Biosynthesis of glycoproteins in Candida albicans: activity of mannosyl and glucosyl transferases.
The enzymes dolichol phosphate glucose synthase and dolichol phosphate mannose synthase (DPMS), which catalyze essential steps in glycoprotein biosynthesis, were solubilized and partially characterized in Candida albicans. Sequential incubation of a mixed membrane fraction with increasing concentrations of Nonidet P-40 released a soluble fraction that transferred glucose from UDP-Glc to dolichol phosphate glucose and minor amounts of glucoproteins in the absence of exogenous dolichol phosphate. Studies with the soluble fraction revealed that some properties were different from those previously determined for the membrane-bound enzyme. Accordingly, the soluble enzyme exhibited a twofold higher affinity for UDP-Glc and a sixfold higher affinity over the competitive inhibitor UMP, and the transfer reaction was fourfold more sensitive to inhibition by amphomycin. On the other hand, a previously described protocol for the solubilization of mannosyl transferases that rendered a fraction exhibiting both DPMS and protein mannosyl transferase (PMT) activities operating in a functionally coupled reaction was modified by increasing the concentration of Nonidet P-40. This resulted in a solubilized preparation enriched with DPMS and nearly free of PMT activity which remained membrane bound. DPMS solubilized in this manner exhibited an absolute dependence on exogenous Dol-P. Uncoupling of these enzyme activities was a fundamental prerequisite for future individual analysis of these transferases.
Arroyo-Flores
B L
BL
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Guanajuato, Gto 36000, México.
Rodríguez-Bonilla
J
J
Villagómez-Castro
J C
JC
Calvo-Méndez
C
C
Flores-Carreón
A
A
López-Romero
E
E
D002176:Candida albicans / Q000201:enzymology* / Q000254:growth & development; D002462:Cell Membrane / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis; D005964:Glucosyltransferases / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis*; D008364:Mannosyltransferases / Q000378:metabolism*; D012995:Solubility
D005656:Fungal Proteins; D006023:Glycoproteins; D005964:Glucosyltransferases; D008364:Mannosyltransferases; C033917:UDPglucose dolicholphosphate glucosyltransferase; C022755:dolichyl-phosphate beta-D-mannosyltransferase