Fig. 4.

AraT assays carried out via the Mt-PrsA-dependent conversion of [¹⁴C]-R5P to p[¹⁴C]Rpp and subsequent chase into a neoglycolipid acceptor. The absolute dependence of the cell wall AraT enzymes on the availability of pRpp was investigated using the neoglycolipid acceptor Ara₂. The basic assay contained (amongst other components described in the Materials and methods section) 0.5 mg of membranes and P60 from M. smegmatis, 2 mM α-d-Araf-(1 → 5)-α-d-Araf-O-(CH₂)₇CH₃, 100,000 cpm [¹⁴C]-R5P, 50 µg of DP and 2 mM ATP. Assays were initiated by the addition of [¹⁴C]-R5P and incubated for 1 h at 37°C. Individual assays carried out contained the addition of no Mt-PrsA, lane 1; 30 µg of Mt-PrsA, lane 2; 30 µg of Mt-PrsA and 100 µg/mL of EMB, lane 3 and 30 µg of Mt-PrsA and 2 mM ADP, lane 4. [¹⁴C]-Araf-linked products were extracted from the assay mix as described in the Materials and methods section and subsequently applied to a silica gel TLC plates, developed in CHCl₃:CH₂OH:H₂O:NH₄OH (65:25:3.6:0.5, v/v/v/v) and visualized by autoradiography from exposure of TLCs to X-ray film (Kodak X-Omat; Lee et al. 1997; Seidel et al. 2007).