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

作　　者：Constanza Liggieri Walter Obregón Sebastián Trejo Nora Priolo 

机构地区：[1]Laboratorio de Investigaci6n de Proteinas Vegetales (LIPROVE), Departamento de Ciencias Biol6gicas, Facultad de Ciencias Exactas,Universidad Nacional de La Plata, C.C. 711, B 1900AVW, La Plata, Argentina [2]Institut de Biotecnologia i de Biomedicina 'Vincent Villar i Palasi', Universidad Autbnoma de Barcelona, 08193 Campus UAB, Bellaterra(Cerdanyola del Vall6s), Barcelona, Spain

出　　处：《Acta Biochimica et Biophysica Sinica》2009年第2期154-162,共9页生物化学与生物物理学报（英文版）

摘　　要：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-phenantrollne, phenyimethanesulfonyl fluoride, and pepstatine. The N-terminal sequence （LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA） showed a high similarity with those of other plant cysteine proteinases. When assayed on N-α-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-α-CBZ-L-Gln-p-nitrophenyl ester as substrate： Km = 0.1634 mM, kcat = 121.48 s^-1, and kcat/Km = 7.4 ×10^5 s^-1/mM.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-phenantrollne, phenyimethanesulfonyl fluoride, and pepstatine. The N-terminal sequence （LPSFVDWRQKGVVFPIRNQGQ CGSCWTFSA） showed a high similarity with those of other plant cysteine proteinases. When assayed on N-α-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-α-CBZ-L-Gln-p-nitrophenyl ester as substrate： Km = 0.1634 mM, kcat = 121.48 s^-1, and kcat/Km = 7.4 ×10^5 s^-1/mM.

关 键 词：Asclepias curassavica ASCLEPIADACEAE cysteine proteinase LATEX LATICIFERS 

分 类 号：Q94[生物学—植物学] Q51