Synthesis of sn-1,2-diacylglycerols by monoacylglycerol acyltransferase from Manduca sexta fat body.
The pathway for the synthesis of sn-1,2-diacylglycerol stimulated by the action of adipokinetic hormone (AKH) in the insect fat body is unknown. Previous results from this laboratory suggested that the hydrolysis of stored triacylglycerol to sn-2-monoacylglycerol followed by the stereospecific acylation of sn-2-monoacylglycerol catalyzed by a monoacylglycerol-acyltransferase (MGAT) could be the major route of AKH-stimulated sn-1,2-diacylglycerol synthesis. Thus, MGAT might represent a key enzyme of this pathway. In this study we characterized the MGAT activity from the Manduca sexta fat body. The activity, which was assayed by acylation of 2-monoolein using radioactive labeled palmitoyl-CoA, was found to be primarily a microsomal enzyme. The products of the acylation of 2-monoolein were 1,2-diacylglycerol (40-50%), 1,3-diacylglycerol (20-30%), and triacylglycerol (30-40%). The presence of triacylglycerol as a product revealed the presence of diacylglycerol-acyltransferase activity in the fat body microsomes. The pH optimum of MGAT activity was 7.0, and the dependence of the activity on the concentration of 2-monoolein showed saturation kinetics. An endogenous MGAT activity, which represented 20% of the maximal activity observed with added substrate, was detected. Optimal concentrations of palmitoyl-CoA ranged between 0.10-0.20 mM. The specific activity of MGAT, measured under optimal conditions, was about 0.6 nmol DG formed/min-mg protein. MGAT activity was greatest with 2-monoolein, and lower activity was observed when a saturated 2-monoacylglycerol was employed. The activity observed with sn-1-monoacylglycerol was lower than that observed with sn-2-monoacylglycerol. AKH did not stimulate MGAT activity, suggesting that either the enzyme is not under hormonal regulation or the monoacylglycerol pathway is not involved in the AKH-stimulated production of sn-1,2-diacylglycerol in the M. sexta fat body.
Arrese
E L
EL
Department of Biochemistry and Center for Insect Science, University of Arizona, Tucson 85721, USA.
Rojas-Rivas
B I
BI
Wells
M A
MA
D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D004075:Diglycerides / Q000096:biosynthesis*; D005216:Fat Body / Q000378:metabolism*; D007301:Insect Hormones / Q000378:metabolism; D018402:Manduca / Q000201:enzymology*; D009842:Oligopeptides / Q000378:metabolism; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives
C011439:1,2-diacylglycerol; D004075:Diglycerides; D007301:Insect Hormones; D009842:Oligopeptides; C044833:adipokinetic hormone; D000217:Acyltransferases; C021802:2-acylglycerol O-acyltransferase; D011761:Pyrrolidonecarboxylic Acid