KINETIC CHARACTERIZATION OF CYSTEINE ENDOPEPTIDASES FROM SOME MILKWEED LATEX

OBREGÓN, D.; LIGGIERI, C.; MORCELLE DEL VALLE, S.; PRIOLO, N.

Pinamar, Pcia. de Buenos Aires, Argentina

Congreso; XLI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular, X Congreso de la Sociedad Panamericana de Bioquímica y Biología Molecular y XX Reunión Anual de la Sociedad Argentina de Neuroquímica; 2005

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB), Panamerican Association for Biochemistry and Molecular Biology (PABMB) y Sociedad Argentina de Neuroquímica (SAN)

KINETIC CHARACTERIZATION OF CYSTEINE ENDOPEPTIDASES FROM SOME MILKWEED LATEX David Obregón, Constanza Liggieri, Susana Morcelle and Nora Priolo. LIPROVE, Depto. de Cs. Biológicas, Fac. de Cs. Exactas, UNLP, Argentina. e-mail: davidobregon@biol.unlp.edu.ar Milkweed latex endopeptidases have been demonstrated to show a strong biochemical similitude to papain. To confirm their similar hydrolytic properties, different synthetic substrates were assayed for some Asclepiadaceae species latex endopeptidases. The synthetic substrates used were: Z-aa p-nitrophenyl esters derivatives, Bz-Phe-Arg p-nitroanilide, Bz-Arg-Arg p-nitroanilide and PFLNA. Preferencies and kinetic parameters (Km, Km/kcat and Vm) were calculated and compared. Z-Gln p-nitrophenyl ester was the most preferred substrate for asclepains (Km 0.0503 mM and 0.1634 mM, resepectively) and araujiains from Araujia hortorum (Km 0.024 mM, 0.238 mM and 0.099 mM, respectively). Likewise, araujiain a II from A. angustifolia also showed the highest preference for the same derivative, whereas araujiain a I, a III and funastrain c II (Km 0.024 mM) from Funastrum clausum for the Z-Ala p-nitrophenyl ester derivative. The allosteric behaviour of araujians from A.. angustifolia prevented the calculation of their kinetic parameters. For PFLNA as substrate, the Km values were: asclepain c I 0.8183 mM, araujiain a II 0.18 mM, araujiain a III 5.14 mM and funastrain c II 0.1011 mM. The other endopeptidases showed no affinity for this substrate. As a conclusion, the endopeptidases studied showed a remarkable preference for two Z-aa p-nitrophenyl ester derivatives: Ala and Gln. The Km values obtained for the PFLNA derivative were in the same order than the one for papain; all these data support the idea that these endopeptidases belong to the papain-like family.