title: Partial purification and characterization of dolichol phosphate mannose synthase from Entamoeba histolytica.
abstract: Dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis, was partially purified from E.histolytica by hydrophobic interaction and affinity chromatography with octyl Sepharose CL-4B and Affi-Gel 501, respectively. Reducing agents, particularly dithiothreitol, positively influenced enzyme activity and stability, indicating a role of sulfhydryl groups on the transferase function. Activity did not depend on phospholipids; however, it was significantly stimulated by phosphatidylethanolamine and to a lower extent by other common phospholipids. Mixtures consisting of activating phospholipids did not exert an additive effect. In vitro phosphorylation with a cAMP-dependent protein kinase resulted in enzyme activation. This alteration was not associated with a change in the K(m) for the substrate but rather with a 2.6-fold increase in V(max). Phosphorylation in the presence of [gamma-(32)P]ATP resulted in strong labeling of two polypeptides, one of which exhibited the molecular mass reported for the enzyme from other organisms. Whether phosphorylation functions in vivo as a mechanism of regulation of dolichol phosphate mannose synthesis in E.histolytica remains to be determined.
authors.affiliation: Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, México.
authors.forename: J C
authors.initials: JC
authors.lastname: Villagómez-Castro
authors.forename: C
authors.initials: C
authors.lastname: Calvo-Méndez
authors.forename: A
authors.initials: A
authors.lastname: Flores-Carreón
authors.forename: E
authors.initials: E
authors.lastname: López-Romero
mesh_terms: D000818:Animals; D004748:Entamoeba histolytica / Q000201:enzymology*; D008364:Mannosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation
chemical_list: D010743:Phospholipids; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase