CHITINASE ACTIVITY IN ENCYSTING ENTAMOEBA-INVADENS AND ITS INHIBITION BY ALLOSAMIDIN

被引:45
作者
VILLAGOMEZCASTRO, JC
CALVOMENDEZ, C
LOPEZROMERO, E
机构
[1] UNIV GUANAJUATO, FAC QUIM, INST INVEST & BIOL EXPTL, APDO POSTAL 187, GUANAIUATO 36000, MEXICO
[2] NATL POLYTECH INST, CTR INVEST & ESTUD AVANZADOS, DEPT GENET & BIOL MOLEC, MEXICO CITY 14, DF, MEXICO
关键词
ENTAMOEBA; ENCYSTMENT; CHITINASE ACTIVITY; ALLOSAMIDIN;
D O I
10.1016/0166-6851(92)90035-I
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Chitinase activity was measured in extracts of Entamoeba invadens cells as a function of time of encystation in axenic conditions using 4-MU(Ch)3 as substrate. Encystment was paralleled by chitinase activity which showed a peak after about 72 h of cultivation where cysts accounted for 63% of cell population. Thereafter, activity fell off rapidly, whereas encystment continued, reaching 80% at the end of the experiment (96 h). Comparison of activity between cysts and the total cell population in 48- and 72-h-old encysting cultures suggested that chitinase may start to accumulate in the pre-cyst forms. About 70% of the enzyme was recovered in the supernatant following low-speed centrifugation of whole extracts. Most of this activity represented soluble chitinase since it was not sedimented by further centrifugation at 105 000 x g. A minor proportion of enzyme activity remained associated to the buffer-washed, high-speed sediment. In addition to 4-MU(Ch)3, chitinase activity was also measured following the hydrolysis of other substrates such as nascent, preformed or colloidal chitin. Like other chitinases, the cyst enzyme preferred nascent over performed chitin as substrate. Digestion of the former yielded GlcNAc and minor amounts of (GlcNAc)2 as products. Allosamidin strongly inhibited hydrolysis of the fluorogenic substrate by the amebic chitinase in vitro with a K(i) of 0.065-mu-M. IC50 values were 0.085-mu-M and 0.16-mu-M at 5-mu-M and 10-mu-M 4-MU(Ch)3, respectively. When added to the axenic medium, the drug markedly retarded encystment though it was partially recovered after longer periods of incubation.
引用
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页码:53 / 62
页数:10
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