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Showing 1981–2000 of 2058 publications.
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Lackmann, Martin; Rajasekariah, Poornima; Iismaa, S. E.; Jones, Graham; Cornish, Coralie J.; Hu, Shengping; Simpson, Richard J.; Moritz, Robert L.; Geczy, Carolyn L.We previously reported the purification and partial amino acid sequence of a novel murine cytokine designated CP-10, which has chemotactic activity for murine polymorphonuclear cells (PMN) and macrophages. The complete cDNA encoding an 88-aminoacid polypeptide has been isolated and the sequence is presented here. Transient transfection of CP-10 cDNA into CV-1 cells confirmed the chemotactic activity of rCP-10 for murine PMN. CP-10 has sequence homology with members of the S100 family of Ca2+-binding proteins with pronounced amino acid sequence similarities within the putative N- and C-terminal Ca2+-binding sites, but differences within their connecting hinge and C-terminal regions. We have confirmed the hypothesis of Kligman and Hilt that functional specificity of individual members of the S100 protein family may reside in the hinge region. A synthetic peptide corresponding to the hinge region of CP-10 (CP-10<inf>42-55</inf>) was compared with native CP-10 in chemotaxis and skin test assays. Native CP-10 had potent activity for phagocytic cells, but not lymphocytes, in vitro (optimal activity, 10-11 to 1013 M) and elicited a sustained recruitment of neutrophils and mononuclear cells over 24 h in vivo. The hinge-region peptide had strong chemotactic activity for murine phagocytic cells (optimal activity, 10-10-10-11 M) but elicited only a transient infiltration of neutrophils over 4 to 8 h after intradermal injection. Results indicate that although the hinge region contributes significantly to the functional specificity of the S100 protein CP-10, sustained cellular recruitment typical of a delayed type hypersensitivity response is apparently dependent on the structural integrity of the protein.
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Dawes, JoanAn iodinated derivative of the low molecular weight dermatan sulphate Desmin 370 was administered to rats by intravenous, subcutaneous and intramuscular routes in conjunction with unlabelled Desmin 370. Following intravenous injection radiolabel was cleared from the plasma according to a tri-exponential function with elimination half-lives of 45 min and 13h; there was a high concentration in tissues, particularly the kidney, compared with plasma. The apparent bioavailability following s.c. injection was 52-65%, but 84-100% after i.m. administration. Undegraded as well as desulphated molecules were identified in plasma, and intact 125I-Desmin 370 comprised >85% of the radiolabel in urine 48h post-injection. Thus intact Desmin 370 persists for a long period after a single injection. The pharmacological significance of these findings, however, remains to be determined.
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Jessup, Wendy K.; Simpson, Jeremy A.; Dean, Roger T.Low-density lipoprotein (LDL) oxidation induced by superoxide radicals generated in a cell-free system could not stimulate the subsequent development of high-uptake LDL during incubation in a medium normally permissive for cell-mediated oxidation. Similarly, LDL oxidative modification by macrophages was not accelerated when extracellular superoxide generation was increased 5-10-fold by stimulation of NADPH oxidase. The NADPH oxidase inhibitor, diphenylene iodonium, did inhibit macrophagemediated medification of LDL, but its effects do not appear to involve superoxide generation. Superoxide dismutase (SOD) was shown to be inappropriate as a test for the involvement of superoxide radicals in cell-mediated oxidation due to its metal-chelating properties and to the development of a pro-oxidant activity by heat inactivation. We conclude that there is presently no secure evidence for the involvement of superoxide radical in macrophage-mediated oxidative modification of LDL. 1993.
