 12, and Met 13. The peptide was synthesized by the Merrifield solid phase method. The circular dichroism spectra of the purified model peptide in water and trifluoroethanol indicated a tendency to form an alpha-helical structure similar to that found for native S-peptide. The model peptide formed a stable complex with ribonuclease S-protein. With 12-fold excess of the peptide, the complex exhibited 36% of the specific activity of fully native ribonuclease S against the substrate cyclic cytidine 2':3'-monophosphate at pH 7.15. The dissociation constant of the model peptide for S-protein was found to be 1.1 x 10(-6) M, compared with 0.1 x 10(-6) M for native S-peptide. Crystals grown of the model peptide-S-protein complex were found to be isomorphous with those of native complex. The activity, stability, and structural integrity of the model complex verify the deductions made about essential sequence information in the NH2-terminal region of ribonuclease. Further, the results emphasize the general usefulness of the conformational approach in designing simplified sequences for other peptides and proteins.
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PMID:Sequence modeling using semisynthetic ribonuclease S. 627 8

Insulin, ribonuclease, papain and collagen solutions saturated with nitrogen, N2O or air were irradiated with doses of 10 to 640 Gy of gamma rays. Protein solutions were also oxidized enzymatically in a system of horse-radish peroxidase: hydrogen peroxide. Column chromatography (Sephadex G-75 or Sephacryl S-200) of treated protein solutions revealed that they contain protein molecular aggregates. Nitrogen saturation of solution before irradiation was most favourable for radiation-induced aggregation of proteins. Fluorescence analysis of protein solutions resulted in detection of dityrosyl structures in irradiated as well as in enzymatically oxidized proteins. Concentration of dityrosine in proteins studied was determined fluorimetrically in their hydrolysates separated on BioGel P-2 column. In irradiated proteins, dityrosine was present almost exclusively in their aggregated forms. In proteins oxidized enzymatically, dityrosine was also present in fractions containing apparently unchanged protein. Mechanisms which could account for differences in the yield of dityrosine formation in radiolysis and in enzymatic oxidation of proteins are suggested.
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PMID:Radiolytic and enzymatic dimerization of tyrosyl residues in insulin, ribonuclease, papain and collagen. 633 34

Previous studies have described the isolation of mutationally altered proteases in Pseudomonas fragi (Noreau, J., and Drapeau, G.R. (1979) J. Bacteriol, 140, 911-916. In the present study, it is shown that one of these proteases cleaves specifically the peptide bonds on the NH2-terminal side of either aspartic acid or cysteic acid residues in oxidized ribonuclease. With myoglobin as the substrate, a similar specificity was observed except that only four out of the six aspartyl bonds present were hydrolyzed.
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PMID:Substrate specificity of a proteolytic enzyme isolated from a mutant of Pseudomonas fragi. 718 96


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