Novel aspartyl endopeptidases from flowers of bull thistle

LUFRANO, D.; VAIRO CAVALLI, S.E.; LIGGIERI, C.; FARO, R.; VERÍSSIMO, P.

San Miguel de Tucumán

Congreso; 45 Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2009

Sociedad Argentina de Bioquímica y Biología Molecular

Plant aspartic proteinases (APs)-EC 3.4.23- have been purified and characterized from a variety of tissues from several species, even though its functions are still unclear. In the flowers the APs are claimed to be involved in interaction pistil-pollen, senescence and stress response. In this work we characterize the APs of flowers of Cirsium vulgare (Savi) Ten. (Asteraceae), also known as bull thistle, as the first step in the study of the potential interaction of the zymogenic form with synthetic and natural membranes. Crude extract from mature flowers obtained at pH 3.0 presented milk clotting activity only inhibited by pepstatin, a specific aspartyl inhibitor. IEF and Zimogram showed an active band towards haemoglobin with a pI of 4. After a two-step-purification procedure, consisting on molecular exclusion (Sephadex G-25) and anionic exchange chromatography (Resource Q), only one significant active fraction was detected of 38 kDa determined by SDS-PAGE. The preproenzyme cDNA was obtained by RT of mRNA from immature flowers followed by PCR, primers used were designed from highly conserved regions among plant APs. At least two sets of clones were cloned in E.coli using a pGEM-Teasyvector and sequenced. Both sequences displayed a great identity with cyprosin precursor, one of the most studied members of the plant APs belonging to the peptidase family A1 (pepsin family).