Catalase http://www.selleckchem.com/products/Y-27632.html activity assays Catalase activity was determined spectrophotometrically as described by Aebi. Cell homogenates were diluted with 50 mM phosphate buffer. The exponen tial decrease of 10 mM hydrogen peroxide was measured at 240 nm in the presence Inhibitors,Modulators,Libraries of cell homogenate. Reaction mixtures without cell homogenate were used as tissue blanks. Glutathione Inhibitors,Modulators,Libraries peroxidase activity determination Glutathione peroxidase activity was determined by the method of Paglia and Valentine. Cell homogenates were diluted with phosphate buffer. Tert butyl hydroperoxide was first reduced by glutathione peroxidase and then recycled by glutathione reductase coupled with nicotinamide adenine dinucleotide phosphate oxidation. The rate of decrease in NADPH was monitored spectrophotometrically at 340 nm as a measure of GPx activ ity.
Glutathione reductase activity determination GR activity was determined using the method of Carlberg and Mannervik. Glutathione reductase is required for the NADPH dependent conversion of oxidized glutath ione to reduced glutathione. Cell homoge nates were diluted Inhibitors,Modulators,Libraries with phosphate buffer. Inhibitors,Modulators,Libraries The exponential decrease of NADPH was measured spectro photometrically at 340 nm in the presence of cell homogenate. Reaction mixtures in which potassium phosphate buffer was used instead of glutathione disulfide in phosphate buffer were used as blanks. Protein determination Protein levels were determined by the Bradford method with Coomassie blue. Two Inhibitors,Modulators,Libraries and a half mil limeters of the diluted reagent were added to 0. 05 ml of a bovine serum albumin standard solution containing 10 to 100 mg ml protein.
This mixture was incubated PF-2341066 at room temperature for 5 to 10 minutes and the optical density was measured at 595 nm. Background Tuberous sclerosis complex is a fairly common inherited tumor suppressor syndrome, characterized by the development of hamartomas in the brain, skin, kid neys, lungs, heart and other organs. There is signifi cant morbidity due to a variety of clinical issues that occur at high frequency including epilepsy, cognitive and or behavioral impairments, kidney disease, pulmonary lym phangioleiomyomatosis, disfiguring facial angiofi bromas, and other manifestations. TSC1 and TSC2, which code for hamartin and tuberin respectively, have been identified as the disease genes of TSC. The two gene products form a tumor suppres sor complex that regulates a conserved cellular signaling pathway that mediates protein synthe sis and cell proliferation. Tuberins GTPase activa tion of Rheb is responsible for the tumor suppressor effect of the tuberin hamartin complex. Rheb in turn directly regu lates the mammalian target of rapamycin complex 1 in the PI3K Akt mTOR pathway.
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