Biochemical analysis of a papain-like protease isolated from the latex of <italic>Asclepias curassavica</italic> L.

Abstract

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain c-II) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain c-II was the minor proteolytic component in the latex, but showed higher specific activity than asclepain c-I, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain c-II displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain c-II is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The Nterminal sequence (LPSFVDWRQKGVVFPIRNQGQCGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-alpha-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-alpha-CBZ-L-Gln-p-nitrophenyl ester as substrate: K(m)=0.1634 mM, k(cat)=121.48 s(-1), and k(cat)/K(m)=7.4 x 10(5) s(-1)/mM.

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

“…9). This experimental pI is similar to papain pI (8.75) and less basic than pI of other Apocynaceae peptidases (Dubey and Jagannadham 2003;Sequeiros et al 2005;Liggieri et al 2009). The estimated molecular mass of araujiain aII is fairly close to the value obtained by mass spectrometry and of the same order of molecular masses of other peptidases from Apocynaceae (Obregón et al 2009a, b;Trejo et al 2009).…”

“…Laticifers not only serve as a repository for natural products but also exhibit unique proteomes. Many latex proteins are thought to have defensive roles in plants, and include proteases, chitinases and pathogenesis-related polypeptides (Hagel et al 2008;Liggieri et al 2009). Over 110 latices of different plant families are known to contain at least one proteolytic enzyme.…”

“…From a pharmaceutical and biotechnological point of view, latex is an important source of plant peptidases used in industry due to their property of being active over wide ranges of temperature and pH (Domsalla and Melzig 2008;Liggieri et al 2009). Applications include the use of these enzymes as meat tenderizers (such as Panol Ò Purified Papain, Liquipanol Ò T100), component of detergent formulations (Khaparde and Singhal 2001), or surfactants synthesis (Morcelle et al 2009) among others.…”

Chemical Analysis of <i>Carica papaya</i> L. Crude Latex

“…Relatively, latex is a milky fluid with a complex mixture of constituents, like proteins, vitamins, carbohydrates, lipids, terpenes, alkaloids, and free amino acids [24]. The presence of certain enzymes like chitinases and proteases in latex vacuoles suggests that they may help plants for defense against pathogens, parasites, and herbivores by attacking the invader once the plant cell is lysed [25].…”

Purification of a novel cysteine protease, procerain B, from Calotropis procera with distinct characteristics compared to procerain

“…j http://www.worthington-biochem.com/pcyp/default.html. k [35]. therapeutic applications and frequently proteases are modified by protein engineering to avoid rapid autolysis.…”