Abstract: AIM: To establish a method for the determination of proteasome activities with fluorescence kinetic assay and evaluate the application in screening proteasome inhibitors.METHODS: The proteasome activities of chymotrypsin-like(CT-L) , trypsin-like (T-L) and peptidyl glutamyl peptide hydrolysing-like (PG-PH-L) were measured with specificity fluorescence peptide substrate Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVT-AMC) , Z-Val-Val-Arg-AMC (ZVVA-AMC) , Z-Leu-Leu-Glu-βNA (ZLLG-βNA) .The conditions of enzyme reaction had been optimised and the technolo-gies were evaluated .The inhibition of the CT-L, T-L and PGPH-L activities of purified 20S proteasome ac-tivity of proteasome extracts were prepared from B16 and Namalwa cells by PS-341 and ZGDHu-1were mea-sured. RESULTS: The optimum pH of enzyme reac-tion was 8.2.The addition of 0.3 g/L SDS buffer only to the assay was required to activate the CT-L of pro-teasome .The K m of CT-L, T-L and PGPH-L activities of proteasome were 3.55, 4.12, 4.88 μmol/L respec-tively and the linearity of reaction curve was up to 20 min.When purified 20S proteasome was used, the as-say for detecting CT-L, T-L and PGPH-L activities was linearly dependent on total protein 100, 120, 100 μg/L in 2 mL assay volume respectively .When the extracts of B16 cell proteasome were detected, the proteinum concentrations were 200 mg/L .The degree of precision in within-run and between-run CVs were (2.25-5.78) %and (3.75-8.42) %respectively .The IC 50 values of the PS -341 and ZGDHu-1 for inhibtion of the CT-L, C-L and PGPH-L activities of purified 20S proteasome were 12.36, 12.42, 24.40 nmol/L and 2.24, 0.92, 0.51 μmol/L respectively.CONCLUSION: The assay provides a reliable and effective method for studying the screen of proteasome inhibitors and it is an useful index in clinical trials with protea-some inhibitors.

Key words: fluorescence kinetic assays, protea-some, inhibitor, Bortezomib, N,N'-di-(m-methylphe-nyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide