Coleman R, Bell R M
J Biol Chem. 1976 Aug 10;251(15):4537-43.
The acyl-CoA:1,2-diacylglycerol acyltransferase (EC 2.3.1.20) activity of isolated fat cells 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 mug 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 has previously been 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 degrees and above. Dependences on acyl-CoAs containing 6 to 18 carbon 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 on diacylglycerols containing fatty acids 6 to 18 carbon 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 to 40 muM for long chain diacylglycerols and 2 to 3 muM for short chain diacylglycerols; these were 75- to 750-fold lower than previously reported. Microsomal diacylglycerol acyltransferase specific activities from isolated cells around 50 nmol/min/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 tissue 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.
通过基于使用1至4微克蛋白质将1,2-[³H]二酰基-sn-甘油转化为三酰甘油的测定，分离的脂肪细胞的酰基辅酶A：1,2-二酰基甘油酰基转移酶（EC 2.3.1.20）活性主要（89%）定位于微粒体亚细胞组分。开发了一种基于将[³H]棕榈酰辅酶A转化为三酰甘油的补充测定方法。这些方法比以前使用的方法灵敏100至1000倍，用于表征微粒体活性。向反应混合物中添加二酰基甘油时分散剂的选择至关重要。在测试的方法中，在乙醇中添加二酰基甘油导致二酰基甘油酰基转移酶活性最高。以前用作分散剂的吐温20严重抑制了该活性。观察到最适pH范围较宽，为7.4至8.0，几种盐使活性提高了2倍以上。该活性在28℃及以上温度下不稳定。使用细菌二酰基甘油研究了对含6至18个碳原子的酰基辅酶A的依赖性。乙酰辅酶A和丁酰辅酶A不是底物。用癸酰辅酶A观察到最高的二酰基甘油酰基转移酶活性，而较长和较短的饱和链的活性较低。油酰辅酶A的最大活性仅为硬脂酰辅酶A的34%。在所使用的酰基辅酶A的临界胶束浓度与二酰基甘油酰基转移酶活性之间未观察到简单关系。用[³H]棕榈酰辅酶A研究了对含6至18个碳原子脂肪酸的二酰基甘油的依赖性。虽然所有测试的1,2-二酰基-sn-甘油都是底物，但二酰基甘油酰基转移酶活性以1,2-二油酰甘油最高。细菌二酰基甘油的最大活性为1,2-二油酰甘油的86%。长链二酰基甘油达到最大速度一半所需的二酰基甘油浓度为20至40微摩尔，短链二酰基甘油为2至3微摩尔；这些浓度比以前报道的低75至750倍。以二油酰甘油和棕榈酰辅酶A为底物时，分离细胞的微粒体二酰基甘油酰基转移酶比活性约为50纳摩尔/分钟/毫克，比以前从脂肪组织报道的高10至50倍，这是典型的。分离脂肪细胞微粒体中的二酰基甘油酰基转移酶比活性比所检查的任何其他组织高17倍。二酰基甘油酰基转移酶似乎对酰基辅酶A和二酰基甘油具有特异性，但由于缺乏在所采用条件下这些底物在溶液中的物理性质数据，难以做出确切解释。
