Partial purification and characterization of beta-mannosyltransferase from suspension-cultured soybean cells.
The beta-mannosyltransferase that catalyzes the synthesis of Man-beta-GlcNAc-GlcNAc-PP-dolichol from GDP-mannose and dolichyl-PP-GlcNAc-GlcNAc was solubilized from microsomes of suspension-cultured soybean cells by treatment with 1.5% Triton X-100 and was purified about 700-fold by chromatography on DEAE-cellulose, hydroxylapatite, and a GDP affinity column. The purified enzyme was reasonably stable in the presence of 20% glycerol and 0.5 mM dithiothreitol. The enzyme required either detergent (Triton X-100 or NP-40) or phospholipid for maximum activity, but the effects of these two were not additive. Thus, either phosphatidylcholine or Triton X-100 could give maximum stimulation. In terms of phospholipid stimulation, both the head group and the acyl chain appeared to be important since phosphatidylcholines with 18-carbon unsaturated fatty acids were most effective. The purified enzyme had a sharp pH optimum of 6.9-7.0 and required a divalent cation. Mg2+ was the best metal ion with optimum activity occurring at 6 mM, but Mn2+ was reasonably effective while Ca2+ was slightly stimulatory. The Km for GDP-mannose was calculated to be 1.7 X 10(-6) M and that for dolichyl-PP-GlcNAc-GlcNAc about 9 X 10(-6) M. The enzyme was inhibited by a number of guanosine nucleotides such as GDP-glucose, GDP, GMP, and GTP, but various uridine and adenosine nucleotides were without effect. The purified enzyme was apparently free of alpha-1,3-mannosyltransferase (and perhaps other mannosyltransferases) and dolichyl-P-mannose synthase since the only product seen from dolichyl-PP-GlcNAc-GlcNAc and GDP-mannose was Man-beta-GlcNAc-GlcNAc-PP-dolichol.(ABSTRACT TRUNCATED AT 250 WORDS)
Kaushal
G P
GP
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
Elbein
A D
AD
D002478:Cells, Cultured; D002845:Chromatography; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D017886:Durapatite; D006150:Guanine Nucleotides / Q000494:pharmacology; D006602:Hexosyltransferases / Q000302:isolation & purification*; D006882:Hydroxyapatites; D007700:Kinetics; D008364:Mannosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D010944:Plants / Q000201:enzymology*; D013025:Glycine max
D006150:Guanine Nucleotides; D006882:Hydroxyapatites; D017886:Durapatite; D006602:Hexosyltransferases; D008364:Mannosyltransferases