Entamoeba histolytica: solubilization and biochemical characterization of dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis.
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 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.
Villagómez-Castro
J C
JC
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, México.
Calvo-Méndez
C
C
Vargas-Rodríguez
L
L
Flores-Carreón
A
A
López-Romero
E
E
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D003902:Detergents / Q000737:chemistry; D004287:Dolichol Monophosphate Mannose / Q000737:chemistry / Q000378:metabolism; D004748:Entamoeba histolytica / Q000201:enzymology*; D006023:Glycoproteins / Q000096:biosynthesis*; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008358:Mannose / Q000378:metabolism; D008364:Mannosyltransferases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology; D015800:Protozoan Proteins / Q000096:biosynthesis*; D012995:Solubility
D003902:Detergents; D006023:Glycoproteins; D009711:Nucleotides; D015800:Protozoan Proteins; D006155:Guanosine Diphosphate Mannose; D008345:Manganese; D004287:Dolichol Monophosphate Mannose; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D008274:Magnesium; D008358:Mannose; D002118:Calcium