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Yen, Tina; Walsh, John D.; Pejler, Gunnar; Berndt, Michael C.; Geczy, Carolyn L.Cytotoxic drugs may potentiate the thrombotic complications in patients with malignancies and platelet function abnormalities have been reported after initiation of cisplatin therapy. This report describes a prolonged activation of platelets over 6?24 h co?culture with peripheral blood mononuclear cells (PBM) by pharmacological doses of cisplatin. Cisplatin had no direct effect on platelets and depended on PBM to produce aggregation which was apparently not mediated by products of the cyclooxygenase or lipoxygenase pathways, by platelet activation factor (PAF) or by thrombin. Although platelet aggregation normally involves the binding of fibrinogen to the ?<inf>3</inf> integrin, GP IIb?IIIa, on activated platelets, the cisplatin?dependent platelet aggregation observed in the co?culture experiments was not inhibited by anti?GP IIb?IIIa monoclonal antibody which blocks fibrinogen?dependent aggregation nor by an adhesive peptide containing the RGDS integrin recognition sequence. Rather, aggregation appeared to involve a novel 140 kD granule membrane protein (GMP?140) mediated mechanism since aggregation was almost completely blocked by Fab fragments of antibody to GMP?140 and was inhibited by fluid?phase GMP?140. At concentrations of cisplatin, adriamycin, and LPS that induced equivalent levels of tissue factor of blood monocytes, prothrombinase activity was significantly greater in cultures containing cisplatin. Prothrombinase activity was dependent on the presence of platelets and the rate of thrombin formation was enhanced by factor Xa generated by the tissue factor?factor VIIa complex. These studies suggest that the vascular and thrombotic complications associated with cisplatin therapy are mediated, at least in part, by platelet activation and aggregation and monocyte procoagulant activity. Copyright 1993, Wiley Blackwell. All rights reserved
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Zammit, Adrian; Pepper, Duncan S.; Dawes, JoanThe profile of proteins bound to immobilised heparins in hirudin-anticoagulated human plasma was analysed. In molar terms, antithrombin III was the most abundant protein bound to therapeutic doses of unfractionated heparin (M(r) = 12,000), whereas heparin cofactor II constituted <1% of the protein bound. Histidine-rich glycoprotein was the only plasma protein likely to influence anticoagulant activity by direct competition with antithrombin III, though significant quantities of complement Factor H, fibrinogen, fibronectin, vitronectin and apolipoprotein B were also detected. Only traces of von Willebrand factor, complement factor I, inter-a-trypsin inhibitor, ?<inf>2</inf>-macroglobulin, serum amyloid P and transferrin were identified, and neither thrombospondin nor platelet factor 4 were measurable. Binding of both antithrombin III and histidine-rich glycoprotein varied with the ratio of heparin to plasma. Clexane (M(r) = 4,500) also bound antithrombin III, but both histidine-rich glycoprotein and vitronectin were quantitatively significant neutralising proteins. Neutralising proteins dominated the binding profile for Oligo H (M(r) = 2,200).
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Dean, Roger T.; Wilcox, Ian; Sullivan, Collin E.; Zwillich, Clifford W.; Grunstein, Ronald R.; Lavie, Peretz; Hedner, Jan Anders[No abstract available]
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Mackinnon, Wanda B.; Dyne, Marlen; Hancock, Rebecca; Mountford, Carolyn E.Chinese hamster ovary (CHO) cell lines are a very popular cell model for a wide range of studies but are often misused experimentally as a substitute for normal cells. Although CHO was originally derived from normal tissue, the cell lines studied here, including the parental wild type, have many characteristics which indicate that they have undergone malignant transformation. Biological properties associated with malignancy were investigated in this study on wild type CHO cells and 4 drug resistant sublines, EOT, Col R-22, Pod R11-6, and Vin R-1. We report evidence of tumorigenicity in experimental animals, invasive capacity, in vivo and in vitro, protease release by 2 of the cell lines, features related to drug resistance in the mutant sublines, and numerical and structural chromosomal abnormalities. 1993 Royal College of Pathologists of Australasia.
