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)

Yukioka, M. (University of Hawaii, Honolulu), and T. Winnick. Synthesis of malformin by an enzyme preparation from Aspergillus niger. J. Bacteriol. 91:2237-2244. 1966.-An enzyme fraction derived from disrupted Aspergillus cells was able to utilize each of the component labeled amino acids of malformin for the synthesis of this cyclic pentapeptide. The process was stimulated by adenosine triphosphate, K(+), and Mg(++), and was optimal at approximately pH 8.5. It was not affected by inhibitors of protein synthesis (ribonuclease, chloramphenicol, puromycin). There is evidence that cysteine, rather than cystine, was incorporated into peptide linkage, so that the disulfide bridge of malformin was formed subsequently. Although only the d isomers of cysteine and leucine occur in the malformin molecule, the l, as well as the d form of these amino acids, was readily utilized by the enzyme preparation. As in the case of several other microbial peptide systems, it appears that the d enantiomorph can arise from the l isomer at an intermediate stage of polypeptide synthesis.
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PMID:Synthesis of malformin by an enzyme preparation from Aspergillus niger. 594 39

Reduced RNase A was reoxidized, and the incorrectly formed disulfide bonds were reshuffled to the native ones by oxidized and reduced glutathiones, as described in the first paper of this series. The intermediates in the regeneration of the disulfide bonds were trapped without any chemical modification and were fractionated on a carboxymethylcellulose column at pH 3.5 with a salt gradient. The elution curves of the partially regenerated RNase A from the carboxymethylcellulose column were obtained by measurement of the absorption at 275 nm and by determination of the SH content (of cysteine residues) and consisted of 11 fractions, G8, G7, G6, G5, G4, G3, G2, G1, G0, N, and F. Some of the fractions were isolated, and their measured molecular weights were consistent with those of monomeric RNase A. Fraction F had a molecular weight between that of the monomer and dimer, so that this fraction could not be identified. The regeneration pathway could be represented in terms of two simple reactions, RNase A(-SH) + GSSG in equilibrium or formed from RNase A(-SSG) + GSH and RNase A(-SH-SSG) in equilibrium RNase A(greater than S2) + GSH, which produced 24 monomeric intermediates (not counting the fully reduced and the native species), which differed from each other in their amino acid composition. These 24 intermediates, plus the fully reduced protein, were assigned to fractions G8--G0 (as indicated in the last column of Table I), with the aid of data from amino acid analysis, SH content, and the elution position on the carboxymethylcellulose column chromatogram. Since the regeneration reaction rapidly reached a preequilibrium among the intermediates and the fully reduced RNase A prior to the rate-limiting steps, i.e., the relative concentrations of the intermediates and fully reduced RNase A became constant with reaction time, the populations of some of the intermediates in preequilibrium were estimated by curve fitting of the elution pattern from the carboxymethylcellulose column chromatogram. The equilibrium constants among the intermediates were calculated from their populations at preequilibrium. These equilibrium constants were "extrapolated" to other intermediates whose populations could not be estimated by curve fitting, and the relative populations of all of the possible intermediates at preequilibrium were thereby represented as a function of the concentrations of reduced and oxidized glutathiones. The regeneration process was also restarted from several of the isolated intermediates, and the resulting distribution of intermediates was consistent with that from which the equilibrium constants were determined, supporting the representation of the regeneration pathways in terms of two simple reactions. Thus, the equilibrium treatment of the regeneration pathways was useful to characterize the preequilibrium state, i.e., to identify the intermediates prior to the rate-limiting steps in the pathways and to estimate their stabilities at preequilibrium at various concentrations of reduced and oxidized glutathiones.
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PMID:Regeneration of ribonuclease A from the reduced protein. Isolation and identification of intermediates, and equilibrium treatment. 626 76

Previous 77Se NMR relaxation time studies established the utility of 77Se NMR spectroscopy in studying low molecular weight (less than 500) selenium-containing molecules. Since the spin rotation and chemical shift anisotrophy mechanisms contributed significantly to the 77Se spin-lattice relaxation in these compounds, it was questionable as to whether the latter mechanism would be efficient enough to enable 77Se resonances to be observed in a reasonable period in high molecular weight selenobiomolecules. Thus, to address this problem, disulfide bonds of ribonuclease-A and lysozyme were reductively cleaved under denaturing conditions, and the resulting 7-8 sulfhydryl groups were treated with a new sulfhydryl group reagent containing selenium, 6,6'-diselenobis(3-nitrobenzoic acid), to give proteins containing covalently attached selenium in the form of selenenyl sulfides. The observation of high resolution 77Se NMR spectra of these proteins under denaturing conditions was accomplished. Five to six 77Se NMR resonances, which fell in a chemical shift range of 14-15 ppm, were observed for each protein and are compared to the chemical shifts of several model selenenyl sulfides derived from cysteine.
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PMID:Demonstration of the feasibility of observing nuclear magnetic resonance signals of 77Se covalently attached to proteins. 627 74

