Arrese E L, Rojas-Rivas B I, Wells M A
Department of Biochemistry and Center for Insect Science, University of Arizona, Tucson 85721, USA.
Arch Insect Biochem Physiol. 1996;31(3):325-35. doi: 10.1002/(SICI)1520-6327(1996)31:3<325::AID-ARCH7>3.0.CO;2-W.
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.
在昆虫脂肪体中，由脂肪动激素（AKH）作用刺激产生的sn-1,2-二酰基甘油的合成途径尚不清楚。该实验室之前的结果表明，储存的三酰基甘油水解为sn-2-单酰基甘油，随后由单酰基甘油-酰基转移酶（MGAT）催化sn-2-单酰基甘油进行立体特异性酰化，可能是AKH刺激的sn-1,2-二酰基甘油合成的主要途径。因此，MGAT可能是该途径的关键酶。在本研究中，我们对烟草天蛾脂肪体中的MGAT活性进行了表征。该活性通过使用放射性标记的棕榈酰辅酶A对2-单油精进行酰化来测定，结果发现它主要是一种微粒体酶。2-单油精酰化的产物为1,2-二酰基甘油（40 - 50%）、1,3-二酰基甘油（20 - 30%）和三酰基甘油（30 - 40%）。三酰基甘油作为产物的存在揭示了脂肪体微粒体中存在二酰基甘油-酰基转移酶活性。MGAT活性的最适pH值为7.0，其活性对2-单油精浓度的依赖性呈现饱和动力学。检测到一种内源性MGAT活性，其占添加底物时观察到的最大活性的20%。棕榈酰辅酶A的最佳浓度范围在0.10 - 0.20 mM之间。在最佳条件下测得的MGAT比活性约为0.6 nmol二酰基甘油形成/分钟-毫克蛋白质。MGAT对2-单油精的活性最高，而使用饱和2-单酰基甘油时活性较低。观察到的sn-1-单酰基甘油的活性低于sn-2-单酰基甘油的活性。AKH不会刺激MGAT活性，这表明要么该酶不受激素调节，要么单酰基甘油途径不参与烟草天蛾脂肪体中AKH刺激的sn-1,2-二酰基甘油的产生。
