Ecto-phosphatase activities on the cell surface of the amastigote forms of Trypanosoma cruzi.
Live Trypanosoma cruzi amastigotes hydrolyzed p-nitrophenylphosphate (PNPP), phospho-amino-acids and 32P-casein under physiologically appropriate conditions. PNPP was hydrolysed at a rate of 80 nmol.mg-1.h-1 in the presence of 5 mM MgCl2, pH 7.2 at 30 degrees C. In the absence of Mg2+ the activity was reduced 40% and we call this basal activity. At saturating concentration 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+ (I50 = 0.43 mM). In the absence of Mg2+ (basal activity) the stimulating half concentration (S0.5) for PNPP was 1.57 mM, while at saturating MgCl2 concentrations the corresponding S0.5 for PNPP for Mg(2+)-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.99 mM. The Mg-dependent PNPP hydrolysis was strongly inhibited by sodium fluoride (NaF), vanadate and Zn2+ but not by tartrate and levamizole. The Mg-independent basal phosphatase activity was insensitive to tartrate, levamizole as well NaF and less inhibited by vanadate and Zn2+. Intact amastigotes were also able to hydrolyse phosphoserine, phosphothreonine and phosphotyrosine but only the phosphotyrosine hydrolysis was stimulated by MgCl2 and inhibited by CaCl2 and phosphotyrosine was a competitive inhibitor of the PNPP hydrolysis stimulated by Mg2+. The cells were also able to hydrolyse 32P-casein phosphorylated on serine and threonine residues but only in the 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-amino acids and phosphoproteins under physiological conditions.
Meyer-Fernandes
J R
JR
Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brasil. Meyer@server.bioqmed.ufrj.br
da Silva-Neto
M A
MA
Soares
M dos S
Mdos S
Fernandes
E
E
Vercesi
A E
AE
de Oliveira
M M
MM
D009597:4-Nitrophenylphosphatase / Q000378:metabolism*; D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology*; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
D015636:Magnesium Chloride; D009597:4-Nitrophenylphosphatase; D002122:Calcium Chloride