FIGURE 5.

HPLC-MS monitoring of the GT MG517 activity. A, reaction monitoring with recombinant E. coli cell extracts (1.3 mg/ml total (solubilized) protein, 0.01 unit based on radiometric assay) with 1 mm UDP-Glc, 1.25 mm DOG. MGD, MGlcDOG m/z 806 [M + Na]⁺; DGD, DGlcDOG m/z 968 [M + Na]⁺; DOG, dioleoylglycerol m/z 644 [M + Na]⁺. B, reaction monitoring with purified GT MG517 (0.1 μm) under initial rate conditions. R1, reaction with 1 mm UDP-Glc, 1.25 mm DOG, where only MGlcDOG product is detected. R2, reaction with 1 mm UDP-Gal, 1.25 mm DOG, where MGalDOG is formed and a trace amount of DGalDOG. Conditions were as follows: 1 mm UDP-Glc or UDP-Gal, 1.25 mm (3.7 mol %) DOG, 12.5 mm (37 mol %) DOPG in 20 mm HEPES, 20 mm CHAPS, 20 mm MgCl₂, pH 8.0, at 35 °C.