Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Unfolded ribonuclease A consists of a mixture of fast- and slow-refolding species. It is generally accepted that the slow-refolding states arise from isomerization of proline residues. We show that unfolding at subzero temperatures may be used to trap the fast-refolding species Uf, since the rate of proline isomerization slows down at a much faster rate than protein unfolding. The unfolding was carried out in 5 M guanidine hydrochloride; at -15 degrees C the protein unfolding process is complete within 30 s and under these conditions there is less than 1.5% proline isomerization. By using ribonuclease in which Tyr-115 was nitrated it was possible to rule out significant isomerization of Pro-114 in the observed slow-unfolding step.
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PMID:Trapping the fast-refolding state of ribonuclease A at subzero temperatures. 334 33

The effect of methanol on the thermal denaturation of ribonuclease A has been investigated over the -40 to 70 degrees C range. The transition was fully reversible to at least 60% (v/v) methanol at an apparent pH of cryosolvent (pH) of 3.0 and was examined at methanol concentrations as high as 80%. The unfolding transition, as monitored by absorbance change at 286 nm, became progressively broader and occurred at increasingly lower temperatures as the alcohol concentration increased. In 50% methanol, increasing the pH from 2 to 6 shifted the transition to higher temperature. A substantial decrease in cooperativity was noted at the more acidic conditions. On the other hand, increasing concentrations of guanidine hydrochloride in 50% methanol caused the transition to shift to lower temperatures with little effect on the cooperativity. The observed effects on the cooperativity of the unfolding transition suggest that methanol and lower temperatures may increase the concentration of partially folded intermediate states in the unfolding of ribonuclease. Comparison of the transition in 50% methanol as determined by absorbance or fluorescence, which monitor the degree of exposure of buried tyrosines and hence the tertiary structure, to that determined by far-UV circular dichroism, which monitors secondary structure, indicated that the major unfolding transition occurred at a higher temperature in the latter case. Thus, the tertiary structure is lost at a lower temperature than the secondary structure. This observation is consistent with a model of protein folding in which initially formed regions of secondary structure pack together, predominantly by hydrophobic interactions, to give the tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the unfolding of ribonuclease A in aqueous methanol solvents. 359 84

A specific high-affinity folate binding protein (FBP) that binds folic acid and folic acid derivatives and that was previously identified in porcine kidney has been purified 50,000-fold using the technique of affinity chromatography. The FBP had a molecular weight of 38,500 daltons and did not appear to aggregate in solution, as has been reported to be the case with folate binding protein from milk. At pH 7.6, the Ka was at least 5 X 10(12)M-1. At pH values greater than 9.5 or less than 5, the binding dramatically decreased. The specificity was determined by an isotopic dilution technique using [3H]folic acid and folic acid analogs and derivatives. The FBP reacted more rapidly with unsubstituted folates, and the number of glutamic acid moieties (N greater than or equal to 1) did not influence binding. Binding of folic acid to the FBP was unaffected by a variety of anions and cations, and 8 M urea, but was disrupted by 6 M guanidine hydrochloride. Proteolytic enzymes irreversibly destroyed binding affinity, but RNase, DNase, phospholipase and neuraminidase had no effect.
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PMID:Properties of a folate binding protein (FBP) isolated from porcine kidney. 372 88

A macromolecule binding 3H-methylcholanthrene (3H-MCA) and 3H-benzo(a)pyrene (3H-BaP) and sedimenting in the 4-5 S region of sucrose gradient (4.5 S) was identified in rat liver cytosol. The binding was displaced by 100-fold molar excess unlabeled ligands whereas 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was ineffective. The dissociation constant for both polycyclic aromatic hydrocarbons (PAHs) was of the order of 10(-8) M or lower. Both 3H-MCA and 3H-BaP bound to 4.5 S in a non covalent manner, since 92% of the bound radioactivity was extractable with ethyl ether. Furthermore the binding was strongly reduced by urea 8 M and by guanidine. HCl 4 M (99 and 70% respectively). Thin layer chromatography of the ethyl ether-solubilized radioactivity showed a peak comigrating with PAHs used as standards. When chromatographed on Sephadex G-200, 4.5 S was eluted as a sharp peak with an apparent molecular weight of 50-60,000 daltons. Enzyme treatment of liver cytosol showed that the 4.5 S binding sites were destroyed by micrococcal nuclease (92% of inhibition). Papain and phosphodiesterase I and II reduced the binding to 50%, whereas DNase I, DNase II, RNase, phospholipase A2 and C and trypsin were ineffective. These data suggest that the PAHs binding macromolecule of rat liver cytosol is a protein associated with a polynucleotide. The binding of both PAHs was enhanced by increasing the incubation temperature, the maximum being reached after 20-30 min at 37 degrees C. After 2.5 min at 65 degrees C, binding sites were completely destroyed. The same temperature-induced "activation" was obtained also by prewarming the cytosol at 37 degrees C in the absence of ligands.
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PMID:Polycyclic aromatic hydrocarbon binding macromolecules. Identification, characterization and temperature activation of a 4.5 S binding nucleoprotein. 406 Feb 44

Poliovirus-infected cells contain a previously unrecognized particle which appears to be an intermediate in virion synthesis and therefore has been named proviron. It sediments at about 125S, contains the three procapsid proteins, VP-0, VP-1, and VP-3, and has 35S viral RNA. It is disrupted both by sodium dodecyl sulfate and EDTA but the RNA resists digestion by ribonuclease. Pulsechase experiments and studies employing the virus-specific inhibitor, guanidine, all indicate that the proviron is formed by combination of newly made RNA with the procapsid. Cleavage of VP-0 to form VP-2 and VP-4 follows formation of the provirion and would be the final step in poliovirus morphogenesis.
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PMID:Morphogenesis of poliovirus. II. Demonstration of a new intermediate, the proviron. 435 64

