DETECTION OF A CYSTEINE PROENZYME IN THE Vasconcellea quercifolia LATEX

TORRES MA JOSÉ; TREJO SEBASTIÁN; NATALUCCI CLAUDIA; LÓPEZ LAURA M I

Puerto Madryn

Congreso; XLVI Reunión Anual de la SAIB; 2010

Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular

The enzymes belonging to the C1A subfamily are synthesized as inactive precursors, with N-terminal propeptides. The propeptides contain characteristic elements which are highly conserved, as the ERFNIN motif and the GXNXFXD heptapeptide. From the latex of Vasconcellea quercifolia were isolated by ion exchange chromatography seven fractions containing cysteine peptidases, one of they showed an additional band of 31 kDa by SDS-PAGE suggesting the presence of the proenzyme. This fraction was incubated for 10 min at 60ºC in buffer pH 4.0, with and without pepsine and in buffer pH 8.5; finally the activity was determined using casein as substrate. The results showed that when the fraction was incubated at pH 8.5 or with pepsin, the caseinolytic activity was increased by 40% and 30% over the control. Additionally, the SDSPAGE analysis showed an increase of the band corresponding to the mature fraction after such treatments. Both results revealed the presence in the latex of a propeptide that can be removed by proteolytic action. This fact was confirmed by the detection of a peptide of 21 amino acids: KNNSYWLGLNVFADMSNDEFK (containing the GXNXFXD heptapeptide), when the tryptic PMF (peptide mass fingerprinting) of the band was compared with the in silico tryptic PMF of papain. Proforms (proenzymes) of proteinases had not been purified from plant latex up to date.