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Acta Biochimica et Biophysica Sinica 2009 41(2):154-162; doi:10.1093/abbs/gmn018
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© The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

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

Constanza Liggieri1,*, Walter Obregón1, Sebastián Trejo2 and Nora Priolo1

1 Laboratorio de Investigación de Proteínas Vegetales (LIPROVE), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, C.C. 711, B1900AVW, La Plata, Argentina
2 Institut de Biotecnologia i de Biomedicina ‘Vincent Villar i Palasi’, Universidad Autònoma de Barcelona, 08193 Campus UAB, Bellaterra (Cerdanyola del Vallès), Barcelona, Spain

* Correspondence address. Tel: +54-0221-423-0121; E-mail: cliggieri{at}biol.unlp.edu.ar


  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 cII) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain cII was the minor proteolytic component in the latex, but showed higher specific activity than asclepain cI, 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 cII 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 cII 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 N-terminal 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: Km = 0.1634 mM, kcat = 121.48 s–1, and kcat/Km = 7.4 x 105 s–1/mM.

Keywords    Asclepias curassavica; Asclepiadaceae; cysteine proteinase; latex; laticifers

Received: October 23, 2008; Accepted: November 19, 2008
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