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)

It is shown that circular dichroism (CD) can distinguish between the S-peptide and the S-protein fragments of RNase S at 225 nm and 235 nm. The conformational source for the strong CD at 225 nm is the S-peptide alpha-helix. The structural assignment of the CD at 235 nm is not clear but it is shown to be largely due to the S-protein moiety. This situation is utilized to monitor the kinetics of pH-induced unfolding and refolding of the two moieties. It is observed that major changes occur both in the fast and slow phases of unfolding as well as refolding. Specifically, the S-peptide alpha-helix unzippering is a fast reaction, followed by slow kinetics only at 235 nm. These latter kinetics parallel the appearance of the slow-folding species commonly attributed to the accumulation of non-native proline isomers. In refolding, a large fraction of the CD of S-protein at 235 nm recovers rapidly. The S-peptide alpha-helix zippers up last. These results are unexpected and their implications for the folding mechanism of ribonuclease are discussed.
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PMID:Kinetic circular dichroism shows that the S-peptide alpha-helix of ribonuclease S unfolds fast and refolds slowly. 659 55

The degree to which amino acid sequence can be simplified with retention of conformational and functional properties has been investigated by semisynthesis using non-covalent fragment complexes of bovine pancreatic ribonuclease as test cases. Based on the ribonuclease S system, a set of synthetic model sequences was defined for the S-peptide (1-20) region which interacted productively with native S-protein (21-124). The most simple sequence, an eicosapeptide containing helix-favoring Ala residues at all positions except Glu 1 and 14, Phe 8, His 12, and Met 13, effected at least 15% of ribonuclease catalytic activity (versus native ribonuclease S) when added to S-protein in saturating amounts. The data for model S-peptides define an alpha-helical framework and specific side chains at positions 8, 12, and 13 as the core of sequence information necessary for S-peptide to effect a productive non-covalent complex with S-protein. Previous ribonuclease fragment studies also were used as a basis for making the productive, non-overlapping complex, (1-15):(21-111):(116-124). Addition of synthetic (1-15) and (116-124) to (21-111) led to a 3 degrees increase in Tm and 4% (versus ribonuclease A) catalytic activity. The three-fragment complex, with the beta-bend residues 112-115 deleted, exhibited significantly lower stability to thermal denaturation than did related two-fragment complexes. The potential use of three-fragment complexes related to the above is discussed for semi-synthetic sequence modeling concomitantly in the N- and C-terminal regions of ribonuclease.
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PMID:Minimum information content and formation of interacting ribonuclease fragment complexes. 682 84

We have tested the feasibility of achieving protease-catalyzed condensation between nonassociating peptide fragments through mediation of a molecular trap. In this study, two subfragments of bovine pancreatic ribonuclease S-peptide, containing residues 1-10 and 11-15, were rejoined by clostripain catalysis to form the 1-15 peptide. The extent of this stereospecific condensation was enhanced by adding ribonuclease S-protein (residues 21-124), which acts as a trap in binding 1-15 but not 1-10 or 11-15 and which thus shifts the equilibrium to favor 1-15 formation. The resultant (1-15) X (21-124) noncovalent complex, defined as [des-16-20]ribonuclease S, was detected by the enzymatic activity characteristic of the naturally derived ribonuclease S complex. Reaction of 1 mM S-protein and 20 mM fragments leads to 80% of the ribonuclease activity expected from the amount of 21-124 present. This indicates that 4% of the fragments 1-10 and 11-15 were condensed, compared to a maximal condensation of 5% based on the amount of trap. The less than theoretical yield is due largely to slow proteolytic degradation of 21-124 to a form which is no longer able to bind the condensation product 1-15. Yields were increased to 15% by addition of further trap. The successful synthesis of 1-15 emphasizes the usefulness of molecular traps to promote stereospecific fragment condensation between nonassociating peptide fragments for the synthesis and semisynthesis of polypeptides.
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PMID:Enzymatic condensation of nonassociated peptide fragments using a molecular trap. 705 21

