Characterization of sialidase from Entamoaeba hystolitica and possible pathogenic role in amebiasis.
Sialidase from Entamoeba histolytica was purified to apparent electrophoretic homogeneity by chromatography on hydroxyapatite, reactive brown agarose, fetuin/agarose and by fast performance liquid chromatography on a MonoQ column. The enzyme had a molecular mass of 65 kDa, as determined by SDS-PAGE. It had an optimum pH of 5.5 and was maximally active at 37 degrees C. It required Ca(2+) and Mg(2+) for activation but was strongly inhibited by Cu(2+), Fe(2+) and Zn(2+). The E. histolytica sialidase exhibited high specificity for Neu5Acalpha2,3lac and methylumbelliferyl-Neu5Ac (4-MU-Neu5Ac) with K(m) values of 0.144 mM and 0.059 mM, respectively. The enzyme was not active against colomic acid and the gangliosides GM(1) and GD(1). It was activated in the presence of lactose and galactose, but was unaffected by glucose, sucrose and mannose. The enzyme was inhibited competitively by 2,3,didehydroneuraminic acid and para-nitro-phenyloxamic acid with inhibition binding constants ( K(i)) of 30 micro M and 185 micro M, respectively. The motility of intact E. hystolytica cells was enhanced about 6-fold in the presence of 0.05-0.1 mM Neu5Acalpha2,3lac, 4-MU-Neu5Ac and fetuin. However, the motility of the parasite was highly diminished when incubated with Neu5Acalpha2en and sialic acid-containing compounds. Lysed E. histolytica trophozoites were found to lack neuraminic acid.
Nok
Andrew J
AJ
Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria. jandrew@skannet.com
Rivera
Windell
W
D000562:Amebiasis / Q000469:parasitology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000472:pathogenicity*; D006863:Hydrogen-Ion Concentration; D008190:Lyases / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D009068:Movement; D009438:Neuraminic Acids / Q000032:analysis; D009439:Neuraminidase / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D000509:alpha-Fetoproteins / Q000378:metabolism
D009438:Neuraminic Acids; D000509:alpha-Fetoproteins; D009439:Neuraminidase; D008190:Lyases; D008274:Magnesium; D002118:Calcium