FIGURE 5.

Conversion of AIP to AI by the membrane fraction of M. thermautotrophicus cell homogenates or homogenates of E. coli pET21a-MTH1691. [¹⁴C]AIP and [¹⁴C]AI were prepared from CDP-archaeol and “[¹⁴C]inositol 1-phosphate” under the conditions described under “Experimental Procedures.” The purified [¹⁴C]AIP (lanes 1, 2, 3, 5, 6, 7, and 8) or [¹⁴C]AI (lane 4) was incubated at 60 °C for 2 h with either enzyme preparation (500 μg protein/30–80 μl), the membranes of M. thermautotrophicus homogenate (lanes 1, 2, and 4), the supernatant fraction of M. thermautotrophicus homogenates (lane 3), the homogenates of E. coli pET21a-MTH1691 (lanes 5 and 6), or the homogenates of E. coli pET21a carrying an empty vector plasmid (lanes 7 and 8) in the presence of 0.1% Triton X-100. Other constituents in the reaction mixture were the same as in the AIP synthase reaction. The enzyme preparation was dissolved in 50 mm phosphate buffer (lanes 1, 4, 5, and 7), in aqueous dithiothreitol solution (lanes 2 and 3), and in Bicine buffer (lanes 6 and 8). After the reaction, lipids were extracted, separated by TLC, and recorded by autoradiography. s.f., solvent front.