SOLUBILIZATION AND ONE-STEP PURIFICATION OF MANNOSYLPHOSPHODOLICHOL SYNTHASE FROM TRICHODERMA-REESEI

Mannosylphosphodolichol synthase (MPD-synthase) (EC 2.4.1.830) catalyz ing formation of MPD from GDPMan and dolichylphosphate (PD) has been p urified from T. reesei cellular membranes almost to homogeneity. Selec tive solubilization of the enzyme was followed by one step purificatio n on Phenyl-Sepharose column. SDS/ PAGE of the purified enzyme fractio n revealed the presence of a protein band of 31 kDa corresponding to t he apparent molecular mass of the MPD-synthase purified from S. cerevi siae [Babczinski, P. et al. (1980) fur. J. Biochem. 105, 509-515; Hase lbeck A. (1989) Eur. J. Biochem. 181, 663-668]. During: solubilization , the enzyme was stabilized by the presence of a lipophilic substrate dolichylphosphate and phospholipids as well as by protease inhibitors. The Phenyl-Sepharose purified enzyme had an absolute requirement for dolichylphosphate and was activated by cAMP dependent protein kinase.