Inactivation of Ribonuclease by the Primary Aqueous Radicals

Mee, Lorna K.; Adelstein, S. James; Stein, Gabriel

doi:10.2307/3573517

Cited by 46 publications

(16 citation statements)

References 28 publications

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“…Since His-12 and His-119 play an important role in the active site, this result explains the high ability in inactivating the enzyme reported in the literature [10]. Moreover, the attack on these residues can be connected to the changes observed in the Met and Tyr band since both Met-13 and Asp-14 H-bonded to Tyr-25 are located near His-12.…”

Section: Resultssupporting

confidence: 57%

“…On the contrary, H • atoms probably attack the enzyme at a more restricted number of sites, perhaps away from the surface and also the far-reaching Tyr can be damaged. A confirm of this behaviour is given by the absence of aggregate formation after H • atom attack [5] , whereas highly aggregated forms of the enzyme have been found under oxidative stress conditions [6,10], since the TyrO • radicals give rise easier to inter-molecular reactions than intra-molecular ones. Since three Tyr "buried" can be probably identified with Tyr-25, -92 and -97, the initial H • radical attack should take place on one or two of them.…”

Section: Resultsmentioning

confidence: 54%

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Structural lesion to ribonuclease A caused by reductive stress: Assessment by Raman spectroscopy

Torreggiani

2008

Spectroscopy

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Abstract. The damages induced by reductive radical stress on bovine pancreatic ribonuclease A (RNAse A) were investigated by Raman spectroscopy. Gamma-irradiation was used to simulate the endogenous formation of reductive species, in particular • H atom that is a simple one-electron equivalent reducing agent. Specific damages occur at sensitive amino acid sites, selectively, rather than indiscriminately, leading to the structure modification of the protein. Sulfur-containing residues (Met and Cys) and aromatic residues are appreciably attacked. In particular, extensive changes in the disulfide bridge conformations are induced as well as conformational changes of the protein secondary structure; a gradual conversion of α-helical to pleated-sheet geometry was evidenced, indicating a higher ability of reducing radicals in denaturing the protein structure compared with that of oxidizing radical species.

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Section: Resultssupporting

confidence: 57%

Section: Resultsmentioning

confidence: 54%

Structural lesion to ribonuclease A caused by reductive stress: Assessment by Raman spectroscopy

Torreggiani

2008

Spectroscopy

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“…USA 78 (1981) 2195 final DNA concentration of 100 ,4g/ml. Samples were contained in Wheaton Hopkins tagging vials (7) and kept at 40C during and after irradiation. Which radicals were present was controlled by irradiation in the presence of appropriate radical scavengers (Table 2).…”

mentioning

confidence: 99%

Predominance of core histones in formation of DNA--protein crosslinks in gamma-irradiated chromatin.

Mee¹,

Adelstein²

1981

Proc. Natl. Acad. Sci. U.S.A.

