Premium
Purification and characterization of the cysteine proteinases in the latex of Vasconcellea spp.
Author(s) -
Kyndt Tina,
Van Damme Els J. M.,
Van Beeumen Jozef,
Gheysen Godelieve
Publication year - 2007
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2006.05592.x
Subject(s) - papain , cysteine , carica , biochemistry , complementary dna , cysteine protease , cysteine proteinase inhibitors , chemistry , microbiology and biotechnology , enzyme , biology , gene , apoptosis , programmed cell death , horticulture , caspase
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 × 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. × 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·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.