Liggieri Constanza, Obregon Walter, Trejo Sebastian, Priolo Nora
Departamento de Ciencias Biologicas, Facultad de Ciencias Exactas, Laboratorio de Investigacion de Proteinas Vegetales, Universidad Nacional de La Plata, La Plata, Argentina.
Acta Biochim Biophys Sin (Shanghai). 2009 Feb;41(2):154-62. doi: 10.1093/abbs/gmn018.
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.
萝摩科的大多数物种通常在乳汁管细胞网络中分泌一种内源性乳状液，其中亚细胞器会大量合成蛋白质和次生代谢产物。一种新的类木瓜蛋白酶内切肽酶（萝摩蛋白酶c-II）已从马利筋（萝摩科）叶柄中提取的乳胶中分离并鉴定出来。萝摩蛋白酶c-II是乳胶中的次要蛋白水解成分，但比之前研究的主要活性组分萝摩蛋白酶c-I具有更高的比活性。这两种酶表现出截然不同的生化特性，证实它们是不同的酶。粗提物通过阳离子交换色谱法（快速蛋白质液相色谱）进行纯化。分离出两个活性组分，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和质谱分析显示为均一的。萝摩蛋白酶c-II的分子量为23,590 Da，pI高于9.3，在pH 9.4 - 10.2时具有最大蛋白水解活性，且热稳定性较差。萝摩蛋白酶c-II的活性受到半胱氨酸蛋白酶抑制剂如E-64的抑制，但不受其他蛋白酶抑制剂如1,10 - 菲咯啉、苯甲基磺酰氟和胃蛋白酶抑制剂的抑制。其N端序列（LPSFVDWRQKGVVFPIRNQGQCGSCWTFSA）与其他植物半胱氨酸蛋白酶的序列高度相似。当以N-α-苄氧羰基氨基酸对硝基苯酯进行测定时，该酶对谷氨酰胺衍生物表现出更高的偏好性。以N-α-苄氧羰基-L-谷氨酰胺对硝基苯酯为底物进行动力学参数测定：K(m)=0.1634 mM，k(cat)=121.48 s(-1)，k(cat)/K(m)=7.4×10(5) s(-1)/mM。
