Entamoeba histolytica:Solubilization and Biochemical Characterization of Dolichol Phosphate Mannose Synthase, an Essential Enzyme in Glycoprotein Biosynthesis

Abstract

Villagómez-Castro, J. C., Calvo-Méndez, C., Vargas-Rodrı́guez, L., Flores-Carreón, A., and López-Romero, E. 1998.Entamoeba histolytica:Solubilization and biochemical characterization of dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis.Experimental Parasitology88, 111–120. Sequential treatment of trophozoite membranes with the nonionic detergents Brij 35 and Igepal CA-630 released a soluble fraction that efficiently catalyzed the transfer of mannose from GDP-Man into a mannolipid that was identified as dolichol phosphate mannose (Dol-P-Man) by several criteria. The transfer reaction occurred only in the presence of exogenously added dolichol monophosphate (Dol-P). Plots of enzyme velocityversusDol-P and GDP-Man concentrations revealed sigmoidal and hyperbolic kinetics, respectively. Values ofS0.5for Dol-P andKmfor GDP-Man were 15 μg/ml and 4.1 μM, respectively. The solubilized fraction failed to transfer the label into other products such as lipid-linked oligosaccharides and glycoproteins. The optimum pH was 7.5–8.0 in potassium phosphate or Tris;shHCl buffers and the enzyme required either Mg2+or Mn2+. The latter was more effective but in a narrower range of concentrations. The transferase was inhibited by a number of nucleotides the strongest being GMP, GDP, and GTP. When assayed in the reverse direction, however, the enzyme catalyzed the transfer of mannose from Dol-P-Man back into GDP-Man as a function of increasing concentrations of GDP. Mg2+was a better activator of the reverse reaction than Mn2+, which reached up to 60% at 2 mM GDP. These results suggest that some of the enzyme catalytic properties may change depending on the direction of the transfer reaction.