Purification and properties of beta-mannosyltransferase that synthesizes Man-beta-GlcNAc-GlcNAc-pyrophosphoryl-dolichol.
The beta-mannosyltransferase that adds mannose, from GDP-mannose, to GlcNAc-GlcNAc-pyrophosphoryl-dolichol, to form Man-beta-GlcNAc-GlcNAc-pyrophosphoryl-dolichol was solubilized from pig aorta microsomal preparations, using 0.5% NP-40, and was purified about 116-fold using conventional methods. The purified enzyme was mostly free of alpha 1,3- or alpha 1,6-mannosyltransferase activities, since Man beta-GlcNAc-GlcNAc-PP-dolichol (PP = pyrophosphoryl) accounted for more than 95% of the product when enzyme was incubated with GDP-[14C]mannose and GlcNAc-GlcNAc-PP-dolichol. Very little Man-beta-GlcNAc-GlcNAc-PP-dolichol was formed when GDP-[14C]mannose was replaced by dolichol-phosphoryl-[14C]mannose, indicating that GDP-mannose was the mannosyl donor. The oligosaccharide portion of this lipid was released by mild acid hydrolysis and was characterized by gel filtration as well as by susceptibility to beta-mannosidase and resistance to alpha-mannosidase. The partially purified enzyme could be stabilized by the addition of 20% glycerol and 0.5 mM dithiothreitol to the buffer, and could be kept in this solution for 5 or 6 days in ice. The enzyme was greatly stimulated by the addition of detergent (NP-40) with optimum activity being observed at 0.1%. However, no stimulation was seen with any phospholipid. The partially purified enzyme had a pH optimum of about 7.0, and showed an almost absolute requirement for Mg2+ with optimal activity occurring at about 5 mM Mg2+. Mn2+ and Ca2+ were only slightly active. The Km for GDP-mannose was about 5 X 10(-7) M and that for GlcNAc-GlcNAc-PP-dolichol about 1 X 10(-6) M. Beta-Mannosyltransferase activity was inhibited competitively by a variety of guanosine nucleotides with GDP and GDP-glucose being most active, but GTP, GMP, guanosine, and periodate-oxidized guanosine were also effective. The enzyme was strongly inhibited by p-chloromercuribenzenesulfonic acid and this inhibition was partially prevented by the addition of dithiothreitol.
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000201:enzymology; D002384:Catalysis; D004795:Enzyme Stability; D006150:Guanine Nucleotides / Q000494:pharmacology; D006602:Hexosyltransferases / Q000302:isolation & purification*; D008364:Mannosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism*; D011105:Polyisoprenyl Phosphate Sugars / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity; D013552:Swine
D006150:Guanine Nucleotides; D011104:Polyisoprenyl Phosphate Oligosaccharides; D011105:Polyisoprenyl Phosphate Sugars; D002731:4-Chloromercuribenzenesulfonate; C038433:dolichol pyrophosphate Man(1-4)-GlcNAc(1-4)GlcNAc; D006602:Hexosyltransferases; D008364:Mannosyltransferases; C035777:chitobiosyldiphosphodolichol beta-mannosyltransferase