ENERGY-DEPENDENT PROTEIN-TRIACYLGLYCEROL INTERACTION IN A CELL-FREE SYSTEM FROM 3T3-L1 ADIPOCYTES

Triacylglycerol is synthesized from the precursors sn-1,2-diacylglycer ol and palmitoyl-CoA in a reaction catalyzed by the microsomal enzyme diacylglycerol acyltransferase (EC 2.3.1.20). lsolated 3T3-L1 adipocyt e microsomal vesicles from cells pulse-labeled with L-[S-35]methionine were found to release microsomal proteins into a low density form dur ing the synthesis of triacylglycerol. The proteins released, which rep resent a subset of those present in the labeled microsomes, include a 62-kDa protein found in high concentration in mature fat droplets. The formation of the triacylglycerol-protein complexes was dependent on t ime and temperature, was not stimulated by cytosol, and required ATP a s well as diacylglycerol and palmitoyl-CoA. Only nucleoside triphospha tes and not non-hydrolyzable analogues could replace ATP in the reacti on. Unlike the enzyme reaction that measures the synthesis of triacylg lycerol, the formation of low density membrane is thus dependent on AT P hydrolysis as well as enzyme substrates. The newly formed, low densi ty particles are selectively enriched in triacylglycerol synthesized d uring the reaction as well as that synthesized prior to the reaction. The cell-free system described thus appears to represent an early adip ogenic event leading to the lipid vacuoles found in mature adipocytes.