Purification and Biochemical Characterization of Asclepain c I from the Latex of Asclepias curassavica L.

Abstract

In this work we report the isolation, purification and characterization of a new protease from latex of Asclepias curassavica L. Crude extract (CE) was obtained by gathering latex on 0.1 M citric-phosphate buffer with EDTA and cysteine with subsequent ultracentrifugation. Proteolytic assays were made on casein or azocasein as substrates. Caseinolytic activity was completely inhibited by E-64. Stability at different temperatures, optimum pH and ionic strength were evaluated by measuring the residual caseinolytic activity at different times after the incubation. CE showed the highest caseinolytic activity at pH 8.5 in the presence of 12 mM cysteine. CE was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by SDS-PAGE, were isolated. The major purified protease (asclepain cI) showed a molecular mass of 23.2 kDa by mass spectrometry and a pI higher than 9.3. The N-terminal sequence showed a high similarity with those of other plant cysteine proteinases. When assayed on N-alpha-CBZ-aminoacid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative. Determinations of kinetic parameter (km and Kcat) were performed with PFLNA.

Kinetic studies on recombinant stem bromelain

“…Hence, there are only minor but noticeable differences in the specificity of recBM and cBM to the substrates. The data obtained in this study is comparable to the kinetic data reported for cysteine proteases including stem bromelain [20,24] granulosain [21], ascepalin [25], hieronymain II [26] and penduliforain I [27]. Based on kinetic data obtained in this study, it can be inferred that the kinetic behavior of recBM (containing His 6x tag, signal and propeptide) is not much different to that of mature cBM.…”

Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from Araujia angustifolia latex

“…Other peptidases of the Apocynaceae family display different substrate specificity. Asclepain cII and asclepain cI from latex of A. curassavica strongly prefer Asp and Tyr derivatives (Liggieri et al 2004. Morrenain bI exhibits strong preference for Ala and Asp derivatives while morrenain bII prefers Asp and Gly, both enzymes isolated from latex of Morrenia brachystephana (VairoCavalli et al 2001(VairoCavalli et al , 2003.…”

“…The effect of pH on enzyme activity of the purified protease was measured with casein (pH range 6.4-10.5) using 10 mM sodium salts of the following ''Good'' buffers: MES, MOPS, TAPS, AMPSO and CAPS (Liggieri et al 2004). …”

The defensive role of latex in plants: detrimental effects on insects

“…Moreover, plant latices are described as rich in small secondary metabolites, non-protein amino acids, enzymatic and non-enzymatic proteins in their serum phase (Yeang et al 2002;Taira et al 2005). Recently, a vast number of cysteine proteases have been described as occurring in many latex-producing plants (Liggieri et al 2004;Morcelle et al 2004a, b). Chitinases, lectins, lipases, oxidative and other hydrolytic enzymes have also been described as occurring in latex fluids (Stirpe et al 1993;Azarkan et al 1997;Giordani et al 1992;Amani et al 2007;Fiorillo et al 2007).…”