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)

Rabbit reticulocyte lysate cleaves the genome-linked protein VPg from foot-and-mouth disease virus (FMDV) RNA. This activity could be reliably monitored since removal of the protein resulted in a change in migration in polyacrylamide gels of the small specific 5' and fragment of the RNA (S fragment). The unlinking activity cleaved the bond between the tyrosine residue of VPg and the RNA to leave a 5' phosphate on the RNA. The 5' sequence of the RNA from which VPg had been removed by rabbit reticulocyte lysate was the same as that of FMDV mRNA isolated from infected cells. VPg released from the RNA was rapidly degraded by the rabbit reticulocyte lysate to material which eluted with the inclusion volume of a Sepharose 6B column and partitioned to the aqueous phase during phenol extraction. The unlinking activity was inhibited by heating the lysate to 56 degrees C, by sodium dodecyl sulfate (SDS), EDTA, and Zn2+ ions but was unaffected by reducing agents, a translation inhibitor, and a number of protease and RNase inhibitors.
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PMID:Removal of the genome-linked protein of foot-and-mouth disease virus by rabbit reticulocyte lysate. 626 21

The relative affinities of all Escherichia coli amino-acyl-tRNAs for E. coli elongation factor (EF) Tu-GTP have been measured by two independent applications of the competition form of the ribonuclease resistance assay. The set of aminoacyl-tRNAs includes at least one tRNA for each of the 20 amino acids as well as purified isoacceptor tRNA species for arginine, glycine, leucine, lysine, and tyrosine. In the first competition study, [3H]Phe-tRNA was used as the competing aminoacyl-tRNA against [14C]aminoacyl-tRNA in the set of all tRNAs; in the second study, [3H]Leu-tRNALeu4 was used as the competing aminoacyl-tRNA. The relative order of aminoacyl-tRNA affinities for EF-Tu-GTP was the same in each study. The results indicate that the affinity of EF-Tu-GTP at 4 degrees C, pH 7.4, is strongest for Gln-tRNA and weakest for Val-tRNA. Both Gly-tRNA and Pro-tRNA bind very strongly to EF-Tu-GTP relative to other aminoacyl-tRNAs. Various models of ternary complex interactions are discussed in light of the new data. Although the properties of the amino acid substituent are primarily responsible for the differences in relative affinities among the noninitiator aminoacyl-tRNAs, the results for the four isoacceptor species of Leu-tRNALeu indicate that the secondary structural features of the tRNA are also influential.
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PMID:Relative affinities of all Escherichia coli aminoacyl-tRNAs for elongation factor Tu-GTP. 637 Sep 98

L.-N. Lin and J.F. Brandts recently proposed a simple model for the folding kinetics of ribonuclease A in which folding intermediates are not detectable. We have tested the basic assumption of the simple model for the major unfolded species, which is produced by a slow isomerization (the "X in equilibrium Y reaction" according to Lin and Brandts) after unfolding. The simple model assumes that in refolding the slow Y----X reaction must occur before any folding can take place. We have measured the Y----X reaction during folding. Tyrosine-detected folding occurs before the Y----X reaction; the difference in rate between the Y----X reaction and folding monitored by tyrosine absorbance becomes large when the stabilizing salt 0.56 M (NH4)2SO4 is added. The simple model predicts that the kinetic properties of the X in equilibrium Y reaction in unfolded ribonuclease are the same as those of tyrosine-detected folding. We find, however, that the kinetics of the X in equilibrium Y reaction in unfolded ribonuclease are independent of urea concentration, whereas the rate of tyrosine-detected folding decreases almost 100-fold between 0.3 and 5 M urea, as reported by Lin and Brandts. We point out that the kinetic properties of the X in equilibrium Y reaction in unfolded ribonuclease are characteristic of proline isomerization.
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PMID:Tests of the simple model of Lin and Brandts for the folding kinetics of ribonuclease A. 646 45

Treatment of hydrochloric acid with sodium sulfite prior to the acid hydrolysis of bovine pancreatic ribonuclease A has been found to suppress the oxidation of cystine, methionine, and tyrosine without adversely affecting the recoveries of other amino acids. Statistical analysis of the results indicated that the assumption of the independence of the mean and the variance, an assumption commonly used in the evaluation of the effects of various treatments, may not be valid in evaluating antioxidants used in the acid hydrolysis of proteins.
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PMID:Sodium sulfite as an antioxidant in the acid hydrolysis of bovine pancreatic ribonuclease A. 649 47

