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)

In a study of factors that influence the remaining secondary structure of reduced chicken eggwhite lysozyme, N-acetyl-D-glucosamine (NAG) and N,N'-diacetylchitobiose (di-NAG) were found to alter the circular dichroic (CD) spectrum of the reduced protein and its carboxymethyl derivative (Cml). Thus, negative ellipticities in the far u.v. were greater in the presence of the analogs, with NAG being the more effective. For Cml, curve fitting analysis of the CD data indicated an increased helical content in the presence of NAG by an average of 3% of the chain length, while beta-structure decreased by an equivalent amount. Other compounds structurally related to NAG produced no similar effects on the CD spectrum of Cml, nor were comparable effects of NAG in evidence on the Cm reduced derivatives of ribonuclease, chymotrypsin, wheat germ agglutinin, or alpha-lactalbumin. The effect therefore appears specific between NAG and Cml. Conversion of the tryptophan residue at Position 62 of Cml to the oxindolealanyl derivative prevented these effects of NAG, and this residue may therefore participate in the interaction. During a 4-day incubation at room temperature, the analog preserved the CD spectrum of Cml as well as its concentration. This effect was nearly specific when compared with other Cm reduced proteins and with other carbohydrates. Only one, N-acetyl mannosamine, was effective in preserving the concentration of Cml, but not the CD spectrum. Since D-glucosamine was entirely without effect on either the CD spectrum of Cml or on its change during incubation, the acetyl group appears essential for the NAG-Cml interaction. The specificity between NAG and Cml is tentatively accounted for in terms of interactions with the primary structure, rather than with the remaining secondary structure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Evidence for interaction of substrate analogs with chicken eggwhite lysozyme after exhaustive reduction of disulfide bonds. 651 17

A temperature-sensitive (t.s.) tRNATrp from Escherichia coli has a single base change from the wild type (w.t.) species, which results in the loss of a base pair at the bottom of the CCA stem of the cloverleaf structure. Thermodynamic studies on this t.s. tRNA show that it is more susceptible to denaturation than the w.t. due to a larger change in the entropy of denaturation. Correlated with this thermodynamic result is the finding that the denatured t.s. tRNA's T psi C loop is more susceptible to digestion by T1 RNase, suggesting that it has greater freedom than the corresponding structure on the denatured w.t. molecule. In contrast, the native form of the t.s. tRNATrp is very similar to the w.t. with regard to aminoacylation, T1 RNase susceptibility, and column chromatographic mobility, despite the fact that it necessarily has one less base pair. In addition, the well known denaturation-dependent shift in column chromatographic mobility, which is observed for both the t.s. and w.t. molecules, depends on a modification in the anticodon loop, since tRNATrp lacking that modification does not shift when denatured. Thus, though it is not usually thought to be implicated, denaturation probably affects the conformation of the anticodon loop. The lethal phenotype of the mutant at high temperature, defective attenuation of the tryptophan biosynthetic operon in the mutant, and some aspects of the denatured state are clarified by these findings.
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PMID:The structure and aminoacylation of a temperature-sensitive tRNATrp (Escherichia coli). 676 36

Cetyltrimethylammonium bromide (CTAB) modified tobacco mosaic virus (TMV) virions so that the intrinsic fluorescence changed, viral infectivity decreased, sensitivity to RNase or UV irradiation increased, and coat protein subunits were released by the addition of Triton X-100. The change in fluorescence emission at 320 nm shifted to 340 nm was observed at 100 micrograms of CTAB per ml. This represents a change in the tryptophan environment inside the virion. At a lower concentration of CTAB, intersubunit contact was weakened, resulting in the release of coat protein subunits and an increase in RNase sensitivity. The release of coat protein took place gradually and two relatively stable intermediates were observed. Increase in UV sensitivity was observed at a lower concentration of CTAB and formation of pyrimidine hydrate was involved in this inactivation. The nature of the minor structural change leading to UV inactivation is discussed.
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PMID:Sensitivity to ultraviolet light of tobacco mosaic virus modified by cetyltrimethylammonium bromide. 732 41

