Protein modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle.
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The accumulation of covalently modified proteins is an important hallmark of biological aging, but relatively few studies have addressed the detailed molecular-chemical changes and processes responsible for the modification of specific protein targets. Recently, Narayanan et al. [Narayanan, Jones, Xu and Yu (1996) Am. J. Physiol. 271, C1032-C1040] reported that the effects of aging on skeletal-muscle function are muscle-specific, with a significant age-dependent change in ATP-supported Ca2+-uptake activity for slow-twitch but not for fast-twitch muscle. Here we have characterized in detail the age-dependent functional and chemical modifications of the rat skeletal-muscle sarcoplasmic-reticulum (SR) Ca2+-ATPase isoforms SERCA1 and SERCA2a from fast-twitch and slow-twitch muscle respectively. We find a significant age-dependent loss in the Ca2+-ATPase activity (26% relative to Ca2+-ATPase content) and Ca2+-uptake rate specifically in SR isolated from predominantly slow-twitch, but not from fast-twitch, muscles. Western immunoblotting and amino acid analysis demonstrate that, selectively, the SERCA2a isoform progressively accumulates a significant amount of nitrotyrosine with age (approximately 3.5+/-0. 7 mol/mol of SR Ca2+-ATPase). Both Ca2+-ATPase isoforms suffer an age-dependent loss of reduced cysteine which is, however, functionally insignificant. In vitro, the incubation of fast- and slow-twitch muscle SR with peroxynitrite (ONOO-) (but not NO/O2) results in the selective nitration only of the SERCA2a, suggesting that ONOO- may be the source of the nitrating agent in vivo. A correlation of the SR Ca2+-ATPase activity and covalent protein modifications in vitro and in vivo suggests that tyrosine nitration may affect the Ca2+-ATPase activity. By means of partial and complete proteolytic digestion of purified SERCA2a with trypsin or Staphylococcus aureus V8 protease, followed by Western-blot, amino acid and HPLC-electrospray-MS (ESI-MS) analysis, we localized a large part of the age-dependent tyrosine nitration to the sequence Tyr294-Tyr295 in the M4-M8 transmembrane domain of the SERCA2a, close to sites essential for Ca2+ translocation. (+info)
Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme.
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Wild-type and site-specific mutants C166S and C166A (Cys-166-->Ser and Cys-166-->Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a=b=c=84 A; alpha=beta=gamma=75 degrees) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility. (+info)
N-isobutyrylcysteine, a donor of systemic thiols, does not reduce the exacerbation rate in chronic bronchitis.
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N-isobutyrylcysteine (NIC), a new thiol compound that is not rapidly hydrolysed to give higher levels of free thiols in the body than N-acetylcysteine (NAC), was used to test if the effect of NAC on exacerbations in chronic bronchitis was an effect of the unhydrolysed thiol compound. Smokers or exsmokers with chronic bronchitis forced expiratory volume in one second (FEV1) >40% and reversibility < or = 10% predicted were treated with oral NIC 300 mg b.i.d. or placebo for 24 weeks. Steroids, NAC, antibiotics, and nonsteroid anti-inflammatory drugs use were restricted. Exacerbations were recorded by a respiratory symptom diary card and the time to onset of the first exacerbation after the start of treatment was measured using life-table analysis. Spirometry was performed at each visit. Six hundred and thirty-seven patients were randomized to treatment with NIC (n=316) or placebo (n=321). NIC did not prolong the time to first exacerbation (life-table analysis, p=0.59) and no increase in FEV1 or forced vital capacity was observed. Altered taste perception, taste loss and anosmia occurred more often in the NIC group (p<0.001). In conclusion, N-isobutyrylcysteine, a N-acetylcysteine-like drug with a greater bioavailability has, contrary to N-acetylcysteine, no effect on exacerbations in chronic bronchitis. This suggests that the effect of N-acetylcysteine on exacerbations in chronic bronchitis is not due to the relatively low free thiol levels (other than glutathione) produced by N-acetylcysteine therapy. (+info)
Improved methods for immunoassay of mycothiol.
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Improved enzyme-linked immunosorbent assay (ELISA) methods have been developed for the determination of femtomole amounts of mycothiol (MSH), the main low-molecular-weight thiol in mycobacteria. The immunoassays utilize an affinity-purified rabbit polyclonal antibody that is highly specific for the pseudodisaccharide moiety of MSH. MSH was first biotinylated by the thiol-specific reagent 3-(N-maleimidopropionyl)biocytin. The MSH-biotin adduct was then captured with immobilized avidin and detected with anti-MSH antibody (biotin-capture ELISA) or was captured with immobilized anti-MSH antibody and detected with alkaline phosphatase-labelled avidin (MSH-capture ELISA). The MSH-capture ELISA was the most sensitive method, measuring as little as 0.3 fmol of MSH. Methods for biotinylating MSH directly from Mycobacterium spp. are described. The MSH-capture ELISA was tested for the detection of M. avium seeded in human urine or cerebrospinal fluid samples and for screening mutant M. smegmatis strains to detect MSH production. (+info)
Chloroplast thioredoxin mutants without active-site cysteines facilitate the reduction of the regulatory disulphide bridge on the gamma-subunit of chloroplast ATP synthase.
