, 2005), voltage-dependent anion channel protein (VDAC) monoclonal antibody was purchased from Calbiochem (San Diego, CA), and phospho-threonine rabbit polyclonal Wortmannin DNA-PK antibody was purchased from Cell Signaling Technology Inc. (Danvers, MA). The 125I-insulin radioimmunoassay kit was obtained from DPC/Siemens Healthcare Diagnostics (Deerfield, IL). INS-1 cells were obtained from the laboratory of Christopher Newgard at Duke University (Durham, NC). Screening for Inhibitors of PTPMT1. The Prestwick library of approximately 1000 small molecules dissolved in Me2SO (Prestwick Chemical Inc., Washington, DC) was screened by using the substrate O-MFP in an in vitro assay (Gottlin et al., 1996) in a 384-well plate format with a Beckman Biomek FX robot (Beckman Coulter, Brea, CA) under conditions where the Z�� score for the assay (Zhang et al.
, 1999) was more than 0.5. In brief, the assay was carried out in a total reaction volume of 40 ��l in a solution consisting of 0.1 M sodium acetate, 0.05 M bis Tris, 0.05 M Tris at pH 5.5, 50 ��M O-MFP substrate, 50 ��M test compound, 5% Me2SO, and 44 nM PTPMT1. The reaction was initiated by addition of enzyme, allowed to proceed for 40 min at room temperature, and then quenched with 40 ��l of 0.4 M NaOH. Product formation was determined by reading fluorescence emission at 520 nm. The 10 compounds showing the most potent inhibition were then subjected to a second round of screening using compound concentrations of both 50 and 5 ��M.
In these assays, product formation was monitored by both fluorescence emission at 520 nm and absorbance at 477 nm to ensure that decreased fluorescence indicated decreased product formation and true inhibition, rather than quenching of the fluorescence signal. Kinetic Studies. After the initial screen, it was determined that the optimal pH for PTPMT1 phosphatase activity with the substrate O-MFP was 7.0. Hence all kinetic experiments were carried out at this pH. Concentration that inhibits response by 50% (IC50) determinations for inhibition of PTPMT1 were carried out by using 44 nM enzyme in a total reaction volume of 100 ��l at pH 7.0 in the buffer system described above. VHR phosphatase assays were carried out by using 21.8 nM enzyme in the same buffer system, except that the pH was adjusted to 6.5, and �� protein phosphatase and T-cell PTP assays were carried out in buffers provided by the supplier using 44.
8 and 7.2 nM enzyme, respectively. All assays were carried out with 50 ��M O-MFP as the substrate at the optimal pH for each enzyme, and product formation was monitored continuously by fluorescence with initial velocity readings used for the analysis. Kinetic characterization of the inhibition Brefeldin_A of PTPMT1 by alexidine dihydrochloride was carried out with 88 nM enzyme to optimize signal and in a total reaction volume of 100 ��l at pH 7.0. PTEN assays were carried out as described previously (Maehama et al., 2000) by using 0.