PURIFICATION AND CHARACTERIZATION OF ALEURAIN - A PLANT THIOL PROTEASE FUNCTIONALLY HOMOLOGOUS TO MAMMALIAN CATHEPSIN-H
PURIFICATION AND CHARACTERIZATION OF ALEURAIN - A PLANT THIOL PROTEASE FUNCTIONALLY HOMOLOGOUS TO MAMMALIAN CATHEPSIN-H
复制标题
DOI：
10.1104/pp.99.3.848
复制
发表时间：
1992-07-01
期刊：
影响因子：
7.4
通讯作者：
ROGERS, JC
中科院分区：
文献类型：
--
作者：
HOLWERDA, BC;ROGERS, JC
大麦（Hordeum vulgare L.）cv Himilaya）糊粉蛋白酶是一种液泡巯基蛋白酶，最初分离为cDNA，与组织蛋白酶H具有65%衍生的氨基酸序列同一性（JC Rogers，D Dean，GR Heck [1985] Proc Natl Acad Sci USA 82：6512-6516）。我们将大麦叶中的糊粉蛋白纯化至同质（>1000倍），并表征其对多种底物的活性。糊粉蛋白最好描述为氨肽酶;它以相似的催化效率水解三种不同的氨肽酶底物，但在水解NH 2封闭的底物类似物和偶氮酪蛋白时效率较低。我们对三种底物（精氨酸4-甲基-7-香豆酰胺、L-精氨酸β-萘酰胺和N-α-苯甲酰基-L-精氨酸β-萘酰胺）的K（m）和k（cat）值以及偶氮酪蛋白的比活性均在之前报道的人组织蛋白酶H对这些底物的活性的三倍范围内（WN Schwartz，Al Barrett [1980] Biochem J 191：487-497）。糊粉蛋白酶还显示出许多其他的相似性组织蛋白酶H，包括电荷形式的异质性，成熟蛋白质的NH 2-末端的位置，和pH活性曲线。糊粉蛋白酶和组织蛋白酶H的相似性质表明这些酶具有植物和动物细胞所需的相似功能。植物系统的可用性可能允许在未来的功能消融实验，以澄清这种酶在高等真核生物中的作用。
Barley (Hordeum vulgare L. cv Himilaya) aleurain is a vacuolar thiol protease originally isolated as a cDNA with 65% derived amino acid sequence identity with cathepsin H (JC Rogers, D Dean, GR Heck [1985] Proc Natl Acad Sci USA 82: 6512-6516). We purified aleurain from barley leaves to homogeneity (>1000-fold) and characterized its activity against a number of substrates. Aleurain is best described as an aminopeptidase; it hydrolyzes three different aminopeptidase substrates with similar catalytic efficiency but is less efficient at hydrolyzing an NH2-blocked substrate analog and azocasein. Our values for K(m) and k(cat) for three substrates (arginine 4-methyl-7-coumarylamide, L-arginine beta-naphthylamide, and N-alpha-benzoyl-L-arginine beta-naphthylamide) and specific activity with azocasein are all within a threefold range of those previously reported for human cathepsin H for these substrates (WN Schwartz, Al Barrett [1980] Biochem J 191: 487-497). Aleurain also shows a number of other similarities to cathepsin H including heterogeneity of charge forms, position of the NH2-terminus of the mature protein, and pH-activity profile. The similar properties of aleurain and cathepsin H suggest that these enzymes have a similar function(s) that is required by both plant and animal cells. The availability of a plant system may permit functional ablation experiments in the future to clarify the role of this enzyme in higher eukaryotes.