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The activity of the chloroplast H+-ATPase (CFoCF1) is regulated by the proton electrochemical membrane potential and the reduction or the formation of the disulphide bridge on the gamma-subunit mediated by chloroplast thioredoxins (Trx). The latter regulation also applies to the water-soluble portion of CFoCF1 (CF1) and includes two successive steps, namely the binding of Trx to CF1 and the subsequent reduction or oxidation of CF1. To study this process thoroughly, a new expression system for spinach Trx-f and Trx-m was designed. In the presence of dithiothreitol (DTT) both forms of the expressed Trx could reduce the disulphide bridge on the gamma-subunit of CF1 and thus activate the ATPase. Trx mutants deficient in the internal, or both, cysteines of the active site were designed to study the details of the interaction. The Trx mutant proteins could still activate CF1-ATPase in the presence of DTT and they also increased the apparent affinity of CF1 for DTT. This implies that the binding of Trx to the CF1 gamma-subunit induces a conformational change facilitating the reduction of the disulphide bridge, and partially explains the high efficiency of Trx as a reductant in vivo. (+info)
Iron-induced oxidative stress in erythrocyte membranes of non-insulin-dependent diabetic Nigerians.
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The presence of higher level of endogenous free radical reaction products in the erythrocyte ghost membrane (EGM) of Non-insulin-dependent diabetes mellitus (NIDDM) subjects compared with that of normal healthy controls has been demonstrated. The EGMs of NIDDM subjects were also shown to be more susceptible to exogenously generated oxidative stress than those of normal healthy individuals. The decreased level of reactive thiol groups in the EGM of NIDDM individuals supported this observation. We propose that the presence of significant levels of non-heme iron in the EGM of NIDDM subjects is an indication of the potential for iron-catalysed production of hydroxy and other toxic radicals which could cause continuous oxidative stress and tissue damage. Oxygen free radicals could therefore be responsible for most of the erythrocyte abnormalities associated with non-insulin-dependent diabetes and could indeed be intimately involved in the mechanism of tissue damage in diabetic complications. (+info)
Fas mediated apoptosis of human Jurkat T-cells: intracellular events and potentiation by redox-active alpha-lipoic acid.
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Activation of caspases is required in Fas receptor mediated apoptosis. Maintenance of a reducing environment inside the cell has been suggested to be necessary for caspase activity during apoptosis. We explored the possibility to potentiate Fas mediated killing of tumor cells by alpha-lipoic acid (LA), a redox-active drug and nutrient that is intracellularly reduced to a potent reductant dihydrolipoic acid. Treatment of cells with 100 microM LA for 72 h markedly potentiated Fas-mediated apoptosis of leukemic Jurkat cells but not that of peripheral blood lymphocytes from healthy humans. In Jurkat, Fas activation was followed by rapid loss of cell thiols, decreased mitochondrial membrane potential, increased [Ca2+]i and increased PKC activity; all these responses were potentiated in LA pretreated cells. PKCdelta played an important role in mediating the effect of LA on Fas-mediated cell death. In response to Fas activation LA treatment potentiated caspase 3 activation by over 100%. The ability of LA to potentiate Fas mediated killing of leukemic cells was abrogated by a caspase 3 inhibitor suggesting that increased caspase 3 activity in LA-treated Fas-activated cells played an important role in potentiating cell death. This work provides first evidence showing that inducible caspase 3 activity may be pharmacologically up-regulated by reducing agents such as dihydrolipoic acid. (+info)
Crystal structure of human serum albumin at 2.5 A resolution.
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A new triclinic crystal form of human serum albumin (HSA), derived either from pool plasma (pHSA) or from a Pichia pastoris expression system (rHSA), was obtained from polyethylene glycol 4000 solution. Three-dimensional structures of pHSA and rHSA were determined at 2.5 A resolution from the new triclinic crystal form by molecular replacement, using atomic coordinates derived from a multiple isomorphous replacement work with a known tetragonal crystal form. The structures of pHSA and rHSA are virtually identical, with an r.m. s. deviation of 0.24 A for all Calpha atoms. The two HSA molecules involved in the asymmetric unit are related by a strict local twofold symmetry such that the Calpha atoms of the two molecules can be superimposed with an r.m.s. deviation of 0.28 A in pHSA. Cys34 is the only cysteine with a free sulfhydryl group which does not participate in a disulfide linkage with any external ligand. Domains II and III both have a pocket formed mostly of hydrophobic and positively charged residues and in which a very wide range of compounds may be accommodated. Three tentative binding sites for long-chain fatty acids, each with different surroundings, are located at the surface of each domain. (+info)