title: Purification and characterization of a new plant endopeptidase isolated from latex of Asclepias fruticosa L. (Asclepiadaceae).
abstract: Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 microg of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45 degrees C, but was quickly inactivated after 5 minutes at 80 degrees C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (Mr = 23,652). The optimum pH range was achieved at 8.5-10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVDWREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices of Asclepiadaceae even when a high degree of homology could be observed with other plant cysteine endopeptidases.
authors.affiliation: Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina.
authors.forename: S A
authors.initials: SA
authors.lastname: Trejo
authors.forename: L M
authors.initials: LM
authors.lastname: López
authors.forename: C V
authors.initials: CV
authors.lastname: Cimino
authors.forename: N O
authors.initials: NO
authors.lastname: Caffini
authors.forename: C L
authors.initials: CL
authors.lastname: Natalucci
mesh_terms: D000595:Amino Acid Sequence; D029062:Apocynaceae / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D007840:Latex; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
chemical_list: D015853:Cysteine Proteinase Inhibitors; D007840:Latex; D010450:Endopeptidases