The Experts below are selected from a list of 324 Experts worldwide ranked by ideXlab platform
Emily Shacter - One of the best experts on this subject based on the ideXlab platform.
-
ROLE OF CARBOHYDRATES IN Oxidative Modification OF FIBRINOGEN AND OTHER PLASMA PROTEINS
Archives of biochemistry and biophysics, 1995Co-Authors: Y.j. Lee, Emily ShacterAbstract:Abstract Oxidative stress is related to the mechanisms of oncogenesis, cell death, and the pathogenesis of many human diseases. Proteins are important targets for Oxidative Modification, and a Western blot assay that can identify individual oxidized proteins in whole tissue extracts has been described. Using that assay, it was found that plasma proteins show different susceptibilities to Oxidative Modification. Here, we examine the possibility that the carbohydrate groups of glycoproteins may contribute to the assessment of protein oxidation by carbonyl assays. We used fibrinogen as a model because it is highly susceptible to Oxidative Modification and contains subunits that are differentially glycosylated. When oxidation-induced carbonyls were measured in fibrinogen subunits by Western blot immunoassay, it was found that the Aα-chains, which contain no associated carbohydrate groups, were most highly oxidized while the Bβ- and γ-chains, which are glycosylated, were oxidized far less. However, no major difference in the oxidation pattern was obtained when fibrinogen was deglycosylated prior to or after exposure to oxidants. This argues against a possible protective role of the carbohydrate moieties in oxidation of the different fibrinogen subunits. Similar results were obtained with purified human immunoglobulin G and transferrin as well as whole plasma. The results show that carbohydrate moieties are not good targets for Oxidative attack by metal-catalyzed oxidation systems. Oxidant-induced carbonyl formation in glycoproteins derives largely, if not entirely, from amino acid oxidation and not from oxidation of carbohydrate groups.
-
Oxidative Modification of fibrinogen inhibits thrombin-catalyzed clot formation.
Free radical biology & medicine, 1995Co-Authors: Emily Shacter, Joy A. Williams, Rodney L. LevineAbstract:Abstract Plasma fibrinogen plays a central role in controlling hemostasis. In an earlier report, we found that fibrinogen is oxidized when whole plasma is treated with a metal-catalyzed oxidation system. These studies show that Oxidative Modification of purified human fibrinogen leads to an exposure-dependent loss of thrombin-induced clot formation. Inhibition of clotting occurred when either metal-catalyzed oxidation or γ-irradiation was employed to generate oxidizing radicals. Both systems caused covalent Modification of fibrinogen, assessed by measuring incorporation of protein carbonyls. Thrombin-catalyzed fibrinopeptide release was normal in irradiated fibrinogen and was only slightly diminished in protein exposed to metal-catalyzed oxidation, indicating that the inhibition of clotting activity was due to impaired fibrin monomer polymerization. Thus, Oxidative Modification of normal fibrinogen causes dysfibrinogenemia and constitutes a novel mechanism for inhibition of thrombosis.
-
differential susceptibility of plasma proteins to Oxidative Modification examination by western blot immunoassay
Free Radical Biology and Medicine, 1994Co-Authors: Emily Shacter, Joy A. Williams, Michael Lim, Rodney L. LevineAbstract:Plasma proteins are exposed to oxidants in a variety of circumstances in vivo, such as during tissue injury and inflammation. In this report, the relative susceptibility of each of the major plasma proteins to Oxidative Modification was assessed by exposing whole plasma to a metal-catalyzed radical generating system and detecting oxidation (protein carbonyl groups) using a novel Western blot immunoassay. Proteins were derivitized with dinitrophenylhydrazine, separated by SDS-gel electrophoresis, and screened with antibodies against dinitrophenyl groups. As little as 1 pmol of protein-associated carbonyls could be detected (100 ng of a 50 kD protein containing 0.5 mol carbonyl/mol protein). Individual plasma proteins were identified by their comigration with standards, crossreactivity with specific antibodies, and by comparison of plasma to serum. Using this approach, we found that plasma fibrinogen was much more susceptible to Oxidative Modification compared to the other major plasma proteins, albumin, immunoglobulins, and transferrin. The results emphasize the utility of this method for studying oxidation of proteins in cell extracts and tissues and indicate that experiments on the effects of oxidation on fibrinogen function are merited.
Rodney L. Levine - One of the best experts on this subject based on the ideXlab platform.
