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- W154851609 abstract "The ..beta..-mannosyl transferase that catalyzes the transfer of mannose from GDP-mannose to GlcNAc-GlcNAc-PP-dolichol (GlcNAc/sub 2/-lipid) to form Man-..beta..-GlcNAc/sub 2/-lipid was solubilized from the microsomal fraction of pig aorta by treatment with 0.5% NP-40. The enzyme was purified about 115-fold using DEAE-cellulose, hydroxylapatite, and epoxy-activated Sepharose. The purified enzyme was free of ..cap alpha.. 1.3 and ..cap alpha..1,6-mannosyl transferases since the only product seen when enzyme was incubated with GDP-(/sup 14/C)-mannose and GlcNAc/sub 2/-lipid was Man-..beta..-GlcNAc/sub 2/-lipid. The oligosaccharide portion of this lipid was released by mild acid hydrolysis and characterized as Man-..beta..-GlcNAc-GlcNAc by gel filtration, as well as susceptibility to ..beta..-mannosidase and resistance to a ..cap alpha..-mannosidase. This partially purified enzyme was stabilized by adding 20% glycerol and 0.5 mM dithiothreitol to the storage buffer. Thus, the transferase was stable for 5 or 6 days at 0/sup 0/ and could be kept for a month at -20/sup 0/. The activity was greatly stimulated by detergent with optimum activity being seen at 0.1% NP-40. However, phospholipids had no effect. The transferase had a pH optimum of 7.0, and showed an almost absolute requirement for Mg/sup + +/, with maximum activity at 5 mM. The K/sub m/ for GDP-mannose was about 5more » x 10/sup -7/ M, and for GlcNAc/sub 2/-lipid about 1 x 10/sup -6/ M. The transferase was competitively inhibited by a variety of guanosine nucleotides.« less" @default.
- W154851609 created "2016-06-24" @default.
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- W154851609 date "1986-05-01" @default.
- W154851609 modified "2023-09-26" @default.
- W154851609 title "Purification of. beta. -mannosyl transferase that synthesizes Man-. beta. -GlcNAc-GlcNAc-PP-dolichol" @default.
- W154851609 hasPublicationYear "1986" @default.
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