Purification and biochemical characterization of lysosomal acid phosphatases (EC 3.1.3.2) from blood stream forms, Trypanosoma brucei brucei.
Three Acid phosphatases (ACP) were isolated and characterized from the lysosomes of blood stream forms of Trypanosoma brucei by a combination of isopynic and differential centrifugation through Ficoll, organic solvent precipitation, ion exchange on DEAE cellulose 52 and size exclusion chromatography on Sephadex G-75 columns. The purified ACP emerged as three distinct peaks (ACP I, ACP II and ACP III) with high specific activities and they moved homogeneously on 12% SDS-PAGE each as a single band with relative molecular weight of 36 kDa, 25 kDa and 45 kDa respectively. The purified enzymes were active at an optimum pH and temperature of 5.5 and 40 degrees C respectively. The enzyme activities appeared to be ACP because their activities were enhanced at low pH values and inhibited by the acid phosphatase inhibitor, sodium fluoride. ACP I and ACP II were sensitive to l-tartrate while ACP III was insensitive to l tartrate. The kinetic analysis of the purified enzyme (ACP I, ACP II and ACP III) determined using para-nitrophenylphosphate as substrate gave KM values of 0.2 mM, 0.15 mM and 0.5 mM. Monofunctional group sulfhydryl group inhibitors; HgCl2, and AgCl2 strongly inhibited the activity of ACP III and millimolar concentrations of dithiothreitol and iodoacetamide activated and inhibited the activity of the ACP III respectively, suggesting the involvement of thiol groups at the active site of the enzyme. Thus, differentiating it from ACP I and ACP II. The implication of these findings in relation to the pathology of trypanosomosis is discussed.
Amlabu
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Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria. ninmac2000@yahoo.com
Nok
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Sallau
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D000135:Acid Phosphatase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002500:Centrifugation, Isopycnic; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000201:enzymology*; D008970:Molecular Weight; D018512:Parasitemia / Q000469:parasitology; D051381:Rats; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D000135:Acid Phosphatase