Transfer RNA (tRNA) has been demonstrated to be present in axons of both invertebrates and the higher vertebrates, but nothing is known of its role in the metabolism of the axon. The present experiments were performed to determine whether tRNA functions in axons as a participant in post-translational protein modification of endogenous proteins. RNA was extracted from the axoplasm of squid giant axons and incubated with a variety of 3H-amino acids, aminoacyl-tRNA synthetases (obtained from squid optic lobe), and an appropriate reaction mixture. All of the amino acids tested were bound to an RNA fraction, but this reaction did not occur when samples were incubated in the presence of ribonuclease or in the absence of axoplasmic RNA. When radioactive RNA was chromatographed by polyacrylamide gel electrophoresis, the radioactivity comigrated with known tRNA markers, suggesting the presence of 3H-aminoacylated tRNA. Aminoacylation of RNA could also be demonstrated by incubating fresh axoplasm with labeled amino acids and a reaction mixture, minus exogenous aminoacyl-tRNA synthetases. These findings indicate the presence in axoplasm of a variety of species of aminoacyl-tRNAs as well as their corresponding synthetase enzymes. In the latter experiment no radioactivity was found associated with the protein fraction. This was also the finding when 3H-aminoacylated tRNA was either injected directly into the axon or incubated with extruded axoplasm. Thus, under the conditions described above, there is no evidence of transfer of amino acids from tRNA to proteins. In other experiments, axoplasm was pooled to a volume of 50 to 100 microliters, homogenized gently, and centrifuged at 150,000 X g for 1 hr. Some of the high speed supernatant was incubated with labeled amino acids and an appropriate reaction mixture, and the remainder was passed through an S-200 Sephacryl column before incubation with the same reaction mixture. There was no incorporation of amino acids into protein in the high speed supernatant fraction. However, in the S-200 purified fraction 3H-labeled Arg, Lys, Tyr, Leu, and Asp were all incorporated into proteins in amounts of 44, 30, 7, 5 and 3.5 times heat-inactivated controls. The reaction is not inhibited by Ca2+ or Ca2+-activated proteases, but appears to be dependent on the presence of tRNA. The addition of amino acids to protein is not protein synthesis since the reactions occurred in a partially purified fraction of the 150,000 X g supernatant, a fraction devoid of ribosomes and free amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Incorporation of 3H-amino acids into proteins in a partially purified fraction of axoplasm: evidence for transfer RNA-mediated, post-translational protein modification in squid giant axons. 655 12

The aromatic region of the NMR spectrum of bovine pancreatic ribonuclease A was analyzed in order to clarify the nature of the microenvironments surrounding the individual histidine, tyrosine, and phenylalanine residues and the interactions with inhibitors. The NMR titration curves of ring protons of six tyrosine and three phenylalanine residues as well as four histidine residues were determined at 37 degrees C between pH 1.5 and pH 11.5 under various conditions. The titration curves were analyzed on the basis of a scheme of a simple proton dissociation sequence and the most probable values were obtained for the macroscopic pK values and intrinsic chemical shifts. The microenvironments surrounding the residues and the effects of inhibitors are discussed on the basis of these results. Based on the titration curves of ring protons, the six tyrosine residues were classified into the following four groups: (1) titratable and different chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (2) titratable but similar chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (3) not titratable and different chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residues), and (4) not titratable and similar chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residue). The resonance signals of ring protons were tentatively assigned to tyrosine and phenylalanine residues. The NMR titration curves of His-48 ring protons were continuous in solution containing 0.2 M sodium acetate but were discontinuous in solution containing 0.3 M NaCl because the NMR signals disappeared at pH values between 5 and 6.5. The effects of addition of formate, acetate, propionate, and ethanol were investigated in order to elucidate the mechanism of the continuity of the titration curves of His-48 in the presence of acetate ion. The NMR signal of His-48 C(2) protons was observed at pH 6 in the presence of acetate and propionate ions but was not observed in the presence of formate ion or ethanol. This indicated that both the alkyl chain and the anionic carboxylate group are necessary for the continuity of the titration curves of His-48 ring protons. Based on the results, the mechanism of the effects of acetate ion is discussed.
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PMID:1H nuclear magnetic resonance titration curves and microenvironments of aromatic residues in bovine pancreatic ribonuclease A. 661 20

