Fig. 1. Design, purification, and activities of recombinant proteins. A: Schematic diagram of recombinant proteins used in this study. Full-length proteins were Them1 (aa 1�C594), THEM1a (aa 1�C607), THEM1b (aa 1�C594), Acot12 (aa 1�C556), … We initially compared the thioesterase selleck compound activity of GST-Them1 and Them1 using long-chain fatty acyl-CoAs (i.e., myristoyl-CoA, palmitoyl-CoA, and stearoyl-CoA).2 shows that the enzymatic activities of Them1 before (left panel) and after (right panel) removal of the GST tag. GST-Them1 demonstrated thioesterase activity using all three substrates but most efficiently hydrolyzed palmitoyl-CoA. Removal of the GST tag from Them1 increased the Km (��M) and Vmax (nmol/min/mg) values of Them1 for palmitoyl-CoA (Km: GST-Them1 0.3 �� 0.02, Them1 7.5 �� 0.

5; Vmax: GST-Them1 14.8 �� 0.6, Them1 38.1 �� 1.8) and myristoyl-CoA (Km: GST-Them1 1.0 �� 0.2, Them1 8.1 �� 0.9; Vmax: GST-Them1 14 �� 0.5, Them1 20.9 �� 1.7) but not stearoyl-CoA (Km: GST-Them1 28.6 �� 6.9, Them1 28.0 �� 4.8; Vmax: GST-Them1 10.3 �� 1.3, Them1 10.3 �� 0.8). On this basis, the GST was removed in subsequent experiments. Oligomerization of Them1 We next used FPLC to examine whether Them1 forms oligomers and to determine the conditions that influence oligomerization (Fig. 2). At 23��C and 4��C, Them1 eluted principally at a molecular weight of 67 kDa, which is consistent with a monomer (Fig. 2A). At 4��C, there was a small second peak associated with the main peak that corresponded to an apparent molecular weight of 112 kDa and suggested the potential presence of dimers.

Upon incubation with palmitoyl-CoA, the elution volume for the entire Them1 protein peak corresponded to an apparent molecular weight of 112 kDa (Fig. 2B), indicative of a Them1 dimer. The same effect was induced by preincubation of Them1 with CoASH but not by myristic acid. Because ATP has been shown to induce tetramerization of Acot12 (17), we tested its effects on Them1. When Them1 was preincubated with ATP (Fig. 2C), we again observed a decrease in elution volume, suggestive of dimerization. The same effect was also observed in the presence ATP-��-S and ADP. In keeping with the observation that it exists as monomer or dimer in the absence of ATP (17), the elution profile of Acot12 under the current experimental conditions (Fig. 2D) was indicative of a monomer with a molecular weight of 67 kDa. A leading shoulder associated with this peak was consistent with a dimer fraction. After incubation with ATP, ATP-��-S, or acetyl-CoA, the elution volume of Acot12 corresponded primarily to an apparent molecular weight of 225 kDa, consistent with a tetramer, but also a smaller peak of 95 kDa apparent AV-951 molecular weight, consistent with a dimer (17). Fig. 2. Oligomerization of Them1.