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:3.1.27.3 (RNase T1)
1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. 3'-Guanylyl-ethanol, 3'-guanylyl-propanol, and 3'-guanylyl-alpha-glycerol were synthesized by ribonuclease N1 [EC 3.1.4.8] using guanosine 2',3'-cyclic phosphate as a phosphate donor and various alcohols as phosphate acceptors. The yields of these phosphodiesters were 15%, 13.5%, 38.2%, respectively, with respect to phosphate donor under the optimum conditions. No phosphodiester was synthesized when 2-propanol was used as a phosphate acceptor. Thus, primary alcoholic hydroxyl groups may be regarded as the preferred phosphate acceptor. 2. 3'-Guanylyl-glucose and 3'-guanylyl-ribose were synthesized using glucose and ribose as phosphate acceptors. Under the optimum conditions, the yields of guanylyl-glucose amounted to 52.0%, while that of guanylyl-ribose was much lower. The guanylyl-glucose can be regarded as 3'-guanylyl-6-glucopyranose, based on the results of periodate oxidation. 3. Neither hydroxyamino acids (serine and threonine) nor N-acetylserinamide could be phosphorylated under the conditions used for the above phosphorylations. 4. 3'-Guanylyl-glycerol obtained as above was hydrolyzed by snake venon phosphodiesterase to produce glycerol 3-phosphate. The latter consisted of L-glycerol 3-phosphate (ca 17%) and the D-isomer (ca. 83%). Ribonuclease N1 thus catalyzes an asymmetric synthesis.
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PMID:Synthesis of various phosphodiesters and phosphomonoesters with ribonuclease N. 18 80

A poliovirus-specific polyuridylic acid [poly(U)] polymerase that copies a polyadenylic acid template complexed to an oligouridylic acid primer was isolated from the membrane fraction of infected HeLa cells and was found to sediment at 4 to 5S on a linear 5 to 20% glycerol gradient. When the poly(U) polymerase was isolated from cells labeled with [(35)S]methionine and was analyzed by glycerol gradient centrifugation and polyacrylamide gel electrophoresis, the position of only one viral protein was found to correlate with the location of enzyme activity. This protein had an apparent molecular weight of 62,500 based on its electrophoretic mobility relative to that of several molecular weight standards and was designated p63. When the poly(U) polymerase was isolated from the soluble fraction of a cytoplasmic extract, the activity was found to sediment at about 7S. In this case, however, both p63 and NCVP2 (77,000-dalton precursor of p63) cosedimented with the 7S activity peak. When the 7S polymerase activity was purified by phosphocellulose chromatography, both p63 and NCVP2 were found to co-chromatograph with poly(U) polymerase activity. The poliovirus replicase complexed with its endogenous RNA template was isolated from infected cells labeled with [(35)S]methionine and was centrifuged through a linear 15 to 30% glycerol gradient. The major viral polypeptide component in a 26S peak of replicase activity was p63, but small amounts of other poliovirus proteins were also present. When the replicase-template complex was treated with RNase T1 before centrifugation, a single peak of activity was found that sedimented at 20S and contained only labeled p63. Thus, p63 was found to be the only viral polypeptide in the replicase bound to its endogenous RNA template, and appears to be active as a poly(U) polymerase either as a monomer protein or as a 7S complex.
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PMID:Poliovirus polyuridylic acid polymerase and RNA replicase have the same viral polypeptide. 21 30

The structural regulation of the access of acrylamide molecules, as quenchers, to the buried tryptophans of a protein can be modelled by a simple gate concept. Such a gate, when open, allows transient exposure of the fluorophore to the quencher molecule in solution. We have previously shown that the observed viscosity dependence of acrylamide quenching process in ribonuclease T1 (RNAse T1) is not reconcilable with the gating mechanism. However, on that occasion, we neglected the effect of changes in the activity of the quencher molecule and the possible presence of static quenching. The experimental observation of a considerable contribution by static quenching and the realization that static quenching might produce dramatic effects in steady state measurements led us to reexamine the question. It is shown that in a gating model the static component can also influence the apparent dynamic quenching. In this paper, we present derived equations for the gated quenching mechanism including possible contributions from the static component. We also carefully remeasured the acrylamide quenching of RNAase T1 as a function of increasing glycerol concentration. Computer simulations were carried out to compare the experimental data set to the generalized model. We reach the conclusion that even the new, quite complex equations fail to predict the qualitative and quantitative features of the observed quenching experiments. We arrived at the conclusion that the fluorophore is never the target of the quencher molecules in solution.
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PMID:Viscosity dependence of acrylamide quenching of ribonuclease T1 fluorescence. The gating mechanism. 154 Jun 39