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Simpson, Jeremy A.; Gieseg, Steven Paul; Dean, Roger T.We have previously shown that exposure of many proteins, and free aromatic amino acids (particularly tyrosine) to free radical fluxes generates a stable activity capable of reducing protein bound and free transition metal ions. Here we define the capacity of several radical generating systems (gamma irradiation of water, UV irradiation, metal-dependent sugar autoxidation and Haber-Weiss systems) to produce protein-bound reducing moieties (PBRedM), and also reducing derivatives of tyrosine. Under the defined conditions of the gamma radiolysis system, reductive activity was generated under both oxic and anoxic irradiations and specific gassing regimes as well as the inclusion of specific radical scavengers established that hydroxyl radicals were responsible. When BSA was irradiated anoxically in the presence of formate a reductive activity related to the exposure of protein thiol groups was generated; all other reductive activities we detected were not thiol-related. Incubations of tyrosinase with BSA or insulin also generated reductive activity. All the conditions we have studied can convert tyrosine into DOPA and we suspect that protein-bound DOPA is the main reductive activity generated on proteins. 1993.
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Sattler, Wolfgang; Stocker, RolandWe have observed recently that high-density lipoproteins (HDL) are the predominant carriers of cholesteryl ester hydroperoxides (CEOOH), the major class of lipid hydroperoxides detectable at nanomolar concentrations in the plasma of healthy fasting humans. The present study investigates the effect of such very low levels of CEOOH in apolipoprotein E-free HDL<inf>3</inf> on lipoprotein particle metabolism and 'selective uptake' of its CE by human Hep G2 cells. Minimal oxidation with aqueous peroxyl radicals had a negligible effect on the binding, internalization and degradation of 125I-labelled HDL<inf>3</inf>. In contrast, with an increasing degree of radical-mediated oxidation of labelled HDL<inf>3</inf>, [3H]cholesteryl linoleate ([3H]Ch18 :2) was taken up at an increasingly greater rate than were 125I-apoproteins. When [3H]cholesteryl linoleate hydroperoxide ([3H]Ch18:2-OOH was incorporated into unoxidized HDL<inf>3</inf> by exchange from donor liposomes, it was taken up at a more than 8-fold higher rate than was incorporated [3H]Ch18:2. The same degree of preferential uptake of oxidized CE was observed when HDL<inf>3</inf> was used that was doubly labelled with [3H]Ch18:2-OOH and cholesteryl [14C]oleate ([14C]Ch18:1). In both situations, uptake of [3H]Ch 18:2-OOH exceeded that of 125I-apolipoprotein A-I some 40-fold. This increased selective uptake of [3H]Ch18:2-OOH from very mildly oxidized HDL<inf>3</inf> was accompanied by a parallel increase in the intracellular levels of labelled free cholesterol. In contrast, lipid hydroperoxides were not detectable within Hep G2 cells, suggesting efficient detoxification of CEOOH by these cells. Neither the increased selective uptake of Ch18:2-OOHs nor the levels of intracellular free cholesterol were influenced by the presence of 50 ?M chloroquine, suggesting extralysosomal hydrolysis of oxidized CEs. These results show that the selective uptake of HDL CEOOH by Hep G2 cells is more efficient than that of unoxidized CE, and support a protective role for rapid selective uptake in the removal of circulating HDL CEOOH.
