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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A unique DNA-binding protein was detected that inhibited DNA degradation induced by bleomycin and was decreased in sera of cancer patients. The protein from normal human serum was purified to homogeneity by ammonium sulfate precipitation and DEAE-cellulose and DNA-cellulose column chromatography. Two-dimensional isoelectric focusing gel electrophoresis revealed a single protein spot with a molecular weight of 64,000 and a pI at pH 5.9. The NH2 terminus was lysine, and the ratio of acidic to basic residues was 1.2. DNA binding was demonstrated by column chromatography, agarose gel electrophoresis, fluorescence quenching, and circular dichroism. The inhibitory activity was abolished by treatment with Pronase but not by RNase or DNase I. FeCl2 caused a partial loss of inhibitory activity. The inhibition of DNA degradation was more effective for breakage induced by bleomycin than neocarzinostatin, macromomycin, or DNase I. Evidence from DNA-binding studies suggests the inhibition is due to binding of the protein to sites on DNA preferred by bleomycin. Thus, the protein could be useful for studies on the mechanism of action of bleomycin and other antitumor agents, the cytotoxic effects of which are due primarily to damage of cellular DNA. The protein was decreased significantly in sera of cancer patients, and its potential use as a diagnostic tool for neoplasias is being investigated further.
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PMID:Inhibition of PM-2 DNA degradation by a human serum protein. 617 27

The guanidinium chloride-unfolded state of ribonuclease A was found to be an equilibrium mixture of slow- and fast-refolding forms of the protein chain, as has been suggested. Both forms appear to have the same spectroscopic observables as judged by the relative changes in fluorescence emission and polarization. The equilibrium between them is thermally dependent, with deltaHapp equal to -1.4 kcal/mol. The activation energy Ea is equal to 18 kcal/mol. These findings are consistent with the proposal that cis-trans isomerism of peptide bonds that are NH2-terminal to proline residues is responsible for the slow phase of RNase A refolding. However, the actual dependence of the magnitude of the slow reaction on initial, prefolding temperature cannot be explained by a model in which the proline configurations of the fast refolding form must be identical to those of the native protein, as has been suggested. Instead, the data reveal that, although the native structure of RNase A contains two cis prolines, cis isomers need not be present in the fast-refolding form in order for folding to occur.
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PMID:Proline isomerism in the protein folding of ribonuclease A. 624 43

3-N-Carboxymethyl-His-12 and 1-N-carboxymethyl-His-119-RNase A bind to the naturally occurring RNase inhibitor, isolated from human placenta, 1.3 and 3.6 times, respectively, more strongly than does native RNase A. Near-ultraviolet circular dichroism measurements indicate that the conformational change which occurs upon carboxymethylation of either of the active site histidine residues appears different from that which the protein undergoes upon binding of substrate of a substrate analogue. Specific carboxymethylation of Lys-41 of RNase A decreased the strength of the interaction between the enzyme and the RNase inhibitor to about 12% of the initial value. The near-UV CD spectra of Cm-Lys-41-RNase A and of acetimidyl-RNase A (9.3 lysines modified) and carbamylated RNase A (3.0 lysines modified), which also have weaker interactions with RNase inhibitor of 25% and 10%, respectively, show a negative [theta]MRW identical to that of native RNase A at 275 nm but are altered in the positive [theta]MRW at 240 nm. The CD measurements suggest that one or more tyrosine residues of RNase A may be involved in the interaction with inhibitor. The effects of pH and salt concentration suggest that a major part of the protein-protein interaction is probably through nonpolar forces. The strengths of interactions between the inhibitor and pancreatic RNases from several species were very similar. Since Tyr-92 is the only tyrosine residue retained in all of the species studied, this residue may have a key role in the nonpolar interaction. The data presented herein suggest that the interaction between RNase A and the inhibitor involves the positively charged epsilon-NH2 group of Lys-41 of RNase A. This interaction could result in the inactivation of the enzyme.
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PMID:The role of lysine-41 of ribonuclease A in the interaction with RNase inhibitor from human placenta. 625 94

The n.m.r. spectra of native S-peptide and of S-peptide II, a derivative obtained after reaction of bovine pancreatic ribonuclease A with 6-chloropurine riboside 5'-monophosphate, both in D2O and in urea-d4, were obtained with a 270 MHz Fourier transform spectrometer. From these spectra it was possible to assign most of the proton resonances of the peptide and the position of the labelling group, the alpha-NH2 of Lys-1, was also deduced.
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PMID:Reaction of bovine pancreatic ribonuclease A with 6-chloropurine riboside 5'-monophosphate. Nuclear magnetic resonance studies of the corresponding S-peptide. 625 20