The reaction of L-3a-hydroxy-1,2,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (Hpi) with methanethiol, ethanethiol, mercaptoethanol or 3-mercaptopropionic acid in warm aqueous acetic acid gives the corresponding 2-thioether derivatives of tryptophan in 50--80% yield (based on Hpi). Better yields may be obtained in 25% trifluoroacetic acid at room temperature. Cysteine reacts with Hpi to give the double amino acid 2-(L-3-alanylthio)-L-tryptophan (tryptathionine), which is a constituent of the highly poisonous cyclopeptides of Amanita phalloides, such as phalloidin. Reaction of a moderate excess of Hpi with cysteine-SH groups of a tripeptide (glutathione) and a protein (reduced ribonuclease) has also been effected, giving the respective S-tryptophanylated peptide or protein. In both cases, reaction occurred specifically with the -SH groups of cysteine and virtually quantitative covalent binding of tryptophan was verified. The extent of the reaction is easily quantitated by spectrophotometry or by amino acid analysis of the content of oxindolylalanine in the hydrolysate with hot 3-N p-toluenesulfonic acid of the S-tryptophanylated peptide or protein. The reaction should be useful in the field of peptide synthesis, providing a simple method for establishing a cross-link between tryptophan and cysteine, as a basic step in the chemical synthesis of toxic peptides of Amanita phalloides.
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PMID:A novel synthesis of 2-thioether derivatives of tryptophan. Covalent binding of tryptophan to cysteine sulfhydryl groups in peptides and proteins. 737 2

Tryptophan residues in ribonuclease from a Rhizopus sp. (RNase Rh) were modified by NBS, H2O2-dioxane, o-nitrophenylsulfenyl chloride (NPS-Cl) and the relation between the extent of modification and enzymatic activity was studied in each case. By extrapolation of the modified tryptophan residue-enzymatic activity curve to a completely inactive state, it was found that modification of 1-2 tryptophan residues is responsible for loss of enzymatic activity. RNase Rh was partly protected from modification by H2O2-dioxane (pH 8.4) and NPS-Cl (pH 3.5) when in the presence of 2'-AMP and the fluorescence emission spectrum of RNase Rh was quenched by adding 2'-AMP. It seems, therefore, that 1 or 2 tryptophan residues are involved in the active site of RNase Rh or are located near the active site. The solvent perturbation difference spectra of RNase Rh were measured using ethylene glycol and D2O as perturbants. The results indicated that 1.2 tryptophan residues for D2O and 1.9 tryptophan residues for ethylene glycol were exposed to the solvents. These data show that about 1.2-1.9 tryptophan residues are exposed to the solvent and their modification causes loss in enzymatic activity.
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PMID:Chemical modification of tryptophan residues in ribonuclease from a Rhizopus sp. 739 Sep 80

Histone H1 contains only one tyrosine and no tryptophan. The intrinsic fluorescence of the tyrosine rises by about 400% as the protein folds from a random coil to a globular structure (Giancotti, V., Fonda, M. and Crane-Robinson, C. (1977) Biophys. Chem. 6, 379-383). Measurements of external quenching by a large variety of quenchers shows very much reduced quenching in the folded state as compared to the disordered. It is concluded that the tyrosine is a buried residue. This is supported by the observation that the fluorescence of modified amino-tyrosyl H1 is similar to that of buried tyrosines in ribonuclease. The classification of tyrosine fluorescence in tryptophan-free proteins (Cowgill, R.W. (1976) in Biochemical Fluorescence Concepts, Vol. 2 to include the case of residues buried in a hydrophobic environment and having a relative quantum yield RTyr, greater than unity.
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PMID:Fluorescence of buried tyrosine residues in proteins. 740 44

Two components having ribonuclease (EC 3.1.27.5) activity were isolated from human milk. Each component of human milk ribonuclease (RNAase) moved at a slightly different rate when electrophoresed on polyacrylamide gel but at the same rate when ultracentrifuged. The major component had a molecular weight of approx. 14 000, an isoelectric point of pH 7.9, and exhibited a broad absorbance maximum between 277 and 281 nm. Human milk RNAase hydrolyzed yeast RNA, poly(cytidylic acid) and poly(uridylic acid) but not DNA, poly(adenylic acid) or poly(guanylic acid). Maximum activity occurred at pH 7.7 and 60 degrees C. Amino acid analysis of the major component revealed a large number of alanine, valine, glycine and aspartic acids but no tryptophan or free sulfhydryl groups. Lysine was the N-terminal amino acid. Tryptic hydrolysis yielded 18 peptides, some of which are similar to those from bovine pancreatic RNAase. Human milk RNAase activity was increased in the presence of NaCl, KCl and sodium citrate and decreased by CaCl(2), MgCl(2), FeSO(4), ZnSO(4) and CuSO(4).
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PMID:Human milk ribonuclease. 741 55