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Grant, Adrienne J.; Jessup, Wendy K.; Dean, Roger T.The location of a protein (soluble or membrane-bound) influences the extent of oxidative damage caused by free radicals. It has been established that after radical attack, soluble proteins can become more susceptible to hydrolysis by individual proteinases than native proteins.1-4 We have now examined the hydrolytic susceptibility following radical attack of a protein that is located within a membrane environment, mitochondrial monoamine oxidase (MAO). After exposure to oxygen radicals generated by gamma irradiation, hydrolysis of sub-mitochondrial particles (SMP) containing MAO was increased in three respects. First, the generation of small fragments of MAO by the proteinases elastase and trypsin, was enhanced. Second, the generation by these enzymes and by phospholipase A2 of non-sedimentable membrane fragments containing MAO was also increased. Third, autolysis of SMP was enhanced. Hence, proteins located within membranes may become more susceptible to enzymatic degradation following oxidative damage. 1993 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
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Bowry, Vincent W.; Stocker, RolandOxidation of human low-density lipoprotein (LDL) is implicated as an initiator of atherosclerosis. ?-Tocopherol (?-TocH) may thus inhibit atherosclerosis because it is the major and most active chain-breaking antioxidant in extracted LDL lipid. Our studies show, however, that ?-TocH can be a strong prooxidant for the LDL itself, i.e., an aqueous dispersion of lipid-bearing particles. Thus, a steady flux (R<inf>g</inf>) of alkylperoxyl radicals (ROO) generated from a water-soluble azo initiator induced lipid peroxidation in LDL which was faster in the presence of ?-TocH than in its absence (for R<inf>g</inf>< 2 nM s?1), insensitive to R<inf>g</inf> and [O<inf>2</inf>], and inhibited by vitamin C, ubiquinol-10 (normally present in fresh LDL), and small phenolic antioxidants but not inhibited by the aqueous radical scavenger uric acid. Furthermore, LDL peroxidation induced by a water- or lipid-soluble azo initiator or by transition metals in Ham's F-10 cell culture medium was accelerated by increasing the concentration of ?-TocH in LDL. We propose that LDL peroxidation is initiated by the reaction of ROO with ?-TocH and that the inability of the ?-Toc formed in this reaction to escape from an LDL particle then forces ?-Toc to propagate a radical chain via its reaction with PUFA lipid (LH) within the particle (?-Toc + LH + O<inf>2</inf> ? ?-TocH + LOO). Termination of a radical chain occurs when a peroxidizing LDL particle captures a second radical from the aqueous medium (ROO + ?-Toc ? nonradical products). Steady-state kinetic analysis of this mechanism yields a theoretical model for tocopherol-mediated peroxidation (TMP) in lipid dispersions which fully explains our findings for LDL. We conclude that peroxidation of LDL lipid can (only) be prevented by agents which eliminate the ?-Toc radical: vitamin C and LDL-associated ubiquinol-10 do so by exporting the radical into the aqueous medium, whereas small phenolic antioxidants (e.g., butylated hydroxytoluene) accelerate the transfer of radicals between particles. The theoretical and practical implications of TMP in LDL, dispersions, and bulk lipids are discussed. 1993, American Chemical Society. All rights reserved.
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Neuil, Ji? Gebicki, Janusz M.; Stocker, RolandExposure of proteins to oxygen-centred radicals results in dramatic changes in their structure, stability and function; properties that have been studied in many laboratories from a qualitative viewpoint. To allow a quantitative evaluation, we subjected aerated solutions of BSA to hydroxyl and superoxide anion radicals generated radiolytically under conditions where all radicals formed reacted with the protein (as judged by maximum damage to BSA). We observed that for each radical generated approx. 15 amino acids were consumed initially. Similar results were found with lysozyme and melittin. Such a massive consumption of BSA's amino acids was not observed when irradiations were carried out under anaerobic conditions. When bilirubin or Trolox (a water-soluble analogue of vitamin E) was added at a 2-fold molar excess over BSA, initial consumption of all measured amino acids, except tryptophan, decreased 4-fold. The total mass of amino acids initially protected (from consumption) exceeded the mass of antioxidants consumed by more than a factor of 20. Such protection of amino acids was not observed when the antioxidant-inactive acetyl Trolox was used. These results suggest that radical-mediated oxidation of proteins can proceed via a previously unrecognized chain reaction that may be inhibited by chain-breaking antioxidants.
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Jessup, Wendy K.; Dean, Roger T.Murine peritoneal macrophages treated with ?-interferon and lipopolysaccharide (activated cells) oxidized low-density lipoprotein (LDL) less readily than unstimulated cells. Activated cells expressed the enzyme nitric oxide synthase, whose activity was measured by the accumulation of nitrite in the culture supernatant. Treatment of activated macrophages with the arginine analogue NG-monomethyl-arginine (NMMA) inhibited nitric oxide synthesis and restored the ability of the cells to oxidize LDL. This treatment had no effect on the ability of unstimulated cells to oxidize LDL. Similarly, LDL oxidation by activated macrophages in arginine-free Ham's F-10 medium was identical to that of unstimulated cells, whereas restoration of arginine to the medium was associated with nitrite secretion and a decline in LDL oxidation by activated cells only. An inverse relationship between nitric oxide synthesis and LDL oxidation was also demonstrated in the presence of diphenylene iodonium, a flavin analogue which is a potent inhibitor of nitric oxide synthase. Thus nitric oxide synthesis appears to mediate the suppression of LDL oxidation which is associated with the activation of mouse macrophages by ?-interferon and lipopolysaccharide. 1993.
