Plasmodium merozoite surface protein-1 (MSP-1) is an essential antigen for the merozoite invasion of erythrocytes. A key challenge to the development of an effective malaria vaccine that can block the erythrocyte invasion is to establish the molecular interaction(s) among the parasite surface proteins as well as with the host cell encoded receptors. In the present study, we applied molecular interactions and proteome approaches to identify PfMSP-1 associated complex on the merozoite surface. Proteomic analysis identified a major malaria surface protein, PfRhopH3 interacting with PfMSP-1(42). Pull-down experiments with merozoite lysate using anti-PfMSP-1 or anti-PfRhopH3 antibodies showed 16 bands that when identified by tandem mass spectrometry corresponded to11 parasite proteins: PfMSP-3, PfMSP-6, PfMSP-7, PfMSP-9, PfRhopH3, PfRhopH1, PfRAP-1, PfRAP-2, and two RAP domain containing proteins. This MSP-1 associated complex was specifically seen at schizont/merozoite stages but not the next ring stage. We could also identify many of these proteins in culture supernatant, suggesting the shedding of the complex. Interestingly, the PfRhopH3 protein also showed binding to the human erythrocyte and anti-PfRhopH3 antibodies blocked the erythrocyte invasion of the merozoites. These results have potential implications in the development of PfMSP-1 based blood stage malaria vaccine.

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Alanine racemase catalyzes the interconversion of L-alanine and D-alanine and plays a crucial role in spore germination and cell wall biosynthesis. In this study, alanine racemase produced by Bacillus anthracis was expressed and purified as a monomer in Escherichia coli and the importance of lysine 41 in the cofactor binding octapeptide and tyrosine 270 in catalysis was evaluated. The native enzyme exhibited an apparent Km of 3 mM for L-alanine, and a Vmax of 295 μmoles/min/mg, with the optimum activity occurring at 37 o C and a pH of 8-9. The activity observed in the absence of exogenous pyridoxal 5 -phosphate suggested that the cofactor is bound to the enzyme. Additionally, the UV-visible absorption spectra indicated that the activity was pH independece, of VV-visible absorption spectra suggests that the bound PLP exists as a protonated Schiff's base. Furthermore, the loss of activity observed in the apoenzyme suggested that bound PLP is required for catalysis. Finally, the enzyme followed non-competitive and mixed inhibition kinetics for hydroxylamine and propionate with a Ki of 160 μM and 30 mM, respectively. [BMB reports 2009; 42(1): 47-52]

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