We report on the effect of the viscogenic agents glycerol and ficoll on the RNase T1 catalyzed turnover of GpA, GpC, GpU, and Torula yeast RNA. For wild-type enzyme, we find that the kcat/Km values for the transesterification of GpC and GpA as well as for the cleavage of RNA are inversely proportional to the relative viscosity of glycerol-containing buffers; no such effect is observed for the conversion of GpU to cGMP and U. The second-order rate constants for His40Ala and Glu46Ala RNase T1, two mutants with a drastically reduced kcat/km ratio, are independent of the microviscosity, indicating that glycerol does not affect the intrinsic kinetic parameters. Consistent with the notion that molecular diffusion rates are unaffected by polymeric viscogens, addition of ficoll has no effect on the kcat/Km for GpC transesterification by wild-type enzyme. The data indicate that the second-order rate constants for GpC, GpA, and Torula yeast RNA are at least partly limited by the diffusion-controlled association rate of substrate and active site; RNase T1 obeys Briggs-Haldane kinetics for these substrates (Km greater than Ks). Calculations suggest that the equilibrium dissociation constants (Ks) for the various GpN-wild-type enzyme complexes are virtually independent of N whereas the measured kcat values follow the order GpC greater than GpA greater than GpU. This is also revealed by the steady-state kinetic parameters of Tyr38Phe and His40Ala RNase T1, two mutants that follow simple Michaelis-Menten kinetics because of a dramatically reduced kcat value (i.e., Km = Ks).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Subsite interactions of ribonuclease T1: viscosity effects indicate that the rate-limiting step of GpN transesterification depends on the nature of N. 190 70

Various agents were tested for their effects on microbial proteases, which activity was monitored by the analysis of cleaved peptide bands in SDS-polyacrylamide gel electrophoresis. Using casein as a substrate, fungal protease (type XIX) was inhibited by the phenyl methyl sulphonyl fluoride, chymostatin, antipain and leupeptin, while bacterial protease (type XXVI) was inhibited by phosphatidyl glycerol, phosphatidyl inositol and sphingosine. MS2 RNA exerted minor inhibition on the bacterial proteolysis of regulatory subunits of cyclic AMP-dependent protein kinase (A-PK). The cleavage of DNA binding protein by both proteases was inhibited, in the presence of MS2 RNA and lambda DNA. In comparison, phosphatidyl serine slightly stimulated the fungal protease on the cleavage of ribonuclease T1. RNA polymerase is a good substrate of the bacterial protease as indicated by the generation of multiple cleaved peptide fragments, whereas alkaline phosphatase is not susceptible to proteolysis.
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PMID:A further study on the regulation of microbial proteases. 222 36

The individual spin-lattice relaxation (SLR) rate constants (Wij) between the lowest triplet sublevels of the lone tryptophan residue buried in the interior of the globular protein ribonuclease T1 have been reported in the temperature range 1.2 to 3.0 K in zero applied magnetic field. The SLR rate constants between spin sublevels exhibit marked anisotropy in their magnitudes and also show appreciable sensitivity to the glycerol content of the aqueous cryogenic matrix. The temperature dependence of SLR suggests that in the temperature range investigated a direct process contributes dominantly to the SLR in this protein.
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PMID:Spin-lattice relaxation in the triplet state of the buried tryptophan residue of ribonuclease T1. 308 81

We have studied the viscosity dependence of the acrylamide quenching of the fluorescence on the internal tryptophan residues in cod parvalbumin and ribonuclease T1, as well as the model systems. N-acetyl-L-tryptophanamide and glucagon. For the latter systems, the apparent rate constant, kq(app), for acrylamide quenching shows a typical diffusion-limited behavior. For parvalbumin and ribonuclease T1, however, the viscosity dependence of kq(app) is quite different. There is little change in the kq(app) values on increasing the bulk viscosity from 1 to 10 cP (by addition of glycerol), but a further increase from 10 to 100 cP results in a significant reduction in the kq(app). Both an unfolding mechanism and a quencher penetration mechanism are considered to explain the results. Only the penetration mechanism is found to be consistent, and our data are interpreted as indicating that the rate-limiting step for quenching goes from being that of diffusion through the protein matrix, at low viscosity, to diffusion through the bulk solvent, at high viscosity. By also considering the Kramers' relationship in fitting our data, we are able to obtain insight regarding the coupling between internal fluctuations in the structure of the protein and motion of the bulk solvent. For parvalbumin and ribonuclease T1, the internal dynamics are found to be very weakly coupled to the bulk.
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PMID:Viscosity dependence of the solute quenching of the tryptophanyl fluorescence of proteins. 310 4