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Dupuis, Marc; Peitsch, Manuel Claude; Hamann, Ute; Stanley, Keith K.; Tschopp, JgComplement protein C9 assembles with C5, C6, C7, C8 on the surface of target cells to form the lytic membrane attack complex (MAC). During MAC assembly and insertion into the target membrane, the hydrophilic, globular C9 partially unfolds to expose a hydrophobic lipid interaction domain. Several copies of amphiphilic C9 subsequently polymerize to form the characteristic ring-like MAC. Using a combined photoaffinity label and computer modeling approach, two amphipathic helices in a segment encompassing the amino acids 293-334 have been predicted to interact with membrane lipids. To elucidate the mechanism of C9 lipid binding and insertion, site-directed mutagenesis was used to change the amphipathic character of the helices. While some conservative amino acid replacements such as Thr<inf>307</inf> by a Leu were tolerated and yielded fully active C9 when expressed in COS cells, successive changes of Leu<inf>305</inf> into Val, Ala, and Glu on the hydrophobic site of the first helix gave rise to only partly or not secreted C9. All non-conservative amino acid replacements introduced on either side of the helices resulted in non-secreted C9 that was subsequently degraded intracellularly, indicating the importance of the correct folding of the presumptive transmembrane domain during biosynthesis. A natural secretion-incompetant mutant was found in which Val<inf>93</inf>, located in the proposed lipid-binding region, was lacking. Taken together, these findings suggest that the high incidence of homozygous C9 deficiencies may be due to a blockage in intracellular transport and secretion due to point mutations in this 'hot spot' region of the molecule. 1992.
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Jessup, Wendy K.[No abstract available]
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Stanley, Keith K.; Howell, Kathryn E.TGN38/41 is a heterodimeric integral membrane protein that cycles between the trans Golgi network and the cell surface. A tyrosine-containing tetrapeptide motif within its cytoplasmic tail is necessary and sufficient for determining its steady-state location in the TGN. Recent results have shown that TGN38/41 plays an essential role in the formation of exocytic vesicles at the TGN by serving as a receptor for complexes of a cytoplasmic protein known as p62, and one of four small GTP-binding proteins, including rab6. For budding to occur, this complex must bind to the cytoplasmic domain of TGN38/41. We propose here that TGN38/41 may couple the segregation of secretory proteins to the budding of exocytic vesicles at the TGN. 1993.
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Bos, Kitty; Wraight, Christopher J.; Stanley, Keith K.Sorting of proteins destined for different plasma membrane domains, lysosomes and secretory pathways takes place in the trans-Golgi network (TGN). TGN38 is an integral membrane protein found in this intracellular compartment. We show that TGN38 contains an autonomous targeting signal within its cytoplasmic domain which determines its intracellular location. Deletion analysis and site-directed mutagenesis of this domain demonstrate that a tyrosine motif homologous to the internalization signal of surface receptors is necessary and sufficient for correct localization. These findings suggest that TGN38 is maintained in the TGN by retrieval from the plasma membrane and employs a different mechanism for retention from that of the transferase enzymes of the trans-Golgi.
