Characterization of cysteine proteases in Malian medicinal plants

Extracts form 10 different Malian medicinal plants with a traditional use against schistosomiasis were investigated for their possible content of proteolytic activity. The proteolytic activity was studied by measuring the hydrolysis of two synthetic peptide substrates Z-Ala-Ala-Asn-NHMec and Z-Phe-Arg-NHMec. Legumain- and papain-like activities were found in all tested crude extracts except those from Entada africana, with the papain-like activity being the strongest. Cissus quadrangularis, Securidaca longepedunculata and Stylosanthes erecta extracts showed high proteolytic activities towards both substrates. After gel filtration the proteolytic activity towards the substrate Z-Ala-Ala-Asn-NHMec in root extract of Securidaca longepedunculata appeared to have Mr of 30 and 97kDa, while the activity in extracts from Cissus quadrangularis was at 39kDa. Enzymatic activity cleaving the substrate Z-Phe-Arg-NHMec showed apparent Mr of 97 and 26kDa in extracts from roots and leaves of Securidaca longepedunculata, while in Cissus quadrangularis extracts the activity eluted at 39 and 20kDa, with the highest activity in the latter. All Z-Phe-Arg-NHMec activities were inhibited by E-64 but unaffected by PMSF. The legumain activity was unaffected by E-64 and PMSF. The SDS-PAGE analysis exhibited five distinct gelatinolytic bands for Cissus quadrangularis extracts (115, 59, 31, 22 and 20kDa), while two bands (59 and 30kDa) were detected in Securidaca longepedunculata extracts. The inhibition profile of the gelatinolytic bands and that of the hydrolysis of the synthetic substrates indicate the cysteine protease class of the proteolytic activities. Several cysteine protease activities with different molecular weights along with a strong variability of these activities between species as well as between plant parts from the same species were observed.