ISOLATION AND CHARACTERIZATION OF FUNASTRAIN C II, A THIOL ENDOPEPTIDASE FROM FUNASTRUM CLAUSUM LATEX

MORCELLE DEL VALLE, S. R.; TREJO, S.A.; CANALS, F.; PRIOLO, N. S.

Bariloche, Argentina

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2003

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

ISOLATION AND CHARACTERIZATION OF FUNASTRAIN C II, A THIOL ENDOPEPTIDASE FROM FUNASTRUM CLAUSUM LATEX   Morcelle del Valle, Susana1,*; Trejo, Sebastián A.1,*; Canals, Francesc2 and Priolo, Nora S.1 1LPROVE, Fac. de Cs. Exactas, UNLP. La Plata, Argentina. 2IIB, UAB, Barcelona, Spain. *CONICET. E-mail: morcelle@biol.unlp.edu.ar   Funastrain c II, a cysteine endopeptidase, was purified and characterized from the latex of Funastrum clausum (Asclepiadaceae). Ultracentrifugation and cation exchange chromatography by FPLC at pH 6.5 were the main purification steps. The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain, and IEF showed that its pI was higher than 9.3. The cysteinic nature of the protease was demonstrated by inhibition with 10 μM E-64, whereas activation was achieved with reductor agents such as 12 mM cysteine and 5 mM DTT. An important percentage of residual caseinolytic activity after incubation at temperatures up to 70ºC was conserved. Funastrain c II enzymatic activity optimum pH varied according to the substrate used: for casein, it was 9.0-10.0, and for the synthetic substrate L-pyroglutamyl-L-leucine- p-nitroanilide (PFLNA) was 6.3-6.8. KM and kcat kinetic parameters were calculated for the synthetic substrates N-α-CBZ-Ala-p -nitrophenyl ester (KM=0.0243 mM,  kcat= 1.5 sec-1) and PFLNA (KM=0.1011 mM,   kcat= 0.86 sec-1). Funastrain c II N-terminal sequence (LPMSVDWRQKGVVSAIRNQGKCGSCWAFSAV) showed a considerable similarity to other proteases isolated from latex of different Asclepiadaceae species, and also to cysteine proteinases belonging to the papain family.