Low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) shares no general sequence homology with other PTPs, although it has an active site sequence motif CXXXXXR and a reaction mechanism identical to those of all PTPs. The main function of this enzyme is the down-regulation of platelet-derived growth factor and insulin receptors. Both human LMW-PTP isoenzymes are inactivated by H 2 O 2 . The enzymes are protected from inactivation by P i , a competitive inhibitor, suggesting that the H 2 O 2 reaction is directed to active site. Analysis of free thiols performed on the inactivated enzymes demonstrates that only two out of the eight LMW-PTP cysteines are modified. Time-course high performance liquid chromatography-electrospray mass spectrometry, together with specific radiolabeling and tryptic fingerprint analyses, enables us to demonstrate that H 2 O 2 causes the oxidation of Cys-12 and Cys-17 to form a disulfide bond. Because both residues are localized into the active site region, this modification inactivates the enzyme. Fluorescence spectroscopy experiments suggest that the fold of the enzyme is modified during oxidation by H 2 O 2 . Because a physiological concentration of H 2 O 2 produces enzyme inactivation and considering that the activity is restored by reduction with low molecular weight thiols, we suggest that oxidative stress conditions and other processes producing hydrogen peroxide regulate the LMW-PTP in the cell.Protein tyrosine phosphorylation in eucaryotes is a key mechanism for cellular control, because it is involved in several processes, such as cellular metabolism, proliferation, differentiation, and oncogenic transformation (1). A fine balancing of cellular protein tyrosine phosphorylation levels is determined by regulating the activities of protein-tyrosine kinases and/or protein-tyrosine phosphatases (PTPs).1 Receptor protein-tyrosine kinases are considered to be the major enzymes regulating mitogenic protein phosphorylation cascades; nevertheless, the presence of SH2 domains in particular PTPs and the receptorlike structure of some membrane PTPs clearly indicate that PTPs are also regulated in the cell. The PTP superfamily consists of four main families: the tyrosine-specific phosphatases, the VH1-like dual specificity phosphatases, the cdc25 phosphatases, and the low molecular weight phosphatases (LMW-PTPs). Despite extremely limited sequence similarity, all share an active site motif consisting of a cysteine and an arginine separated by five residues (CXXXXXR, where X is any amino acid). All PTPs have identical catalytic mechanism, which involves the formation of a cysteinyl-phosphate intermediate (2).Recent papers from our laboratory have demonstrated that LMW-PTP is involved in the regulation of cellular signaling started by the activation of PDGF and insulin receptors (3-5). In fact, the overexpression of the wild type enzyme in NIH/3T3 cells causes decrease of cellular growth rate and of phosphorylation level of the PDGF receptor (3). Furthermore, the overexpression in ...

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Regular physical activity is associated with a reduced risk of coronary heart disease, as it probably modifies the balance between free-radical generation and antioxidant activity. On the other hand, however, acute physical activity increases oxygen uptake and leads to a temporary imbalance between the production of reactive oxygen and nitrogen species (RONS) and their disposal: this phenomenon is called oxidative stress. Proteins are one of the most important oxidation targets during physical exercise and carbonylation is one of the most common oxidative protein modifications. In cells there is a physiological level of oxidized proteins that doesn't interfere with cell function; however, an increase in oxidized protein levels may cause a series of cellular malfunctions that could lead to a disease state. For this reason the quantification of protein oxidation is important to distinguish a healthy state from a disease state. Several studies have demonstrated an increase of carbonylated plasma proteins in athletes after exercise, but none have identified targets of this oxidation. Recently a process of protein decarbonylation has been discovered, this may indicate that carbonylation could be involved in signal transduction. The aim of our research was to characterize plasma protein carbonylation in response to physical exercise in trained male endurance athletes. We analyzed by proteomic approach their plasma proteins at resting condition and after two different kinds of physical exercise (PE). We used 2D-GE followed by western blot with specific antibodies against carbonylated proteins. The 2D analysis identified Haptoglobin as potential protein target of carbonylation after PE. We also identified Serotransferrin and Fibrinogen whose carbonylation is reduced after exercise. These methods have allowed us to obtain an overview of plasma protein oxidation after physical exercise.