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Tri acyl glycerol synthesis in isolated fat cells studies on the microsomal di acyl glycerol acyl transferase ec 2.3.1.20 activity using ethanol dispersed di acyl glycerols


Journal of Biological Chemistry 251(15): 4537-4543
Tri acyl glycerol synthesis in isolated fat cells studies on the microsomal di acyl glycerol acyl transferase ec 2.3.1.20 activity using ethanol dispersed di acyl glycerols
The acyl-CoA:1,2-diacylglycerol acyltransferase (EC 2.3.1.20) activity of isolated fat cells [rat] was predominantly (89%) localized to the microsomal subcellular fraction by assays based on the conversion of 1,2-[3H]diacyl-sn-glycerol to triacylglycerol using 1 to 4 .mu.g of protein. A complementary assay based on the conversion of [3H]palmitoyl-CoA to triacylglycerol was developed. These methods, 100 to 1000 times more sensitive than those previously employed, were used to characterize the microsomal activity. The choice of dispersing agent for addition of diacylglycerol to the reaction mixture was crucial. Addition of diacylglycerol in ethanol resulted in the highest diacylglycerol acyltransferase activity of the methods tested. Tween 20, which was previously employed as the dispersing agent, severely inhibited the activity. A broad pH optimum from 7.4 to 8.0 was noted and several salts stimulated the activity more than 2-fold. The activity was unstable at temperatures of 28.degree. and above. Dependences of acyl-CoAs containing 6-18 C atoms were investigated using bacterial diacylglycerol. Acetyl- and butyryl-CoA were not substrates. Highest diacylglycerol acyltransferase activities were observed with decanoyl-CoA and lower activities were noted with longer and shorter saturated chains. Maximal activity with oleoyl-CoA was only 34% of that seen with stearoyl-CoA. No simple relationship between the critical micellar concentrations of the acyl-CoAs employed and diacylglycerol acyltransferase activity was observed. The dependences of diacylglycerols containing fatty acids 6-18 C atoms in length were investigated with [3H]palmitoyl-CoA. While all the 1,2-diacyl-sn-glycerols tested were substrates, diacylglycerol acyltransferase activity was highest with 1,2-dioleoylglycerol. Maximum activity with the bacterial diacylglycerol was 86% that with 1,2-dioleoylglycerol. The diacylglycerol concentrations required for half-maximal velocity were 20-40 .mu.M for long chain diacylglycerols and 2-3 .mu.M for short chain diacylglycerols; these were 75 and 750-fold lower than previously reported. Microsomal diacylglycerol acyltransferase specific activities from isolated cells around 50 nmol/min per mg, 10 to 50-fold higher than previously reported from adipose tissue, were typical using dioleoylglycerol and palmitoyl-CoA as substrates. Diacylglycerol acyltransferase specific activities were 17-fold higher in microsomes from isolated fat cells than any other tissues examined. The diacylglycerol acyltransferase appears to have specificity with respect to acyl-CoAs and diacylglycerols, but a definitive interpretation is limited by the lack of data on the physical properties of these substrates in solution under the conditions employed.


Accession: 006839855



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