Entamoeba histolytica: solubilization and biochemical characterization of dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis

Experimental Parasitology
Julio C Villagómez-CastroEverardo López-Romero

Abstract

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 velocity versus Dol-P and GDP-Man concentrations revealed sigmoidal and hyperbolic kinetics, respectively. Values of S0.5 for Dol-P and K(m) for GDP-Man were 15 micrograms/ml and 4.1 microM, 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/HCl 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 ...Continue Reading

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Citations

Apr 9, 2005·Parasite Immunology·Rafael Campos-Rodríguezp, Adriana Jarillo-Luna
Jan 5, 2010·Experimental Parasitology·Clara E Santacruz-TinocoEverardo López-Romero
Jun 13, 2009·Experimental Parasitology·Yolanda Terán-FigueroaCarlos Calvo-Méndez
May 26, 2005·Experimental Parasitology·Milena SalgadoRosana Sánchez-López
Sep 30, 1999·Experimental Parasitology·S Zamarripa-MoralesE López-Romero
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Jun 6, 2000·Molecular and Biochemical Parasitology·M A RodríguezE Orozco
Feb 22, 2003·Experimental Parasitology·Pablo Arteaga-NietoCarlos Calvo-Méndez

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