GSH, but not GSSG, inhibits the reactivation by phosphate ion of ribonuclease activity inactivated by urea or guanidine. The effects of GSH are rather slow and pretreatment of ribonuclease with urea is a requisite for the inhibitory action of GSH on enzyme reactivation. GSH is more effective in urea than in guanidine and its action is greatly enhanced by EDTA. An optimum pH of about 9.0 was found for the inhibitory effect of GSH. Titration of the thiol groups formed after inactivation of ribonuclease by GSH strongly suggests that the reduction of only one disulphide linkage is involved. The reduction of this bond is sufficient to completely abolish the enzymic activity.
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PMID:Effect of glutathione on ribonuclease. 496 74

Specific antibodies to digoxin were isolated from antisera of sheep immunized with a digoxin-human serum albumin conjugate. The antibody was purified by adsorption to an immunoadsorbent, synthesized by coupling a ouabain-ribonuclease conjugate to bromoacetyl-cellulose, followed by elution with 25 mM ouabain. Ouabain was dissociated from antibody by denaturation in 6 M guanidine. The renatured antibody bound 1.6 mol of digoxin per mol and had an association constant of 1.6 x 10(8) M(-1). At near-stoichiometric concentrations, either purified antibody to digoxin, or its papain-digested product (Fab-Fc), reversed digoxin-induced: (a) inhibition of (86)Rb transport in human erythrocytes, (b) increase in developed tension in isolated guinea-pig atrial strips, and (c) ventricular tachycardia in intact dogs, and also corrected digoxin-induced automaticity in isolated guineapig atrial strips.
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PMID:The isolation of digoxin-specific antibody and its use in reversing the effects of digoxin. 528 75

The effect of guanidine on the replication of the group A arboviruses, Sindbis virus, and Semliki Forest virus (SFV) was studied. Guanidine rapidly, but reversibly, inhibited SFV ribonucleic acid (RNA) synthesis. The synthesis of all species of viral RNA was inhibited, but that of ribonuclease-resistant forms was least affected. This inhibition occurred when the drug was added at any point during the log phase of virus growth. The growth of SFV was also markedly inhibited, but Sindbis virus growth was unimpaired. Infection of guanidine-treated cells with the viruses together resulted in a significant inhibition of the yields of both. It appears that, in the case of Sindbis virus, viral RNA is ordinarily produced in such excess that inhibition of its synthesis does not reduce virus yields. In the case of SFV, guanidine also markedly distorts the pattern of RNA synthesis by greatly decreasing the production of the 26S interjacent RNA form. This may account for the observed inhibition of SFV growth in the presence of guanidine.
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PMID:Basis for variable response of arboviruses to guanidine treatment. 553 10

Earlier studies have indicated the marked resistance of two pronase endopeptidases to denaturation in high concentrations of urea or guanidine hydrochloride (Siegel, S., and Awad, W. M., Jr. (1973) J. Biol. Chem. 248, 3233--3240). One component has only a single residue of lysine and the other has none. The consideration arose that lysine-containing peptide segments may be less stable than those containing arginine because of the fluctuations of the side groups of the former residue. The small epsilon amino groups may not be able to sustain solvation of the hydrophobic arm in an aqueous medium. Arginine residues have shorter hydrophobic arms, larger hydrophilic groups, and higher pKa values and, thus may be less motile than lysine. The hypothesis was tested by guanidination of seven globular proteins (bovine carbonic anhydrase, chymotrypsinogen, alpha-lactalbumin, serum albumin, ribonuclease, hen egg lysozyme, and horse heart cytochrome c). Conversion of lysine residues to homoarginine was between 90 and 99%. Tritium-hydrogen isotope exchange revealed that all proteins except lysozyme demonstrated reduced out-exchange after guanidination. The results with lysozyme were not unexpected since only this protein has a high arginine to lysine ratio. These findings suggest that high arginine to lysine ratios contribute to protein stability.
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PMID:Stabilization of proteins by guanidination. 625 87

The reaction of ribonuclease (RNase) with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (1,5-IAENS) yields a derivative in which one fluorescent group is covalently attached to the protein. Several arguments suggest that the chemical modification has occurred at the enzyme active site: (i) 1,5-IAENS should have the same specificity as iodoacetamide, i.e., carboxymethylate one histidine of the active site; (ii) the derivatized protein is enzymatically inactive; (iii) in the native state of the protein, the fluorescent group is (almost) completely protected from the aqueous solvent; (iv) this group has no motions other than those of the protein. The fluorescence properties of the derivatized RNase change markedly upon unfolding induced by guanidine hydrochloride (Gdn . HCl), as seen from fluorescence intensity, maximum emission wavelength, and polarization measurements. Upon Gdn . HCl-induced unfolding, the fluorescent group is transferred from a nonpolar to a highly polar environment. Dynamic fluorescence measurements show also that unfolding results in a markedly increased mobility of the fluorescent label with respect to its proteic environment. These results are compared to those of Young & Potts (1963) [Young, D. M., & Potts, J. T. (1963) J. Biol. Chem. 238, 1995--2002], who studied the fluorescence properties of a surface-labeled derivative of RNase.
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PMID:Fluorescent probe of ribonuclease A conformation. 627 83


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