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Chromatin and a subunit ofchromatin containing a complex of DNA and the core histones-H2A, H2B, H3, and H4-have been prepared from cultured Chinese hamster cells.Comparison of the formation of radiation-induced DNA-protein crosslinks in whole chromatin with that in the DNA-core histone complex has demonstrated that the core histones are the specific proteins involved in crosslinking. y irradiation of the chromatin subunit in the presence of radical scavengers has shown the hydroxyl radical to be the most effective aqueous radical intermediate for the promotion of crosslinking and the solvated electron and superoxide radical to be essentially ineffective.DNA-protein crosslinks are formed when whole cells or mixtures of DNA and cell protein in vitro are subjected to y or UV irradiation (1,2 It is generally accepted that, in eukaryotic cells,,nuclear DNA is organized as nucleosomes, consisting of a core region, containing the histon-es H2A, .H2B, H3, and H4, wrapped around with DNA; these core regions are joined by-linker DNA, probably. associated with H1 histone. Isolated together with the 'DNA and histones in purified chromatin are nonhistone chromosomal proteins (NHCP), probably consisting of as many as 100 structural, enzymic, and regulatory proteins, of which 15-20 are major constituents.Chromatin and a subunit of chromatin containing a complex of DNA and the core histones have been isolated from cultured Chinese hamster cells. On the basis ofcomparing radiation-induced crosslinking in the whole chromatin and in the chromatin subunit, we have now identified the core histones as the specific chromosomal proteins predominantly involved in crosslinking to DNA. Furthermore, y irradiation of the chromatin subunit in the presence of radical scavengers has confirmed the efficacy of the hydroxyl radical for the promotion of formation of crosslinks, and the inefficacy ofthe solvated electron and superoxide radical (3). MATERLALS AND METHODSCell Culture and Labeling. Chinese hamster lung fibroblasts (V79-753 cell line from J. A. Belli, Harvard Medical School) were grown as described (4). Eagle's minimal essential medium was supplemented with 15%.fetal bovine serum, 4% NCTC-109 medium (M. A. Bioproducts, Walkersville, MD), 2 mM L-glutamine, 0.1 mM of each nonessential amino acid, penicillin at 50 units/ml, and streptomycin at 50 Ag/ml. Cells were grown as monolayers in 150-cm2 plastic flasks at 370C in an atmosphere of 5% CO2. They were grown for two generations (18 hr) prior to harvesting in a medium containing [methyl-3H]thymidine (New England Nuclear) at.a concentration of0.25 ,uCi/ml (1 Ci = 3.7 x -101 becquerels) to label the DNA, and in some experiments, in a medium containing L-[U-'4C]lysine (New En-gland Nuclear) at a concentration of 0.5 ,Ci/ml to label the proteins.Preparation of Chromatin and a Chromatin Subunit. The isolation of chromatin from the cell pellet has been described in detail (4). Nuclei were prepared.by the method ofHymer and Kuff (5), in which cells were suspended in hypotonic sucrose b...

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“…[9][10][11][12] In this respect, the fate of the radical species derived from protein damage has been the subject of many investigations. In bovine pancreatic ribonuclease A (RNase A), chosen by us as a model for this study, sulfur-containing residues and aromatic amino acids have been reported to be the main sites of radical attack and are likely to be involved in the enzyme activity; 13,14 in addition, a specific damage of sulfur moieties of RNase A was connected to the release of low-molecular-weight thiols. 13,15 The aim of this study was to elucidate the possible role of sulfur-containing residues of proteins in the damage of other biomolecules by using a RNase A/liposome system under -irradiation in the absence of oxygen.…”

Section: Introductionmentioning

confidence: 99%

Investigation of radical‐based damage of RNase A in aqueous solution and lipid vesicles

Torreggiani¹,

Tamba²,

Manco

et al. 2005

Biopolymers

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The gamma-irradiation of bovine pancreatic ribonuclease A (RNase A) in aqueous solution were investigated at different doses by vibrational spectroscopy as well as enzymatic assay, electrophoresis, and HPLC analysis. Both functional and structural changes of the protein were caused by attack of H(*) atoms and (*)OH radicals. In particular, Raman spectroscopy was shown to be a useful tool in identifying conformational changes of the protein structure and amino acidic residues that are preferential sites of the radical attack (i.e., tyrosine and methionine). After partial structural changes by the initial radical attack, the internal sulfur-containing amino acid residues were rendered susceptible to transformation. By using the biomimetic model of dioleoyl phosphatidyl choline vesicle suspensions containing RNase A, the damage to methione residues could be connected to a parallel alteration of membrane unsaturated lipids. In fact, thiyl radical species formed from protein degradation can diffuse into the lipid bilayer and cause isomerization of the naturally occurring cis double bonds. As a consequence, trans unsaturated fatty acids are formed in vesicles and can be considered to be markers of this protein damage.

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Inactivation of Ribonuclease by the Primary Aqueous Radicals

Cited by 46 publications

References 28 publications

Structural lesion to ribonuclease A caused by reductive stress: Assessment by Raman spectroscopy

Structural lesion to ribonuclease A caused by reductive stress: Assessment by Raman spectroscopy

Predominance of core histones in formation of DNA--protein crosslinks in gamma-irradiated chromatin.

Investigation of radical‐based damage of RNase A in aqueous solution and lipid vesicles

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