Purification and characterization of the cysteine proteinases in the latex of Vasconcellea spp.
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.
Kyndt
Tina
T
Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Belgium.
Van Damme
Els J M
EJ
Van Beeumen
Jozef
J
Gheysen
Godelieve
G
D000595:Amino Acid Sequence; D001937:Brassica / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification*; D018076:DNA, Complementary / Q000378:metabolism; D019143:Evolution, Molecular; D007840:Latex / Q000737:chemistry*; D008969:Molecular Sequence Data; D010802:Phylogeny; D014176:Protein Biosynthesis; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D007840:Latex; D020033:Protein Isoforms; D003546:Cysteine Endopeptidases