Ecto-phosphatase activities on the cell surface of the amastigote forms ofTrypanosoma cruzi

Live Trypanosoma cruzi amastigotes hydrolyzed p-nitrophenylphosphate (PNPP) , phospho-amino-acids and P-32-casein under physiologically appropriate con ditions. PNPP was hydrolysed at a rate of 80 nmol.mg(-1).h(-1) in the prese nce of 5 mM MgCl2, pH 7.2 at 30 degrees C. In the absence of Mg2+ the activ ity was reduced 40% and we call this basal activity. At saturating concentr ation of PNPP, half-maximal PNPP hydrolysis was obtained with 0.22 mM MgCl2 . Ca2+ had no effect on the basal activity, could not substitute Mg2+ as an activator and in contrast inhibited the PNPP hydrolysis stimulated by Mg2 (I-50 = 0.43 mM). In the absence of Mg2+ (basal activity) the stimulating half concentration (S-0.5) for PNPP was 1.57 mM, while at saturating MgCl2 concentrations the corresponding S-0.5 for PNPP for Mg2+-stimulated phospha tase activity (difference between total minus basal phosphatase activity) w as 0.99 mM. The Mg-dependent PNPP hydrolysis was strongly inhibited by sodi um fluoride (NaF), vanadate and Zn2+ but not by tartrate and levamizole. Th e Mg-independent basal phosphatase activity was insensitive to tartrate, le vamizole as well NaF and less inhibited by vanadate and Zn2+. Intact amasti gotes were also able to hydrolyse phosphoserine, phosphothreonine and phosp hotyrosine but only the phosphotyrosine hydrolysis was stimulated by MgCl2 and inhibited by CaCl2 and phosphotyrosine was a competitive inhibitor of t he PNPP hydrolysis stimulated by Mg2+. The cells were also able to hydrolys e P-32-casein phosphorylated on serine and threonine residues but only in t he presence of MgCl2. These results indicate that in the amastigote form of T.: cruzi there are at least two ectophosphatase activities, one of which is Mg2+ dependent and can dephosphorylate phospho-aminoacids and phosphopro teins under physiological conditions.