-
Oxidative Modification of Glutamine Synthetase
2011Co-Authors: Rodney L. LevineAbstract:The first step in the proteolytic degradation of bacterial glutamine synthetase is a mixed function oxidation of one of the 16 histidine residues in the glutamine synthetase subunit (Levine, R. L. (1983) J. Biol. Chem. 258, 11823-11827). A model system, consisting of oxygen, a metal ion, and ascorbic acid, mimics the bacterial system in mediating the Oxidative Modification of glutamine synthetase. This model system was studied to gain an understanding of the mechanism of oxidation and of factors which control the susceptibility of the enzyme to oxidation. Availability of substrates and the extent of covalent Modification of the enzyme (adenylylation) interact to modulate susceptibility of the enzyme to oxidation. This interaction provides the biochemical basis for physiologic regulation of intracellular proteolysis of glutamine synthetase. The Oxidative Modification requires hydrogen peroxide. While the reaction may involve Fenton chemistry, the participation of free radicals, superoxide anion, and singlet oxygen could not be demonstrated.
-
Carbonic Anhydrase III. Oxidative Modification IN VIVO AND LOSS OF PHOSPHATASE ACTIVITY DURING AGING
The Journal of biological chemistry, 1995Co-Authors: Elisa Cabiscol, Rodney L. LevineAbstract:Oxidative Modification of DNA, lipids, and proteins occurs as a consequence of reaction with free radicals and activated oxygen. Oxidative Modification of total cellular proteins has been described under many pathologic and experimental conditions, but no specific proteins have been identified as in vivo targets for Oxidative Modification. Utilizing an immunochemical method for detection of Oxidatively modified proteins, we identified a protein in rat liver that was highly oxidized. It was purified to homogeneity and identified as carbonic anhydrase, isozyme III. Its characteristics match those previously described for a protein that was lost during aging of the rat, senescence marker protein-1. Carbonic anhydrase III was purified from rats aged 2, 10, and 18 months, and the proteins were characterized. All three preparations were highly Oxidatively modified as assessed by their carbonyl content. The enzyme has three known catalytic activities, and the specific activities for carbon dioxide hydration and for ester hydrolysis decreased during aging by approximately 30%. However, the third activity, that of a phosphatase, was virtually lost during aging. While the physiologic role of carbonic anhydrase III is unknown, we suggest that it functions in an oxidizing environment, which leads to its own Oxidative Modification.
-
Oxidative Modification of fibrinogen inhibits thrombin-catalyzed clot formation.
Free radical biology & medicine, 1995Co-Authors: Emily Shacter, Joy A. Williams, Rodney L. LevineAbstract:Abstract Plasma fibrinogen plays a central role in controlling hemostasis. In an earlier report, we found that fibrinogen is oxidized when whole plasma is treated with a metal-catalyzed oxidation system. These studies show that Oxidative Modification of purified human fibrinogen leads to an exposure-dependent loss of thrombin-induced clot formation. Inhibition of clotting occurred when either metal-catalyzed oxidation or γ-irradiation was employed to generate oxidizing radicals. Both systems caused covalent Modification of fibrinogen, assessed by measuring incorporation of protein carbonyls. Thrombin-catalyzed fibrinopeptide release was normal in irradiated fibrinogen and was only slightly diminished in protein exposed to metal-catalyzed oxidation, indicating that the inhibition of clotting activity was due to impaired fibrin monomer polymerization. Thus, Oxidative Modification of normal fibrinogen causes dysfibrinogenemia and constitutes a novel mechanism for inhibition of thrombosis.
-
differential susceptibility of plasma proteins to Oxidative Modification examination by western blot immunoassay
Free Radical Biology and Medicine, 1994Co-Authors: Emily Shacter, Joy A. Williams, Michael Lim, Rodney L. LevineAbstract:Plasma proteins are exposed to oxidants in a variety of circumstances in vivo, such as during tissue injury and inflammation. In this report, the relative susceptibility of each of the major plasma proteins to Oxidative Modification was assessed by exposing whole plasma to a metal-catalyzed radical generating system and detecting oxidation (protein carbonyl groups) using a novel Western blot immunoassay. Proteins were derivitized with dinitrophenylhydrazine, separated by SDS-gel electrophoresis, and screened with antibodies against dinitrophenyl groups. As little as 1 pmol of protein-associated carbonyls could be detected (100 ng of a 50 kD protein containing 0.5 mol carbonyl/mol protein). Individual plasma proteins were identified by their comigration with standards, crossreactivity with specific antibodies, and by comparison of plasma to serum. Using this approach, we found that plasma fibrinogen was much more susceptible to Oxidative Modification compared to the other major plasma proteins, albumin, immunoglobulins, and transferrin. The results emphasize the utility of this method for studying oxidation of proteins in cell extracts and tissues and indicate that experiments on the effects of oxidation on fibrinogen function are merited.
