期刊
JOURNAL OF PROTEIN CHEMISTRY
卷 20, 期 6, 页码 469-477出版社
SPRINGER/PLENUM PUBLISHERS
DOI: 10.1023/A:1012502412612
关键词
Asclepias fruticosa; Aselepiadaceae; latex; milkweed; plant endopeptidases
Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 mug 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 degreesC, but was quickly inactivated after 5 minutes at 80 degreesC. 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 (M-r = 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 (LPDSVD-WREKGVVFPIRNQGK) 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.
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