Although increased BAT concentrations of fatty acyl-CoAs and decreased thioesterase activities of BAT homogenates suggested that Them1 functions as an Acot in vivo (4), the enzymatic characteristics of mouse Them1 and the two alternatively spliced isoforms of human THEM1, herein tech support referred to as THEM1a (synonym BFIT1) and THEM1b (synonym BFIT2), remain unknown. The current study was designed to systematically explore key properties of purified recombinant Them1/THEM1 and to correlate these with measurements of acyl-CoA thioesterase activity in subcellular fractions of BAT and liver from Them1+/+ and Them1?/? mice. Our results demonstrate that Them1 forms a homodimer that preferentially hydrolyzes long-chain fatty acyl-CoAs and that the START domain contributes toward optimizing this activity.

MATERIALS AND METHODS Reagents Acyl-CoA thioesters, myristic acid, ATP, the nonhydrolyzable ATP analog adenosine 5��-[��-thio]triphosphate (ATP-��-S), ADP, CoASH, and 5,5��-dithiobis-(2-nitrobenzoic acid) were purchased from Sigma-Aldrich (St. Louis, MO). The plasmid pCMV-SPORT6-Them1 was obtained from Invitrogen (Grand Island, NY), pCMV6-XL5-BFIT1 was from OriGene (Rockville, MD), pGEX-KG was from Lablife (Northfield, MN), and pCR4-TOPO-BFIT2 and pCR4-TOPO-Acot12 were from Open Biosystems (Lafayette, CO). A glutathione S-transferase (GST)-PCTP expression plasmid (pGEX-KG-PCTP) was as described (10). Phusion High-Fidelity DNA Polymerase was from Thermo Scientific (Waltham, MA).

Molecular cloning The open reading frames of full-length and truncated Them1, THEM1a, THEM1b, and Acot12 were amplified using PCR based on template plasmids, digested with restriction enzymes, and cloned into the expression vector pGEX-KG according to supplementary Table I. Expression and purification of recombinant proteins Expression plasmids were transformed into the Escherichia coli strain BL21 (DE3). Bacteria were grown in LB, and protein expression was induced using 2 mM isopropyl ��-D-thiogalactopyranoside followed by shaking at 23��C for 24�C72 h. Bacteria were pelleted by centrifugation and lysed using 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM EDTA, and 0.5% Triton X-100 plus protease inhibitors (Protease Inhibitor Cocktail Tablets; Roche, Basel, Switzerland). Lysates were sonicated 6 �� 10 s (Fisher Sonic Dismembrator Model 300; Fisher Scientific, Pittsburgh, PA), rotated at 4��C for 1 h, and centrifuged at 11,000 g for 20 min.

If not used immediately, supernatants were frozen at ?80��C. GST fusion proteins were purified by FPLC using a GST affinity column (GSTrap HP Cilengitide column; GE Healthcare, Waukesha, WI). Briefly, lysates were applied to the column after equilibration with 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 (pH 7.3) and then washed with 5 column volumes of the same buffer. GST-fusion proteins were eluted using 10 mM reduced glutathione (50 mM Tris-HCl at pH 8.0).