Trypanosoma evansi sialidase: surface localization, properties and hydrolysis of ghost red blood cells and brain cells-implications in trypanosomiasis.
A membrane-bound sialidase was isolated from blood stream (BS) Trypanosoma evansi partially purified and characterized. The enzyme is a glycosyl phosphatidyl inositol (GPI) membrane anchored protein. It was solubilized from T. evansi cells recovered from infected camel blood by detergent treatment with Triton CF 54 and partially purified by a series of chromatography steps. The enzyme was optimally active at pH 5.5 and 37 degrees C. It had a KM and Vmax values of 4.8 x 10(-6) M and 3.75 x 10(-6) mol/min x mg protein with Neu5Acalpha2, 3lac as substrate respectively. The KM and Vmax values with fetuin (4-nitrophenyl-oxamic acid) as substrate were 2.9 x 10(-2) M and 4.2 x 10(-3) mol/min x mg protein in the same respect. Kinetic analysis with methly umbelliferyl sialate (MU-Neu5Ac) gave KM and Vmax values of 0.17 mM and 0.84 mmol/min x mg protein respectively. The T. evansi SD could hydrolyse internally linked sialic acid residues of the ganglioside GM2, but was inactive towards colomic acid, and NeuSAc2, 6. lac. When ghost red blood cell (RBC) was used as substrate, it desialylated the RBC in the following order of efficiency; mouse, rat, camel, goat, and dog. Similarly, cerebral cells isolated from BalbC mouse was desialylated by the T. evansi SD. Inhibition studies using 2-deoxy-2, 3 didehydro-N-acetyl neuraminic acid (NeuAc2, 3en) against MU-Neu5Ac revealed a competitive inhibition pattern with Ki of 5.8 microM. The enzyme was also inhibited non-competitively by parahydroxy oxamic acid (pHOA), and competitively by N-ethylmaleimide and N-bromosuccinate with Ki values of 25, 42, and 53 microM, respectively. It was activated by Mg2+ ion and inhibited by Cu2+ and Zn2+.
Nok
Andrew J
AJ
Department of Biochemistry, Ahmadu Bello University Zaria, Nigeria. jandrew@skannet.com
Nzelibe
Humphrey C
HC
Yako
Sarah K
SK
D000818:Animals; D001921:Brain / Q000469:parasitology*; D002162:Camelus; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000469:parasitology*; D006461:Hemolysis; D006868:Hydrolysis; D007700:Kinetics; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014345:Trypanosoma / Q000201:enzymology* / Q000302:isolation & purification; D014352:Trypanosomiasis / Q000201:enzymology* / Q000662:veterinary
D004791:Enzyme Inhibitors; D009439:Neuraminidase