We have investigated the effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease. The sensitivity of nonglycosylated RNase A to trypsin and chymotrypsin was compared with three glycosylated species of RNase B which differed with respect to the size of the carbohydrate chain. Two forms of glycosylated RNase B were isolated by concanavalin A-Sepharose affinity chromatography, and each was shown to contain a single carbohydrate chain composed of GlcNAc2Man1 (RNase B") or GlcNAc2Man5-8 (RNase B). A third form (RNase B'), with oligosaccharide composed of GlcNAc2Man4, was prepared by partial digestion of RNase B with alpha-mannosidase. Fully glycosylated RNase B was found to be 6-10 times more resistant to trypsin digestion than nonglycosylated RNase A. RNase B' and B", with intermediate chain sizes, were 3.0- and 1.3-fold more resistant to trypsin digestion than RNase A, respectively. With chymotrypsin, however, differences in rates of digestion were much less marked, with a maximum difference of 3-fold between RNase A and B. In addition, we found that the specificity of the primary trypsin (Arg 33-Asp 34 bond) or chymotrypsin (Tyr 25-Cys 26 bond) cleavage site was not affected by the presence or size of the oligosaccharide chain. These results are consistent with the view that the size of the oligosaccharide chain and its proximity to the primary or rate-limiting cleavage site are important for expression of the carbohydrate protection against proteolytic degradation, which thus appears to be mediated by steric hindrance.
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PMID:Effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease. 663 Jan 85

The small positive ellipticity near 239 nm in the CD spectrum of RNase has been investigated as a function of pH. Theoretical calculations using CD parameters representing buried or exposed tyrosine residues have been carried out. A comparison of the theoretical calculations with experimental data suggests that the changes in the band's intensity, as a function of pH, arise mainly from electronic transitions associated with the tyrosine residues. The buried tyrosine residues are the major contributors to the ellipticity in this region at neutral pH. At higher pH contributions from exposed residues are also observed.
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PMID:A study on the positive ellipticity in the circular dichroism of ribonuclease A. 666 2

A ribonuclease (RNase) activity, RNase "XlaI," responsible for the excision of intervening sequences from two yeast transfer ribonucleic acid (tRNA) precursors, pre-tRNA(Tyr) and pre-tRNA(3Leu), has been purified 54-fold from nuclear extracts of Xenopus laevis oocytes. The RNase preparation is essentially free of contaminating RNase. A quantitative assay for RNase XlaI was developed, and the reaction products were characterized. RNase XlaI cleavage sites in the yeast tRNA precursors were identical to those made by yeast extracts (including 3'-phosphate and 5'-hydroxyl termini). Cleavage of pre-tRNA(3Leu) by RNase XlaI and subsequent ligation of the half-tRNA molecules do not require removal of the 5' leader or 3' trailer sequences.
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PMID:Ribonuclease "XlaI," an activity from Xenopus laevis oocytes that excises intervening sequences from yeast transfer ribonucleic acid precursors. 676 1

Exposure of ribonuclease (EC 3.1.27.5) to 5% trichloroacetic acid solution is found to partially inactivate the enzyme. This inactivation is a function of time of exposure to trichloroacetic acid and reaches a plateau of about 45% residual activity. Higher concentrations of trichloroacetic acid lead to greater inactivation. Physicochemical properties such as sedimentation coefficient, gel-filtration behaviour and polyacrylamide gel electrophoresis of the trichloroacetic acid-treated enzyme remain unaffected as compared to the untreated enzyme. However, spectrophotometric titration of the trichloroacetic acid-treated enzyme revealed that one of the three 'buried' groups of tyrosine is exposed to the outside surface of the molecule. Near ultraviolet CD spectra supported these observations. Far ultraviolet CD spectra suggested some refolding of the enzyme after trichloroacetic acid treatment. Immunological determinants on the molecule remain unaltered upon trichloroacetic acid treatment. It is concluded that the exposed tyrosine group may be causing a conformational change in the protein and this change may be indirectly responsible for the observed reduction in the activity after trichloroacetic acid treatment.
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PMID:Denaturation studies on bovine pancreatic ribonuclease. Effect of trichloroacetic acid. 683 Aug 11


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