Triacylglycerol synthesis in isolated fat cells. Evidence that the sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities are dual catalytic functions of a single microsomal enzyme.
The acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) (glycerol-P acyltransferase) and acyl-CoA:dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) (DHAP acyltransferase) activities were investigated in vitro in order to evaluate the quantitative contribution of the glycerol-P and DHAP pathways for the synthesis of triacylglycerols in isolated fat cells and to test the hypothesis that these two activities may be dual catalytic functions of a single enzyme. More than 85% of both acyltransferase activities was associated with the microsomal subcellular fraction. The microsomal glycerol-P acyltransferase activity showed an apparent Km of 8 muM for glycerol-P with a Vmax of 15.6 nmol/min/mg, while the DHAP acyltransferase activity showed an apparent Km of 40 muM for DHAP with a Vmax of 9.7 nmol/min/mg. Glycerol-P was a competitive inhibitor (Ki = 7.2 muM) of the DHAP acyltransferase, and DHAP was a competitive inhibitor (Ki = 92 muM) of the glycerol-P acyltransferase. The two acyltransferase activities showed virtual identity in their pH dependence, acyl-CoA chain length dependence, thermolability, and inactivation by N-ethylmaleimide. Trypsin, detergents, collagenase, phospholipases, and various salts and organic solvents also had similar effects on both activities. Taken as a whole, the data strongly suggest that the microsomal glycerol-P and DHAP acyltransferase activities actually represent dual functions of a single enzyme. Calculations based on the above kinetic constants and previously reported glycerol-P and DHAP pools in adipocytes suggest that the in vivo ratio of glycerol-P to DHAP acylation should be greater than 24:1.
D000217:Acyltransferases / Q000378:metabolism*; D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D003840:Deoxycholic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology; D014280:Triglycerides / Q000096:biosynthesis*
D014280:Triglycerides; D003840:Deoxycholic Acid; D011092:Polyethylene Glycols; D000217:Acyltransferases; D005992:Glycerol-3-Phosphate O-Acyltransferase; D005033:Ethylmaleimide