The degradation of S--S bonds in 0.2 M-NaOH at 25 degrees C was studied for a series of proteins and simple aliphatic disulphide compounds, by using cathodic stripping voltammetry, ion-selective-electrode potentiometry, spectrophotometry and ultrafiltration. The disulphide bonds that dissociated in 0.2 M-NaOH were usually those that are solvent accessible and that can be reduced by mild chemical reductants. Some unexpected differences were found between similar proteins, both in the number of S--S bonds dissociated and in their rates of decomposition. Chymotrypsin has one S--S bond attacked, whereas chymotrypsinogen and trypsinogen have two. Ribonuclease A has two S--S bonds dissociated, but ribonuclease S and S-protein have three. Denaturation in 6 M-guanidine hydrochloride before alkaline digestion caused the loss of an additional S--S bond in ribonuclease A and insulin, and increased the rate of dissociation of the S--S bonds of some other proteins. The initial product of S--S bond dissociation in dilute alkali is believed to be a persulphide intermediate formed by a beta-elimination reaction. This intermediate is in mobile equilibrium with bisulphide ion, HS-, and decomposes at a mercury electrode or in acid solution to yield a stoichiometric amount of sulphide. Rate constants and equilibrium constants were measured for the equilibria between HS- and the intermediates involved in the alkaline dissociation of several proteins. Elemental sulphur was not detected in any of the protein digests. It is suggested that formation of HS- from a persulphide intermediate involves a hydrolysis reaction to yield a sulphenic acid derivative. The small polypeptides glutathione and oxytocin gave only a low yield of persulphide, and their alkaline decomposition must proceed by a mechanism different from that of the proteins.
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PMID:Degradation of protein disulphide bonds in dilute alkali. 721 43

A hybrid RNase S', consisting of synthetic S-peptide of rat ribonuclease and bovine S-protein, was studied by 1H n.m.r. to determine the effects of the many N-terminal amino acid replacements in rat S-peptide as compared to bovine S-peptide. As judged from the aromatic resonances, the conformation of the hybrid RNase S' is essentially identical to the conformation of bovine RNase S'. Notably, the active-site histidines 12 and 119 were not affected by the substitutions in the S-peptide, confirming earlier findings that the catalytic properties of naturally occurring RNases are modulated by the S-protein part of the molecule only. However, the resonances of Tyr-25 and His-48, which in RNase A are involved in a pH-dependent conformational transition, appeared to be different in the hybrid RNase, demonstrating that amino acid replacements may influence the structure of the protein locally.
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PMID:Nuclear magnetic resonance study of a hybrid of bovine and rat ribonuclease. 744 54

Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisin-catalyzed semisynthesis starting from synthetic RNase 1-20 peptides and S-protein (RNase 21-124). The lysyl residue at position 1 was replaced by alanine, whereas Lys-7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl-3',5'-adenosine and yeast RNA. When Lys-7 was replaced by S-methyl-cysteine or S-carboxamido-contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A-RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys-1, Cys-7-methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol-1. Modifications that introduced a positive charge in position 7 (S-aminoethyl- or S-ethylpyridyl-cysteine) led to much smaller losses. The replacement of Lys-1 resulted in a 4-kJ mol-1 loss in binding energy. S-protein bound to RI with Ki = 63.4 pM, 800-fold weaker than RNase A. This corresponded to a 16-kJ mol-1 difference in binding energy. The results show that the N-terminal portion of RNase A contributes significantly to binding of ribonuclease inhibitor and that ionic interactions of Lys-7 and to a smaller extent of Lys-1 provide most of the binding energy.
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PMID:Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor. 800 61