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Braidotti, Giovanna; Borthwick, Iain A.; May, Brian K.The housekeeping enzyme 5-aminolevulinate synthase (ALAS) regulates the supply of heme for respiratory cytochromes. Here we report on the isolation of a genomic clone for the rat ALAS gene. The 5?-flanking region was fused to the chloramphenicol acetyltransferase gene and transient expression analysis revealed the presence of both positive and negative cis-acting sequences. Expression was substantially increased by the inclusion of the first intron located in the 5?-untranslated region. Sequence analysis of the promoter identified two elements at positions -59 and -88 bp with strong similarity to the binding site for nuclear respiratory factor 1 (NRF-1). Gel shift analysis revealed that both NRF-1 elements formed nucleoprotein complexes which could be abolished by an authentic NRF-1 oligomer. Mutagenesis of each NRF-1 motif in the ALAS promoter gave substantially lowered levels of chloramphenicol acetyltransferase expression, whereas mutagenesis of both NRF-1 motifs resulted in the almost complete loss of expression. These results establish that the NRF-1 motifs in the ALAS promoter are critical for promoter activity. NRF-1 binding sites have been identified in the promoters of several nuclear genes encoding mitochondrial proteins concerned with oxidative phosphorylation. The present studies suggest that NRF-1 may co-ordinate the supply of mitochondrial heme with the synthesis of respiratory cytochromes by regulating expression of ALAS. In erythroid cells, NRF-1 may be less important for controlling heme levels since an erythroid ALAS gene is strongly expressed and the promoter for this gene apparently lacks NRF-1 binding sites.
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Suarna, Cacang; Hood, Ross L.; Dean, Roger T.; Stocker, RolandThe antioxidant activity of tocotrienols toward peroxyl radicals was compared with that of other natural lipid-soluble antioxidants in three different systems by measuring the temporal disappearance of antioxidants and the formation of lipid hydroperoxides. In homogeneous solution, the initial rates of consumption of the various antioxidants, assessed by competition experiments between pairs of antioxidants for radicals, decreased in the order: ubiquinol-10 ? ubiquinol-9 ? ?-tocopherol ? ?-tocotrienol ? ? lycopene ? ?-tocopherol ? ?-tocotrienol. Following in vitro incubation of human plasma with ?-tocotrienol, this form of vitamin E was present in all classes of lipoproteins isolated from the supplemented plasma. Dietary supplementation of rats and humans with a tocotrienol-rich preparation resulted in a dose-dependent appearance of ?- and ?-tocotrienols in plasma and all circulating lipoproteins, respectively. Exposure of such enriched rat plasma to aqueous peroxyl radicals resulted in simultaneous consumption of the ?- and then ?-isomers of vitamin E. The sequence of radical-induced consumption of antioxidants in freshly isolated, in vitro and in vivo tocotrienol-enriched low density lipoprotein (LDL) was again ubiquinol-10 ? ?-tocotrienol ? ?-tocopherol ? carotenoids ? ?-tocopherol ? ?-tocotrienol. Under conditions where radicals were generated at constant rates, the rate of lipid hydroperoxide formation in LDL was not constant. It proceeded in at least three stages separated by the phase of ubiquinol-10 consumption and, subsequently, that of ?-tocopherol/ ?-tocotrienol. Our results show that dietary tocotrienols become incorporated into circulating human lipoproteins where they react with peroxyl radicals as efficiently as the corresponding tocopherol isomers. 1993.
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Gieseg, Steven Paul; Simpson, Jeremy A.; Charlton, Timothy Stuart; Duncan, Mark William; Dean, Roger T.Proteins and aromatic amino acids previously exposed to hydroxyl radicals reduced cytochrome c, free iron, and copper ions. A major product of hydroxyl radical addition to tyrosine is 3,4-dihydroxyphenylalanine (DOPA), which has these reducing properties. The reduction of nitro blue tetrazolium by radical-damaged protein was consistent with the generation of quinones in the protein. By acid hydrolysis followed by high-performance C<inf>18</inf> reversed-phase liquid chromatography we have shown that hydroxyl radical-damaged proteins contain significant amounts of protein-bound DOPA (PB-DOPA). The authenticity of the DOPA measured was confirmed by gas chromatography-mass spectrometry. PB-DOPA was also generated enzymatically using mushroom tyrosinase, which catalyzes the hydroxylation of tyrosine residues. By comparing the levels of DOPA in radical-damaged or enzyme-treated protein with that of cytochrome c reduction, we show that PB-DOPA is a major source of the observed reducing activity. PB-DOPA may have a role in the replenishment of reduced transition metal ions involved in free radical generating systems in vivo. 1993, American Chemical Society. All rights reserved.
