Purification and characterization of dolichyl-P-mannose:Man5(GlcNAc)2-PP-dolichol mannosyltransferase.
The dolichyl-P-mannose:dolichyl-PP-heptasaccharide alpha-mannosyltransferase (2.4.1.130), which catalyzes the transfer of mannose from dolichyl-P-mannose to the Man5(GlcNAc)2-PP-dolichol acceptor glycolipid, was solubilized from pig aorta microsomes with 0.5% NP-40 and purified 985-fold by a variety of conventional methods. The partially purified enzyme had a pH optimum of 6.5 and required Ca2+, at an optimum concentration of 8-10 mM, for activity. Mn2+ was only 20% as effective as Ca2+, and Mg2+ was inhibitory. The mannosyltransferase activity was also inhibited by the addition of EDTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The enzyme was quite specific for dolichyl-P-mannose as the mannosyl donor and Man5(GlcNAc)2-PP-dolichol as the mannosyl acceptor. The Km values for dolichyl-P-mannose and the acceptor lipid Man5(GlcNAc)2-PP-dolichol were 1.8 and 1.6 microM. On Bio-Gel P-4 columns and by HPLC, the radiolabeled oligosaccharide formed during incubation of dolichyl-P-[14C]mannose and unlabeled Man5(GlcNAc)2-PP-dolichol with the purified enzyme behaved like Man6(GlcNAc)2. This octasaccharide was susceptible to digestion by endoglucosaminidase H, indicating that the newly added mannose was attached to the 6-linked mannose in an alpha 1,3-linkage. This linkage was further confirmed by acetolysis of the oligosaccharide product [i.e., Man6(GlcNAc)2], which gave a labeled disaccharide as the major product (greater than 90%).
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Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
Kaushal
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Pan
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Elbein
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D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000201:enzymology; D002412:Cations / Q000494:pharmacology; D002855:Chromatography, Thin Layer; D004287:Dolichol Monophosphate Mannose / Q000737:chemistry / Q000302:isolation & purification*; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D008364:Mannosyltransferases / Q000737:chemistry / Q000302:isolation & purification*; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D013379:Substrate Specificity; D013552:Swine
D002412:Cations; D004287:Dolichol Monophosphate Mannose; D004492:Edetic Acid; D008364:Mannosyltransferases; C066968:dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichol alpha-1,3-mannosyltransferase