3'-Terminal fragments present in a partial ribonuclease T1 digest of RNA 4 of alfalfa mosaic virus were selectively bound to virions of the same virus by incubation in 10 mM sodium phosphate and 1 mM EDTA, pH 7.0. The virions with the associated fragments were separated from the remainder of the digest by velocity gradient centrifugation. When the incubation was performed in 27.7 mM Na2HPO4 and 6.2 mM citric acid, pH 6.8, plus 10% glycerol, much more, but less specific, binding was obtained. However, in this case the terminal fragments present in unbound material of the digest were almost exclusively 5' termini. The four RNA species species of alfalfa mosaic virus have an extensive 3'-terminal homology. Therefore, it is expected that specific virion binding can be used to obtain 3'-terminal fragments of different lengths of the three genome RNA species (RNAs 1, 2, and 3) of alfalfa mosaic virus and, possibly, of related viruses. Such fragments are of great importance for the study of the specific 3'-terminal interaction with coat protein subunits which is thought to be responsible for the activation of the viral genome.
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PMID:Preferential binding of 3'-terminal fragments of alfalfa mosaic virus RNA 4 to virions. 677 2

Our recent equilibrium dialysis studies showed that proteins are able to interact preferentially with acrylamide (Punyiczki et al. (1993) Biophys. Chem. 47, 9-19). The presence of considerable amounts of acrylamide--albeit weakly bound--in the protein volume, coupled with the failure of a simple gating model of quenching to rationalise viscosity dependence of the quenching of tryptophan (Trp) fluorescence in Ribonuclease T1 (RNase T1) has prompted us to explore a new model, the two-phase model for quenching. According to this model, the dynamic quenching is accomplished by quencher molecules already in the protein phase at the moment of excitation. Some of the molecules may, at this moment, form an encounter complex with the fluorophore and thus be responsible for the observed static contribution. We use the rate equation derived from our model to study the viscosity dependence of acrylamide quenching of Trp fluorescence in RNase T1. The model allows us to separate co-solvent effects: the chemical effect on the protein and on the distribution of quencher molecules between the bulk and the protein phases and, further, the viscosity effect due to coupling between the bulk viscosity and the local friction affecting intramolecular fluctuations of the protein matrix. We express local friction in terms of bulk viscosity, eta, and a coupling constant kappa (friction = eta kappa). Addition of glycerol up to 65% is characterised by a kappa of 0.50. The viscosity dependence of the apparent bimolecular quenching constant is a combination of two compensating effects: changes in chemical activity and changes in patterns of structural fluctuations.
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PMID:Coupling between external viscosity and the intramolecular dynamics of ribonuclease T1: a two-phase model for the quenching of protein fluorescence. 794 83

Alterations in flexibility of monomeric proteins induced by hydrostatic pressure in the predenaturational range (< or = 3 kbar) were probed through the decay kinetics of tryptophan phosphorescence. With apoazurin, ribonuclease T1, wild-type and V67G mutant and phosphoglycerate kinase, pressure effects on the triplet lifetime (tau) and the amplitudes of multicomponent decays emphasize that subtle changes in conformation are ubiquitous. With apoazurin the increase in tau attests to a tightening of the protein core that is enhanced at high temperature. On the contrary, tau decreases with ribonuclease T1, wild-type and mutant, and with phosphoglycerate kinase, indicating that pressure induces a greater flexibility to protein regions in proximity to the surface of the macromolecule. For phosphoglycerate kinase the decrease in tau and the parallel increase in fluorescence intensity and red-shift of the fluorescence spectrum unveil an "unfolding" like transition with midpoint pressures of 1.1 kbar at 5 degrees C and 1.6 kbar at 25 degrees C. Evidence that unfolding of the C-domain of this protein is, however, less than complete is provided by a delta G zero that is about half of that obtained by denaturation in guanidine hydrochloride and also by the ability of this structure to undergo conformational drift. In 70% glycerol, pressure effects on tau of apoazurin are attenuated while for ribonuclease T1 there is a reversal of the tendency with a pronounced increase in tau. With phosphoglycerate kinase glycerol abolishes entirely the "unfolding" transition and all hysteresis effects. A consistent picture of these findings is provided in terms of the location of the probe and of the opposing effects that pressure exerts on protein flexibility by reducing internal cavities and increasing the hydration of the polypeptide.
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PMID:Pressure effects on protein flexibility monomeric proteins. 808 48


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