Assessment and Characterization of Ca2+ - ATPase expression in selected isolates and clones of Plasmodium Falciparum

O M Bolaji, T C Happi, E A Bababunmi

Abstract


Ca2+-ATPase expression in 15 selected isolates from malaria patients at the University College Hospital (UCH) Ibadan and two cloned strains (W2-chloroquine resistant, D6-chloroquine sensitive) of P.falciparum was assessed using spectrophotometric assay method. The kinetics of activity of Ca2+ - ATPase in three isolates (NCP 14, NCP5, NCP1) and two clones (W2, D6) also assessed. 12% SDS – PAGE analysis of total proteins in one isolate (NCP14) and two clones (W2, D6) was also investigated. All the selected isolates and the two cloned strains exhibited measurable Ca2+-ATPase activity. The Ca2+ - ATPase activity in cloned strain D6 (6.50 + 0.74mmolPi/min/mg protein) was higher than in cloned strain W2 (3.93 + 0.61mmolPi/min/mg protein.  The Ca2+-ATPase activity in isolates from malaria patients varied widely (1.95 + 0.74 – 21.56 +1.43mmolPi/min/mg protein). The kinetic constants obtained for the two cloned strains showed that clone W2 had a higher Vmax (Vmax = 363mmolPi/min/mg protein) than clone D6 (Vmax = 74mmolPi/min/mg protein). All the isolates and the two cloned strains showed similar affinity for ATP (Km ~ 10mM). Scan of SDS-PAGE gel of total proteins in the isolate and cloned strains showed the presence of oligopeptide bands of molecular weights range of 148-176 KDa; 116-123 KDa respectively. These suggest the presence of predicted polypeptide of Ca2+ - ATPase nature of molecular weight estimate of 139 KDa. The study agrees with previous findings that Ca2+-ATPase is functionally expressed in P.falciparum, The study also indicates that Ca2+ - ATPase functional expression may vary with isolate or clone but the ATP binding mechanism to the enzyme is similar in all isolates and clones of P.falciparum. The study further suggests a possible association between acquisition of chloroquine resistance and Ca2+- ATPase functional expression in P.falciparum.

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