Yufei Hua - One of the best experts on this subject based on the ideXlab platform.
-
Oxidative Modification of soy protein by peroxyl radicals
Food Chemistry, 2009Co-Authors: Caimeng Zhang, Xiangzhen Kong, Yufei HuaAbstract:Abstract Oxidative Modification of soy protein by peroxyl radicals generated in a solution containing 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH) under aerobic condition was investigated. Incubation of soy protein with increasing concentration of AAPH resulted in gradual generation of protein carbonyl derivatives and loss of protein sulphydryl groups. Circular dichroism spectra indicated that exposure of soy protein to AAPH led to loss of α-helix structure. Effect of oxidation on tertiary structure was demonstrated by surface hydrophobicity and tryptophan fluorescence. Surface hydrophobicity steadily decreased, accompanied by loss and burial of some tryptophan residues, indicating that soy protein gradually aggregated. The results of the size exclusion chromatogram (SEC) implied that incubation caused an AAPH-dose-dependent increase of fragmentation and aggregation of oxidised soy protein. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) indicated that non-disulphide linkages were involved in aggregate formation, and β-conglycinin was more vulnerable to peroxyl radicals than glycinin.
Gerald J. Brook - One of the best experts on this subject based on the ideXlab platform.
-
Hypocholesterolemia and cancer: tumor cells induce enhanced LDL uptake by non-tumor cells and stimulate Oxidative Modification of LDL
Pathophysiology, 1999Co-Authors: Bianca Fuhrman, Efrat Wolfovitz, Uri Cogan, Gerald J. BrookAbstract:Abstract Hypocholesterolemia has been reported in patients with disseminated cancer. In this study we evaluated the effect of tumor cells on two processes that lead to enhanced clearance from plasma of low density lipoprotein (LDL), the main carrier of cholesterol: (1) LDL uptake by non-tumor cells; (2) LDL Oxidative Modification. Incubation of control normal cells (bovine smooth muscle cells or human skin fibroblasts) with conditioned medium (CM) from ras mutated fibroblasts (RAS + ) resulted in a 49 and 31% increase in the cellular LDL uptake, respectively, in comparison to cells incubated with CM from non-mutated fibroblasts (RAS − ). Serum samples derived from patients with disseminated cancer also promoted cellular uptake of LDL by J-774 A.1 macrophages by up to twofold. Cellular mediated Oxidative Modification of LDL, measured as TBARS formation, was significantly higher when LDL was incubated with RAS + , or with MCF-7 tumor cells under Oxidative stress, in comparison to LDL incubated with non-tumor cells. Furthermore, LDL incubated with MCF-7 tumor cells had undergone physicochemical Modifications, as evaluated by a decrease in the lipoprotein fluidity by 26%, when compared to the fluidity of LDL that was incubated with J-774 A.1 cells. In conclusion the results indicate that tumor cells induce accelerated LDL uptake by non-tumor cells and stimulate LDL Oxidative Modification. Both processes may result in hypocholesterolemia.
Caimeng Zhang - One of the best experts on this subject based on the ideXlab platform.
-
Oxidative Modification of soy protein by peroxyl radicals
Food Chemistry, 2009Co-Authors: Caimeng Zhang, Xiangzhen Kong, Yufei HuaAbstract:Abstract Oxidative Modification of soy protein by peroxyl radicals generated in a solution containing 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH) under aerobic condition was investigated. Incubation of soy protein with increasing concentration of AAPH resulted in gradual generation of protein carbonyl derivatives and loss of protein sulphydryl groups. Circular dichroism spectra indicated that exposure of soy protein to AAPH led to loss of α-helix structure. Effect of oxidation on tertiary structure was demonstrated by surface hydrophobicity and tryptophan fluorescence. Surface hydrophobicity steadily decreased, accompanied by loss and burial of some tryptophan residues, indicating that soy protein gradually aggregated. The results of the size exclusion chromatogram (SEC) implied that incubation caused an AAPH-dose-dependent increase of fragmentation and aggregation of oxidised soy protein. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) indicated that non-disulphide linkages were involved in aggregate formation, and β-conglycinin was more vulnerable to peroxyl radicals than glycinin.
