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TAME (tosyl-L-arginine methyl ester, 200 μM) shows inhibitory effect on APC activation when added to mitotic Xenopus extract. TAME (1-200 μM) inhibits Cdc20 association with mitotic APC and also inhibits Cdh1 association with interphase APC. TAME significantly inhibits crosslinking of the IR peptide to Cdc27 and Cdc16 but only slightly reduces crosslinking to Cdc23 and Apc7 at 20 µM; TAME strongly inhibits crosslinking to all APC subunits at 200 µM. TAME (200 µM) also inhibits binding of wild type Cdc20 to the APC, but not binding of a ΔIR mutant. TAME (200 μM) induces Cdc20 dissociation from the APC in mitotic Xenopus extract calls for APC-dependent ubiquitination. TAME induces Cdc20 dissociation from the APC by promoting Cdc20 ubiquitination, but the dissociation can be suppresses by Cyclin B1. TAME terminates cyclin B1 ubiquitination prematurely. TAME interacts with ATP by β and γ phosphate and the adenine ring of ATP. TAME in combination with the Mg(II) ion accelerates the ATP hydrolysis process.