Table 3.

Enzymatic transfer of mannose from C₁₀ donors

Mannosyl donor	[3H]mannose transferred to acceptor (pmol/min/mg membrane protein)
	Man5GlcNAc2-P-P-dolichol (Lec15)	mannan (M. luteus)
[3H]MPC	1.60	21.0
[3H]MPN	0.04 (2.5%)	18.2 (86.7%)

To assay LLO synthesis, enzymatic reactions included microsomes (0.4 mg of protein) from Lec15 cells (which accumulate acceptors for MPD-dependent mannosyltransferases), 40 mM Tris-HCl (pH 7.4), 4 mM CaCl₂, and either [³H]MPC (100 cpm/pmol) or [³H]MPN (22 cpm/pmol) in a total volume of 0.025 ml. Following incubation at 37°C for 5 min, the amount of labeled mannose transferred into LLO was determined (Rush et al., 1993). To assay the Micrococcus luteus mannosyltransferases, reaction mixtures contained micrococcal membranes (0.22 mg of protein), 20 mM Na-PIPES (pH 6.6), 10 mM MnCl₂, and either [³H]MPC or [³H]MPN (25 cpm/pmol) in a total volume of 0.02 ml. Following incubation for 10 min at 21°C, the amount of radiolabeled mannose incorporated into mannan was determined (Rush et al., 1993). The results for both donors are expressed as picomoles of mannose transferred per minute per milligram of membrane protein, and the results for MPN are expressed as a percentage of the MPC activity.