We have examined the equilibrium unfolding of Escherichia coli ribonuclease HI (RNase H), a member of a family of enzymes that cleaves RNA from RNA:DNA hybrids. A completely synthetic gene was constructed that expresses a variant of the wild-type sequence with all 3 cysteines replaced with alanine. The resulting recombinant protein is active and folds reversibly. Denaturation studies monitored by circular dichroism and tryptophan fluorescence yield coincident curves that suggest the equilibrium unfolding reaction is a 2-state process. Acid denaturation, however, reveals a cooperative transition at approximately pH 1.8 to a partially folded state. This acid state can be further denatured in a reversible manner by the addition of heat or urea as monitored by either CD or tryptophan fluorescence. Analytical ultracentrifugation studies indicate that the acid state of RNase H is both compact and monomeric. Although compact, the acid state does not resemble the native protein: the acid state displays a near-UV CD spectrum similar to the unfolded state and binds to and enhances the fluorescence of the dye 1-anilinonaphthalene, 8-sulfonate much more than either the native or unfolded states. Therefore, the acid state of E. coli RNase H has the characteristics of a molten globule: it retains a high degree of secondary structure, remains compact, yet does not appear to contain a tightly packed core.
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PMID:Equilibrium unfolding of Escherichia coli ribonuclease H: characterization of a partially folded state. 783 2

Fluorescence techniques have been used to investigate the interaction of bovine 70-kDa heat shock cognate protein (Hsc 70) with small molecular weight peptides and myo-inositol monophosphatase. The emission properties of Hsc 70 remain invariant upon addition of ATP. The results of steady-state fluorescence indicate that the tryptophan residues of Hsc 70 are exposed to the rapidly relaxing aqueous solvent. Binding of residues 1-20 of ribonuclease A (RNase S-peptide) to Hsc 70 causes protein fluorescence quenching which was used to determine a dissociation constant Kd = 2.7 microM for the binary Hsc 70.RNase S-peptide complex. The octapeptide corresponding to the NH2-terminal portion of sickle cell hemoglobin recognizes Hsc 70 and binds with a Kd = 9.3 microM. Binding of RNase S-peptide to Hsc 70 produces a small enhancement of ATPase activity. Unfolded myo-inositol monophosphatase, tagged with the fluorescent probe 5-[2-(2-iodoacetamido)ethylamino]-1-naphthalenesulfonic acid, recognizes Hsc 70; the formation of a stable complex was detected by steady-state emission anisotropy measurements. The rate and extent of recovery of catalytic activity of unfolded myo-inositol monophosphatase is not influenced by Hsc 70. It is suggested that interaction of Hsc 70 with unfolded proteins in the cell may be able to delay the formation of misfolded structures.
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PMID:Interaction of 70-kDA heat shock cognate protein with peptides and myo-inositol monophosphatase. 827 5

Methotrexate (MTX) dose-escalation studies were conducted in two inbred lines of FVB/N transgenic mice expressing distinct drug-resistant dihydrofolate reductases (DHFRs) and in animals transplanted with transgenic marrow. Survival of animals expressing a tryptophan-31 variant DHFR transgene was only slightly improved over that of normal animals, and survival of tryptophan-31 variant DHFR marrow transplant recipients was indistinguishable from that of normal animals (at a MTX dose of 4 mg/kg i.p. daily). In contrast, extended survival was observed for animals expressing an arginine-22 variant (Arg22) DHFR transgene, with the last three of eight animals in this group succumbing at a final MTX dose of 14 mg/kg i.p. daily. Survival was slightly reduced for normal animals transplanted with Arg22 marrow. Interestingly, demise of animals in both Arg22 groups was not associated with the profound drop in hematocrit levels usually observed in MTX-treated animals. These animals were instead characterized by severe atrophy of the gastrointestinal tract, whereas hematocrit levels and marrow histology were relatively normal. Kidney pathology (mesangiocapillary glomerulopathy) was also observed in Arg22 marrow recipients but not in Arg22 transgenics, consistent with expression of the drug-resistance gene in kidney tissues of the transgenics, as demonstrated by ribonuclease protection analysis. Immediate dose-response studies in Arg22 marrow transplant recipients defined a maximum tolerated dose of 4 mg/kg/day MTX, 2 to 3 times that of animals transplanted with normal marrow or of normal untransplanted animals. These results define the extent of chemoprotection afforded by drug-resistant DHFR expression and serve to identify alternate sites of toxicity in animals administered the higher levels of MTX afforded by drug-resistant DHFR expression in the marrow.
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PMID:Methotrexate dose-escalation studies in transgenic mice and marrow transplant recipients expressing drug-resistant dihydrofolate reductase activity. 881 32


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