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

C-peptide, which contains the 13 NH2-terminal residues of RNase A, shows partial helix formation in water at low temperature (1 degree C, pH 5, 0.1 M NaCl), as judged by CD spectra; the helix is formed intramolecularly [Brown, J. E. & Klee, W. A. (1971) Biochemistry 10, 470-476]. We find that helix stability depends strongly on pH: both a protonated histidine (residue 12) and a deprotonated glutamate (residue 9 or 2 or both) are required for optimal stability. This information, together with model building, suggests that the salt bridge Glu-9- ... His-12+ stabilizes the helix. Formation of the helix is enthalpy driven [van't Hoff delta H, - 16Kcal/mol (1 cal = 4.18 J)] and the helix is not observed above 30 degrees C. Proton NMR data indicate that several side chains adopt specific conformations as the helix is formed. These results have two implications for the mechanism of protein folding. First, they indicate that short alpha-helices, stabilized by specific side-chain interactions within the helix, can be stable enough in water to function as folding intermediates. Second, they suggest that similar experiments with peptides of controlled amino acid sequence could be used to catalogue the intrahelix interactions that stabilize or destabilize alpha-helices in aqueous solution. These data might provide the code relating amino acid sequence to the locations of alpha-helices in proteins.
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PMID:A salt bridge stabilizes the helix formed by isolated C-peptide of RNase A. 628 28

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

A complex of RNase A with a transition-state analog, uridine vanadate, has been studied by a combination of neutron and x-ray diffraction. The vanadium atom occupies the center of a distorted trigonal bipyramid, with the ribose oxygen O2' at the apical position. Contrary to expectations based on the straightforward interpretation of the known in-line mechanism of action of RNase, nitrogen NE2 of histidine-12 was found to form a hydrogen bond to the equatorial oxygen O8, while nitrogen NZ of lysine-41 makes a clear hydrogen bond to the apical oxygen O2'. Nitrogen ND1 of histidine-119 appears to be within a hydrogen-bond distance of the other apical oxygen, O7. Two other hydrogen bonds between the vanadate and the protein are made by nitrogen NE2 of glutamine-11 and by the amide nitrogen of phenylalanine-120. The observed geometry of the complex may necessitate reinterpretation of the mechanism of action of RNase.
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PMID:Active site of RNase: neutron diffraction study of a complex with uridine vanadate, a transition-state analog. 657 1

Dimers of bovine pancreatic RNase A give nonhyperbolic saturation curves for the substrate of the second, rate-limiting step of the reaction. Under the same conditions, the monomeric native enzyme shows Michaelis-Menten kinetics. Naturally dimeric bovine seminal RNase, which has been found to give nonhyperbolic saturation curves, loses this property upon monomerization. It is proposed that when RNase monomers are arranged in a quaternary structure, they assume a conformation which enables them to be modulated in their catalytic activities. A correlation is suggested between this effect and the quaternary structure proposed for both of these dimeric ribonucleases, in which composite active sites are generated by the mutual exchange of the NH2-terminal ends of the two monomers.
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PMID:Relationships between nonhyperbolic kinetics and dimeric structure in ribonucleases. 669 91

The complex of elongation factor Tu with GTP (EF-Tu.GTP) reacts with N or epsilon -bromoacetyl-lys-tRNA ( or epsilon BrAcLys-tRNA) to form a functional covalently linked complex (XLTC). The site of cross-linking must be near the site on EF-Tu.GTP that binds the aminoacyl moiety of aminoacyl transfer ribonucleic acid (AA-tRNA). For identification of this site, a nanomole of purified XLTC prepared from or epsilon BrAc[(14)C]Lys-tRNA was digested first with RNase A and then with trypsin, and the peptides were resolved by high-performance liquid chromatography using a c8 reverse-phase column. A single peptide contained 80% of the label. The amino acid composition of this peptide was identical with that of residues 59-74 in EF-Tu. The NH2-terminal sequence of the peptide was determined to be Fly-Ile-Thr-Ile, which are residues 59-62 in EF-Tu. The modified amino acid was identified as pi - (carboxymethyl)histidine, which establishes that His-66 is at or near the AA-tRNA binding site on EF-Tu.GTP.
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PMID:Identification of a histidine residue near the aminoacyl transfer ribonucleic acid binding site of elongation factor Tu. 691 6


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