Partial purification and characterization of .beta.-mannosyltransferase from suspension-cultured soybean cells

Abstract

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)

Glucomannan synthesis in pea epicotyls: The mannose and glucose transferases

“…The transferase from the pea internodes, unlike the soybean enzyme, did not require divalent cations for its activity nor did EDTA and EGTA alter its activity. The K~ value for the mannan synthesis is different from that reported for the Man-13-GlcNAc-GlcNAc-PP-dolichol formation brought about by the preparation reported by Kaushal and Elbein (1987).…”

“…However, in the present study, no lipid linked mono-or oligosaccharides were found in the incubations during the synthesis. Our enzymic preparations do not have the characteristics of the mannosyl transferase isolated from soybean cells that was shown to be involved in the formation of the mannose oligosaccharides attached to dolichol, and hence to glycoproteins (Kaushal and Elbein 1987). Triton X-100 inhibited the activity of the mannosyl transferase described here whereas this detergent was used to solubilize and activate the enzyme from soybean cells.…”