Bovine seminal ribonuclease (BS-RNase) is an unusual homolog of RNase A. Isolated from bulls as a dimer, BS-RNase has special biological properties including antispermatogenic, antitumor and immunosuppressive activities. The structural bases for these properties are unknown. Four forms of BS-RNase were isolated after folding and air oxidation of the denatured and reduced protein produced in Escherichia coli: two dimers (M = M and M x I, where x signifies an active site composed of residues from both subunits) and two monomers (M and I). Considerable ribonuclease activity was generated by air oxidation of an equimolar mixture of two inactive mutant proteins ([H12D]BS-RNase and [H119D]BS-RNase) prepared by site-directed mutagenesis. This activity came from a dimer (M x I) with a composite active site. 1H-NMR spectroscopy revealed that this dimer contained one correctly folded subunit (M), and one incorrectly folded subunit (I). Form I, which is a poor catalyst, was activated by ribonuclease S-protein, suggesting that the C-terminal portion of I was not folded properly. Electrospray-ionization mass spectrometry and sulfhydryl group titration indicated that I contains a single oxidized sulfhydryl group, which cannot participate in a disulfide bond. These results show that quaternary structure in BS-RNase is attained by the initial formation of two monomers, M and I, which then combine with another M to form M = M and M x I, respectively. Adventitious oxidation can thus lead to the formation of a misfolded but active enzyme (M x I).
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PMID:A misfolded but active dimer of bovine seminal ribonuclease. 807 30

S-peptide (residues 1-20) and S-protein (residues 21-124) are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S-peptide binds S-protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S-peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a four-residue sequence recognized by factor Xa protease. The target was beta-galactosidase. The interaction between the S-peptide portion of the fusion protein and immobilized S-protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S-protein. S-peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (> or = 15 residues), a tunable affinity for ligand (Kd > or = 10(-9) M), and a high sensitivity of detection (> or = 10(-16) mol in a gel).
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PMID:Ribonuclease S-peptide as a carrier in fusion proteins. 845 73

From a filamentous phage library displaying random hexapeptides, we selected clones displaying peptides that bind S-protein, a 104-amino-acid (aa) fragment of bovine pancreatic ribonuclease (RNase). The selected peptides show a sequence motif, (F/Y)NF(E/V)(I/V)(L/V), that bears little resemblance to S-peptide, a 20-aa fragment of RNase that is S-protein's natural ligand. One of the displayed peptides, YNFEVL, was synthesized chemically and shown by isothermal titration calorimetry to bind S-protein with a dissociation equilibrium constant of 5.5 microM at 25 degrees C, an affinity comparable to that of previously studied S-peptide variants. The YNFEVL peptide is an antagonist of S-peptide, in that it blocks the ability of S-peptide to restore enzyme activity to S-protein. The S-protein/S-peptide system preserves the essential features of a pharmacologically significant receptor/hormone couple, and the S-peptide antagonist can therefore be regarded as a new RNase-specific 'drug'. This work illustrates the potential value of phage display libraries for discovering novel classes of pharmaceuticals.
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PMID:A ribonuclease S-peptide antagonist discovered with a bacteriophage display library. 850 58

In this paper the thermal denaturation of ribonuclease S, the product of mild digestion of ribonuclease A by subtilisin, is deeply investigated by means of DSC and CD measurements. It results that at whatever pH in the range 4-7.5 the process if fully reversible but not well represented by the simple two-state N<-->D transition. Actually, a two-state model that considers both unfolding and dissociation, NL<-->D + L*, well accounts for the main features of the process: the tail present in the low-temperature side of DSC peaks and the marked dependence of denaturation temperature on protein concentration. This mechanism is strictly linked to the exact stoichiometry of RNase S. An excess of the protein component of RNase S, the so-called S-protein, shifts the system toward a more complex behavior, that deserves a separate treatment in the accompanying paper [Graziano, G., Catanzano, F., Giancola, C., & Barone, G. (1996) Biochemistry 35, 13386-13392]. The thermodynamic analysis leads to the conclusion that the difference in thermal stability between RNase S and RNase A is due to entropic effects, i.e., a greater conformational flexibility of both backbone and side chains in RNase S. The process becomes irreversible at pH 8.0-8.5, probably due to side-reactions occurring at high temperature. Finally, the influence of phosphate ion on the stability of RNase A and RNase S at pH 7.0 is studied and explained in terms of its binding on the active site of ribonuclease. The analysis enables us to obtain an estimate of the apparent association constant and binding enthalpy also.
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PMID:Temperature-induced denaturation of ribonuclease S: a thermodynamic study. 887 5


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