This paper is concerned with the pathways for the regeneration of RNase A from the reduced protein by a mixture of GSSG and GSH. Experimental work on the regeneration has led to the identification of several different pathways, depending on the concentrations of GSH and GSSG, and an energetic analysis has provided information about the stabilities of the various intermediates. The equilibrium and kinetic data for the regeneration process have led to two models of protein-folding pathways. The intermediates in the regeneration process were trapped without chemical modification, and were fractionated on a carboxymethyl-cellulose column. The regeneration pathway(s) could be represented in terms of two simple reactions (Eqs. (1) and (2)). The system rapidly reaches a pre-equilibrium among the intermediates prior to the rate-limiting steps, and the concentrations of the intermediates (and hence the equilibrium constants among them) were determined. The regeneration process was also re-started from several of the isolated intermediates, and led to the predicted distribution of intermediates in the pre-equilibrium. Kinetic data, obtained by following the time dependence of the regain of enzymatic activity, together with the distributions of the intermediates at pre-equilibrium, led to the identification of the rate limiting steps, which differed according to the concentrations of GSH and GSSG. The relative apparent standard state conformational chemical potentials of the intermediates were estimated by using data for the apparent equilibrium constants (among the species in pre-equilibrium) and for the redox potentials of cysteine/cystine and GSH/GSSG. The two models deduced from the equilibrium and kinetic data are designated as growth-type and rearrangement-type models. In the growth-type model, nucleation of the native-like structure occurs in the folding process, in the rate-limiting step(s), and subsequent folding around the nucleation sites proceeds smoothly to form the native disulfide bonds and conformation. In the rearrangement-type model, proper nucleation does not occur in the folding process; instead, non-native interactions play a significant role in the folding pathways and lead to metastable intermediate species. Such non-native interactions must be disrupted or rearranged to nucleate the native interactions (in the rate limiting step(s)) for the protein to fold. Other protein foldings, reported in the literature, can be shown to conform to this model.
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PMID:Multiple pathways for regenerating ribonuclease A. 639 20

Lysozyme, ribonuclease and insulin were exposed to dry heating for 1 to 24 h at temperatures between 80 and 180 degrees C. Amino acid analyses of the heated samples showed that most of the amino acids are stable up to 120 degrees C. Initially, at higher temperatures, an almost rectilinear decrease took place which reached a critical stage at 160 degrees C. Nonpolar aliphatic, acidic and aromatic amino acids were all relatively stable (maximum loss less than 20% after 24 h at 180 degrees C). The lability of the other amino acids increased in the order proline, arginine, histidine, cysteine, threonine, lysine, tryptophan, serine, and methionine. Methionine was 86% decomposed after 24 h at 180 degrees C. Loss of trinitrobenzene sulfonic acid-reactive lysine ("available lysine") reached 20% at 100 degrees C and essentially 100% after 24 h at 180 degrees C. Maximum loss in weight during heating was 11%, although maximum protein loss was between 20 and 35%. Reaction orders and activation energies were estimated for some of the amino acid losses. Of the atypical amino acids ("hot spots") lysinoalanine, allo-isoleucine and ornithine that were detected, only lysinoalanine is useful as an indicator to detect amino acid damage after dry heating.
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PMID:Model studies on the heating of food proteins. Amino acid composition of lysozyme, ribonuclease and insulin after dry heating. 641 75

The primary structure of Penicillium brevicompactum guanyl-specific RNase was determined. The enzyme consists of 102 amino acid residues, Mr 10801. The 4 cysteine residues of the RNase are linked in pairs by disulfide bonds: Cys2-Cys10, Cys6-Cys101. P. brevicompactum RNase structure is similar to RNase T1; the degree of homology is 66%.
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PMID:Amino acid sequence and S-S bonds of Penicillium brevicompactum guanyl-specific ribonuclease. 643 69

The C epsilon H proton resonance of His-12 of reduced cysteine S-sulfonated bovine pancreatic ribonuclease A exhibits a nonlinear temperature dependence of the chemical shift in its 1H-NMR spectrum at an apparent pH of 3.0. At temperatures below ca. 35 degrees C, the temperature dependence of the chemical shift of the His-12 C epsilon H resonance is opposite in sign to those of His-48, His-105, and His-119. At temperatures above ca. 35 degrees C, the temperature dependence of the chemical shift of the His-12 C epsilon H resonance is similar to those of the other three His C epsilon H resonances. These data indicate the existence of an equilibrium between locally ordered and locally disordered environments of His-12 in the sulfonated protein at temperatures below ca. 35 degrees C. The ordered and disordered conformations interconvert at a rate that is fast relative to the 1H-NMR chemical shift time scale--i.e., the locally ordered structure has a lifetime of much less than 7 msec. These results demonstrate that short- and medium-range interactions can define short-lived local structures under conditions of temperature and solution composition at which the native protein structure is stable. Furthermore, they demonstrate the utility of reduced derivatives of disulfide-containing proteins as model systems for the identification of local structures that may play a role as early-forming chain-folding initiation structures.
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PMID:Local structure involving histidine-12 in reduced S-sulfonated ribonuclease A detected by proton NMR spectroscopy under folding conditions. 658 14

