Purification and topographical location of tegumental alkaline phosphatase from adult Schistosoma mansoni.
Alkaline phosphatase, a marker for tegumental membranes of Schistosoma mansoni, was extracted using Triton X-100 from membranes purified by sucrose density gradient centrifugation. The enzyme activity was purified 6 800-fold over parasite homogenates and 118-fold over the tegumental membranes released when parasites were incubated in phosphate-buffered saline. Purification of the solubilised enzyme was achieved by binding to a Con A agarose affinity column, gel filtration of the eluted glycoproteins, and Blue affigel chromatography. The purified enzyme was shown to consist of a single glycosylated polypeptide Mr 65 000 on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Enzyme activity was associated with a possible tetramer, Mr 260 000 on gel filtration. Activity was associated with a band Mr 130 000 in sodium dodecyl sulphate-polyacrylamide gel electrophoresis run in the absence of reducing agents. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the absence of reducing agents, the size of the enzyme was shown to be similar in cercariae, schistosomula, adult schistosomes and their eggs, but it was smaller than the activity (Mr 145 000) extracted from host liver and intestinal microsomal membranes. The topography of the enzyme in the schistosome tegument was investigated by using surface radio-labelling reagents followed by its purification. The enzyme could be radio-iodinated only with difficulty in adult worms. Bolton and Hunter reagent, used at high concentration and for prolonged time periods, resulted in labelling of enzyme activity and the Mr 65 000 polypeptide subunit was also iodinated under these extreme conditions. It was concluded that the enzyme is not exposed at the schistome's surface, and is probably buried in the tegumental membrane network.
D000469:Alkaline Phosphatase / Q000032:analysis / Q000302:isolation & purification*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D007422:Intestines / Q000201:enzymology; D007457:Iodine Radioisotopes; D008647:Mesocricetus; D008970:Molecular Weight; D012550:Schistosoma mansoni / Q000201:enzymology*
D007457:Iodine Radioisotopes; D000469:Alkaline Phosphatase