Detection, purification and characterisation of a secretory alkaline phosphatase from Onchocerca species.
An Onchocerca secretory alkaline phosphatase (E.C. 3.1.3.1) of molecular weight 90 kDa when in crude extract, but which dimerises to about 180 kDa upon purification, was detected, purified and characterised. The enzyme was found to be secreted by both O. ochengi and O. volvulus worms. It was shown to be of Onchocerca origin by Western blotting with bovine onchocerciasis sera and by its time-dependent release in cultures. The O. ochengi enzyme was purified to near homogeneity by a combination of polyethylene glycol precipitation, DEAE-cellulose chromatography and preparative electrophoresis. About 0.96 mg of the active enzyme was purified from 48.4 mg of the crude parasite-released products, giving a purification fold of 71.45 and a yield of 8.7%. The purified enzyme exhibited a typical Michaelis-Menten kinetics with optimum activity on p-nitrophenylphosphate (p-NPP) at pH 10.2. Its apparent K(m) for p-NPP was 0.56+/-0.03 mM and it required Mg(2+) and dithiothreitol (DTT) for stability throughout its purification. Sodium dodecyl sulphate at 2% (w/v) did not inhibit the enzyme activity, but apparently stabilised it during freezing. Inorganic phosphate inhibited the enzyme competitively with an apparent inhibition constant (K(i)) of 3.33+/-0.04 mM, whereas l-phenylalanine inhibited it in a mixed way with a K(i) of 3.18+/-0.03 mM. While contributing to the understanding of metabolism in Onchocerca, the present apparently unique enzyme which is likely to serve in the nutrition of the parasite could be further characterised as a macrofilaricide target or diagnostic marker in onchocerciasis.
Cho-Ngwa
Fidelis
F
Biotechnology Unit, Department of Biochemistry and Microbiology, Faculty of Science, University of Buea, P.O. Box 63, Buea, Cameroon.
Mbua
Eric Ngalle
EN
Nchamukong
Kingsley G
KG
Titanji
Vincent P K
VP
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange; D003067:Coenzymes / Q000494:pharmacology; D003451:Cryoprotective Agents / Q000494:pharmacology; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D004586:Electrophoresis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009596:Nitrophenols / Q000378:metabolism; D009854:Onchocerca / Q000201:enzymology*; D009943:Organophosphorus Compounds / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D003067:Coenzymes; D003451:Cryoprotective Agents; D004791:Enzyme Inhibitors; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010710:Phosphates; C008644:nitrophenylphosphate; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D000469:Alkaline Phosphatase; D008274:Magnesium; D004229:Dithiothreitol