Kyndt Tina, Van Damme Els J M, Van Beeumen Jozef, Gheysen Godelieve
Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Belgium.
FEBS J. 2007 Jan;274(2):451-62. doi: 10.1111/j.1742-4658.2006.05592.x.
Latex of all Vasconcellea species analyzed to date exhibits higher proteolytic amidase activities, generally attributed to cysteine proteinases, than the latex of Carica papaya. In the present study, we show that this higher activity is correlated with a higher concentration of enzymes in the latex of Vasconcellea fruits, but in addition also results from the presence of other cysteine proteinases or isoforms. In contrast to the cysteine proteinases present in papaya latex, which have been extensively studied, very little is known about the cysteine proteinases of Vasconcellea spp. In this investigation, several cDNA sequences coding for cysteine proteinases in Vasconcellea x heilbornii and Vasconcellea stipulata were determined using primers based on conserved sequences. In silico translation showed that they hold the characteristic features of all known papain-class cysteine proteinases, and a phylogenetic analysis revealed the existence of several papain and chymopapain homologues in these species. Ion-exchange chromatography and gel filtration procedures were applied on latex of V. x heilbornii in order to characterize its cysteine proteinases at the protein level. Five major protein fractions (VXH-I-VXH-V) revealing very high amidase activities (between 7.5 and 23.3 nkat x mg protein(-1)) were isolated. After further purification, three of them were N-terminally sequenced. The observed microheterogeneity in the N-terminal and cDNA sequences reveals the presence of several distinct cysteine proteinase isoforms in the latex of Vasconcellea spp.
迄今为止分析的所有番木瓜属物种的乳汁都表现出比番木瓜乳汁更高的蛋白水解酰胺酶活性，这种活性通常归因于半胱氨酸蛋白酶。在本研究中，我们表明这种较高的活性与番木瓜属果实乳汁中酶的较高浓度相关，但此外也源于其他半胱氨酸蛋白酶或同工型的存在。与番木瓜乳汁中已被广泛研究的半胱氨酸蛋白酶不同，关于番木瓜属物种的半胱氨酸蛋白酶知之甚少。在本研究中，使用基于保守序列的引物确定了番木瓜属植物和刺果番木瓜属植物中编码半胱氨酸蛋白酶的几个cDNA序列。电子计算机模拟翻译表明它们具有所有已知木瓜类半胱氨酸蛋白酶的特征，系统发育分析揭示了这些物种中存在几种木瓜蛋白酶和糜蛋白酶同源物。为了在蛋白质水平上表征其半胱氨酸蛋白酶，对番木瓜属植物的乳汁进行了离子交换色谱和凝胶过滤程序。分离出了五个显示出非常高酰胺酶活性（7.5至23.3 nkat x mg蛋白质-1）的主要蛋白质组分（VXH-I-VXH-V）。进一步纯化后，对其中三个进行了N端测序。在N端和cDNA序列中观察到的微异质性揭示了番木瓜属物种乳汁中存在几种不同的半胱氨酸蛋白酶同工型。
