caa a22 4500 46582630X CHVBK 20180323112150.0 cr unu---uuuuu 170327e19901101xx s 000 0 eng 10.1007/BF00691724 doi (NATIONALLICENCE)springer-10.1007/BF00691724 Horst Michael Biochemistry Section, Division of Basic Medical Science, School of Medicine, Mercer University, Macon, GA 31207, USA aut Isolation of a crustacean n -acetyl- d -glucosamine-1-phosphate transferase and its activation by phospholipids [Elektronische Daten] [Michael Horst] 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. Springer-Verlag, 1990 Chitin nationallicence GlcNAc-1-P transferase nationallicence Phospholipid nationallicence Biosynthesis nationallicence Artemia nationallicence SDS : sodium dodecyl sulfate nationallicence PMSF : phenyl methanesulfonylfluoride nationallicence HEPES : 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid nationallicence GlcNAc : N-acetyl-d-glucosamine nationallicence Dol-PP-GlcNAc : dolichol pyrophosphate N-acetyl-d-glucosamine nationallicence Dol-P-man : dolichol-phosphate-mannose nationallicence Dol-PP- (GlcNAc)2 : dolichol-pyrophosphate-di-N- acetylchitobiose nationallicence DMSO : dimethylsulfoxide nationallicence C:M (2:1) : chloroform:methanol (2:1) nationallicence C:M:W (10:10:3) : chloroform:methanol:water (10:10:3) nationallicence GlcNAc-1-P : N-acetyl-d-glucosamine-1-phosphate nationallicence Dol-P : dolichol phosphate nationallicence EGTA : ethylene glycol bis (b-aminoethyl ether)-NNN′N′ tetraacetic acid nationallicence Journal of Comparative Physiology B Springer-Verlag 159/6(1990-11-01), 777-788 0174-1578 159:6<777 1990 159 360 https://doi.org/10.1007/BF00691724 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00691724 text/html Onlinezugriff via DOI NATIONALLICENCE 100 1- Horst Michael Biochemistry Section, Division of Basic Medical Science, School of Medicine, Mercer University, Macon, GA 31207, USA aut NATIONALLICENCE 773 0- Journal of Comparative Physiology B Springer-Verlag 159/6(1990-11-01), 777-788 0174-1578 159:6<777 1990 159 360 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer