Glycobiology, 2000, Vol. 10, No. 12 1311-1316
© 2000 Oxford University Press
Partial purification and characterization of dolichol phosphate mannose synthase from Entamoeba histolytica
2Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, México, and 3Departamento de Genética y Biología Molecular, CINVESTAV del IPN, Apartado Postal 14–740, México, D.F. 07000, México
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 Km for the substrate but rather with a 2.6-fold increase in Vmax. Phosphorylation in the presence of [
-32P]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.
1 To whom correspondence should be addressed at: IIBE, Facultad de Química, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36000, México