Isolation of a crustaceann-acetyl-d-glucosamine-1-phosphate transferase and its activation by phospholipids

Summary Then-acetyl-d-glucosamine-1-phosphate: dolichol phosphate transferase fromArtemia has been partially purified and characterized. The enzyme is solubilized from crude microsomes using Triton X-100, and after detergent removal appears to be associated with phospholipids. Using dolichol phosphate and UDP-n-acetyl-d-glucosamine as substrates, the enzyme catalyzes the formation of dolichol-pyrophosphate-n-acetyl-d-glucosamine. the product identity has been verified by TLC and paper chromatography following mild acid hydrolysis. Under the incubation conditions used only one product is made, i.e., Dol-p-p-GlcNAc. The formation of product is linear with increasing amounts of added protein and with time of incubation. The enzyme requires magnesium ions for activity. Activity of the enzyme is stimulated 6-fold by exogenous dolichol phosphate and is also stimulated by added phospholipids, with optimal activity being obtained in the presence of mixtures of phosphatidylcholine and phosphatidylglycerol. Enzymatic activity is not increased upon addition of GDP-mannose or dolichol phosphate mannose. The enzyme is rapidly inactivated by exposure to several detergents, including Triton X-100 and deoxycholate. The activity is inhibited by tunicamycin and by the purified B₂ homologue of this antibiotic. Other antibiotic inhibitors such as diumycin and polyoxin D have little effect on the enzyme. Both the microsomal and solubilized enzyme preparations are inactivated by 70% upon treatment with phospholipase A₂; activity may be restored by addition of phospholipids. Following hydrophobic interaction chromatography on Phenyl Sepharose, gel filtration chromatography on Sepharose CL-4B indicated that the enzyme, purified 81-fold, contained phophatidylcholine and phosphatidyl-ethanolamine.Abbreviations SDS sodium dodecyl sulfate - PMSF phenyl methanesulfonylfluoride - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - GlcNAc N-acetyl-d-glucosamine - Dol-PP-GlcNAc dolichol pyrophosphate N-acetyl-d-glucosamine - Dol-P-man dolichol-phosphate-mannose - Dol-PP- (GlcNAc)₂ dolichol-pyrophosphate-di-N- acetylchitobiose - DMSO dimethylsulfoxide - C:M (2:1) chloroform:methanol (2:1) - C:M:W (10:10:3) chloroform:methanol:water (10:10:3) - GlcNAc-1-P N-acetyl-d-glucosamine-1-phosphate - Dol-P dolichol phosphate - EGTA ethylene glycol bis (b-aminoethyl ether)-NNNN tetraacetic acid