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The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent [3H]-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the alpha-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment alpha 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the alpha-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the alpha-chain that assign the amino-terminal segment alpha 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified [3H]DDF-labeled residues, which are conserved in muscle and neuronal alpha-chains but not in the other subunits, may be directly involved in agonist binding.

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The process by which small proteins fold to their native conformations has been intensively studied over the last few decades. In this field, the particular chemistry of disulfide bond formation has facilitated the characterization of the oxidative folding of numerous small, disulfide-rich proteins with results that illustrate a high diversity of folding mechanisms, differing in the heterogeneity and disulfide pairing nativeness of their intermediates. In this review, we combine information on the folding of different protein models together with the recent structural determinations of major intermediates to provide new molecular clues in oxidative folding. Also, we turn to analyze the role of disulfide bonds in misfolding and protein aggregation and their implications in amyloidosis and conformational diseases.2

Oxidative stress has been implicated in dysfunctional mitochondria in diabetes. Tyrosine nitration of mitochondrial proteins was observed under conditions of oxidative stress. We hypothesize that nitration of mitochondrial proteins is a common mechanism by which oxidative stress causes dysfunctional mitochondria. The putative mechanism of nitration in a diabetic model of oxidative stress and functional changes of nitrated proteins were studied in this work. As a source of mitochondria, alloxan-susceptible and alloxan-resistant mice were used. These inbred strains are distinguished by the differential ability to detoxify free radicals. A proteomic approach revealed significant similarity between patterns of tyrosine-nitrated proteins generated in the heart mitochondria under different in vitro and in vivo conditions of oxidative stress. This observation points to a common nitrating species, which may derive from different nitrating pathways in vivo and may be responsible for the majority of nitrotyrosine formed. Functional studies show that protein nitration has an adverse effect on protein function and that protection against nitration protects functional properties of proteins. Because proteins that undergo nitration are involved in major mitochondrial functions, such as energy production, antioxidant defense, and apoptosis, we concluded that tyrosine nitration of mitochondrial proteins may lead to dysfunctional mitochondria in diabetes.Protein tyrosine nitration is a common post-translational modification occurring under conditions of oxidative stress in a number of diseases (1, 2). Diabetes is a state of oxidative stress (3, 4). The studies on diabetic mitochondria suggest that diabetes causes dysfunctional mitochondria (5-8). Furthermore, the studies that associate altered free radical status with impaired mitochondrial function provide evidence of protein tyrosine nitration in mitochondria exposed to oxidative stress (9 -13), including diabetic mitochondria (14). These observations may have important implications for the pathogenesis of diabetes if the protein targets of nitration, functional consequences of nitration, and pathways for the increase of protein tyrosine nitration in mitochondria were established.Over the past several years, substantial evidence has been accumulated that major pathways of protein tyrosine nitration in vivo include peroxynitrite (ONOO Ϫ ) and heme peroxidasedependent reactions (1). A recent development points to the common nitrating species (nitrogen dioxide, ⅐ NO 2 ) formed from both pathways, which is responsible for the most nitrotyrosine generated (15, 16). Despite some progress in assessing the putative mechanism of in vivo nitration, the biological relevance of protein tyrosine nitration remains unclear. Very little is known about which specific proteins undergo nitration and whether disease-associated nitration contributes to the appearance of complications or whether it is merely a biomarker, reflecting the presence of complications.Our hypothesis is that nitration of m...