title: Characterization and partial purification of a novel mannosylphosphodolichol phosphodiesterase from chicken liver microsomes.
abstract: Mannosylphosphodolichol phosphodiesterase, which catalyzes the release of mannose from mannosylphosphodolichol, was solubilized from chicken liver microsomes by treatment with the non-ionic detergent, Emulgen 909. The enzyme was partially purified using ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration on Sepharose 6B. The enzyme showed absolute requirement for sulfhydryl reducing agents. The enzyme activity was stimulated by the addition of CaCl2 and Emulgen 909 and exhibited a pH optimum around 5.3. The Km value for mannosylphosphodolichol was found to be 0.43 microM. The activity was competitively inhibited by dolichyl phosphate and dolichol and the Ki value for dolichyl phosphate was estimated to be 12.5 microM. The purified preparation had no activity toward N-acetylglucosaminyldiphosphodolichol, glucosylphosphodolichol, mannose 1-phosphate, or artificial substrates for mannosidases, glucosidases, acid phosphatase, and acid phosphodiesterase. A heat-stable factor which stabilizes the mannosylphosphodolichol phosphodiesterase was separated from the enzyme by DEAE-cellulose chromatography. It was precipitated by trichloroacetic acid and not extracted into lipid solvents. The separation resulted in the complete loss of the enzyme activity and the restoration of the activity was not observed when the factor was added back to the enzyme solution.
authors.affiliation: Institute for Enzyme Research, University of Tokushima School of Medicine, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Tomita
authors.forename: Y
authors.initials: Y
authors.lastname: Motokawa
mesh_terms: D000818:Animals; D002645:Chickens; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
chemical_list: D010727:Phosphoric Diester Hydrolases; C054533:dolichyl-phosphate-mannose phosphodiesterase