Refolding of dimeric porcine cytosolic or mitochondrial malate dehydrogenases and of tetrameric pig heart and skeletal muscle lactate dehydrogenases (containing 5-7 cysteine residues), as well as reformation of the four cystine cross-bridges of bovine pancreatic ribonuclease, were studied in the presence of reduced and oxidized glutathione (GSH and GSSG). At the intracellular GSH level (5 mM) reduced ribonuclease can be reoxidized by 0.01-0.5 mM GSSG (pH 7.4) both at 20 degrees C and 37 degrees C. In this physiological range of GSSG concentrations and pH, the dehydrogenases show at least partial reactivation. With GSSG concentrations greater than 5 mM, reactivation is found to be completely inhibited for all the enzymes given. The results show that at the intracellular level of GSH and GSSG, thiol groups in reduced, unfolded ribonuclease are oxidized to form intramolecular cystine cross-bridges, while thiol groups of typical cysteine enzymes, such as lactate and malate dehydrogenase, remain in their reduced state during refolding. The rate of reactivation of lactate dehydrogenase (porcine muscle) is not affected by GSSG. In the case of ribonuclease, increasing concentrations of GSSG increase the rate of reactivation: At 20 degrees C, the halftime of the correct disulfide bond formation varies from approximately equal to 80 h in the presence of 0.01 mM GSSG to approximately equal to 10 h in the presence of 0.25 mM GSSG. A further increase in the rate of reactivation at higher GSSG concentrations is accompanied by a decrease in yield. Reactivation of ribonuclease is also observed at the low glutathione level found in blood plasma (5-25 microM GSH).
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PMID:Influence of glutathione on the reactivation of enzymes containing cysteine or cystine. 661 43

An enzyme complex is a multifunctional catalytic unit that efficiently associates substrates with functionally related enzymes. The enzyme complex provides for the cellular regulation of enzymatic activities by physical interaction of the proteins with each other and by prior alteration of one enzyme's substrate by a related enzyme. Such regulatory abilities may go awry in neoplasia. Components of the protein biosynthetic machinery, such as aminoacyl-tRNA synthetases, have been thought to exist freely in the cytoplasm. However, high-molecular-weight enzyme complexes with aminoacyl-tRNA synthetase activities have been found in mammalian cells. We have been the first to report that the mammalian cell enzymes responsible for modification of tRNA occur in enzyme complexes (molecular weight 900000 daltons) associated with aminoacyl-tRNA synthetases and that the activities of these enzymes differ in normal and leukemic cells. Thus the enzymes responsible for the methylation of tRNA occur in enzyme complexes that provide efficient maturation of tRNA and possible regulation of protein synthesis. In FLC cells a unique enzyme complex composed of tRNA-methyltransferase and aminoacyl-tRNA synthetase activities has also been shown to contain a specific ribonuclease activity and a cysteine-tRNA sulfurtransferase activity. Sulfurtransferase activity has been characterized and optimized for its tRNA and cysteine substrates and mercaptoethanol and cation cofactors. Abnormal activity of this enzyme during neoplasia could result in improper acylation of tRNA and/or infidelity of coding by tRNA. Specific RNase is important in the sizing of percursor tRNA into mature tRNA. Results showed that this sizing was dependent upon the presence of the enzyme complex and the length of the incubation time. Many of the 20 aminoacyl-tRNA synthetases are also found in the complex. Electron microscopy has verified the subunit nature of the complex, seen previously by density gradient centrifugation and gel filtration. Three subunits, each of 300 000 daltons, comprise a complex approximately 200 A in diameter.
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PMID:Processing of tRNA is accomplished by a high-molecular-weight enzyme complex. 684 94

The aminoacylation of rat liver tRNA with selenocysteine was studied in tissue slices and in a cell-free system with [75Se]selenocysteine and [75Se]selenite as substrates. [75Se]Selenocysteyl tRNA was isolated via phenol extraction, 1 M NaCl extraction and chromatography on DEAE-cellulose. [75Se]Selenocysteyl tRNA was purified on columns of DEAE-Sephacel, benzoylated DEAE-cellulose and Sepharose 4B. In a dual-label aminoacylation with [35S]cysteine, the most highly purified 75Se-fractions were greater than 100-fold purified relative to 35S. These fractions contained less than 0.7% of the [35S]cysteine originally present in the total tRNA. When [35Se]selenocysteyl tRNA was purified from a mixture of 14C-labeled amino acids, over 97% of the [14C]aminoacyl tRNA was removed. The [75Se]selenocysteine was associated with the tRNA via an aminoacyl linkage. Criteria used for identification included alkaline hydrolysis and recovery of [75Se]selenocysteine, reaction with hydroxylamine and recovery of [75Se]selenocysteyl hydroxamic acid and release of 75Se by ribonuclease. The specificity of [75Se]selenocysteine aminoacylation was demonstrated by resistance to competition by a 125-fold molar excess of either unlabeled cysteine or a mixture of the other 19 amino acids in the cell-free selenocysteine aminoacylation system.
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PMID:Identification of a selenocysteine-specific aminoacyl transfer RNA from rat liver. 692 51


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