Purification and characterization of the alkaline phosphatase from Echinococcus granulosus cyst membranes.
The purification to homogeneity and the characterization of Echinococcus granulosus alkaline phosphatase (AP; EC 3.1.3.1) from hydatid cyst membranes are described. After n-butanol extraction, the parasite enzyme was sequentially purified by affinity chromatography on concanavalin A-sepharose followed by gel filtration. The purified protein (210 kDa) had a tetrameric structure composed of 4 56-kDa subunits. Its isoelectric point (4.8) and its kinetic parameters were determined (Km = 0.24 +/- 0.05 mmol/L; Vm = 173 +/- 21 nmol/min/mg protein for p-nitrophenylphosphate). The parasite enzyme differed from the host liver enzyme in its thermal stability, optimum reaction temperature, optimum pH, and catalytic parameters, but not in its apparent molecular weight. Furthermore, sera from patients infected with E. granulosus recognized the parasite AP on immunoblots, whereas uninfected controls were negative. These results as well as the role of this enzyme in the host-parasite relationship emphasize its potential importance as a diagnostic and prognostic antigen in the monitoring of hydatid infection.
Lawton
P
P
Laboratoire de Parasitologie, Faculté de Pharmacie, Université Claude-Bernard, Lyon, France.
Sarciron
E
E
Petavy
A F
AF
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D000909:Antibodies, Helminth / Q000276:immunology; D004443:Echinococcosis / Q000276:immunology / Q000469:parasitology; D004446:Echinococcus / Q000201:enzymology* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D012756:Sheep; D012757:Sheep Diseases / Q000469:parasitology; D013696:Temperature
D000909:Antibodies, Helminth; D007106:Immune Sera; D000469:Alkaline Phosphatase