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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.

1976; Elsevier BV; Volume: 251; Issue: 18 Linguagem: Inglês

10.1016/s0021-9258(17)33118-6

1083-351X

David M. Schlossman, Robert M. Bell,

Metabolomics and Mass Spectrometry Studies

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 K, of 8 FM for glycerol-P with a V,,,,, of 15.6 nmol/min/mg, while the DHAP acyltransferase activity showed an apparent K, of 40 PM for DHAP with a V,,,,, of 9.7 nmol/min/mg.Glycerol-P was a competitive inhibitor (K, = 7.2 FM) of the DHAP acyltransferase, and DHAP was a competitive inhibitor (K, = 92 pM) 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 uiuo ratio of glycerol-P to DHAP acylation should be greater than 24:l.'The abbreviations used are: glycerol-P, sn-glycerol 3-phosphate; DHAP, dihydroxyacetone phosphate.