Characterization of an oligosaccharide-pyrophosphodolichol pyrophosphatase activity in yeast.

Use of Triton X-114 allowed us to develop a new method to separate hydrophilic oligosaccharidic material from hydrophobic oligosaccharide pyrophosphodolichols (oligosaccharide-PP-Dol). Taking advantage of this procedure we characterize, in yeast microsomal membranes, an enzymic activity that hydrolyses oligosaccharide-PP-Dol into oligosaccharidic material. H.p.l.c. analysis together with alkaline-phosphatase- and endo-N-acetyl-beta-D-glucosaminidase-susceptibility demonstrate that the oligosaccharidic released material is mainly composed of oligomannosides containing a chitobiose phosphate at the reducing end. The enzymic activity requires bivalent cations and is inhibited by pyrophosphate, NAD+ and bacitracin. As other, commercially available, pyrophosphatases have no action on lipid intermediates, the described pyrophosphatase activity appears to be the specific enzyme for oligosaccharide-PP-Dol. This enzymic splitting of the pyrophosphate bond might be the primary event in the catabolism of lipid intermediates.