Pillai M G, Certik M, Nakahara T, Kamisaka Y
Applied Microbiology Department, National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki, 305-8566, Japan.
Biochim Biophys Acta. 1998 Jul 31;1393(1):128-36. doi: 10.1016/s0005-2760(98)00069-1.
Triacylglycerol (TG) biosynthetic enzymes were characterized in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora. When the membrane or lipid body fraction of this fungus was incubated with [14C]oleoyl-CoA without adding exogenous acyl acceptors, radioactivity was incorporated predominantly into TG, indicating that diacylglycerol acyltransferase (DGAT) used endogenous diacylglycerol to incorporate [14C]oleoyl-CoA into TG. Adding glycerol 3-phosphate or lysophosphatidic acid increased radiolabeled phosphatidic acid (PA) in the membrane fraction, which reflected the presence of glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT). Label accumulation did not occur in lysophosphatidic acid when glycerol 3-phosphate was added, suggesting that GPAT was rate-limiting in sequential acylation. In the lipid body fraction, adding lysophosphatidic acid similarly increased radiolabeled PA, whereas adding glycerol 3-phosphate caused much lower increase in radiolabeled PA. Quantitative assays for GPAT, LPAAT, phosphatidic acid phosphatase (PAP), and DGAT essentially confirmed the results obtained from [1-14C]oleoyl-CoA incorporation; LPAAT had the highest activity in the membrane and lipid body fractions, GPAT was significantly lower in the lipid body fraction, and DGAT was much higher in the lipid body fraction. GPAT and LPAAT in the membrane fraction had a strong preference toward oleoyl-CoA as a substrate over palmitoyl-CoA. Results indicate that TG biosynthetic enzymes had different subcellular distribution with the sequence of enrichment in the lipid body fraction, i.e., GPAT<LPAAT approximately PAP<DGAT. This may reflect a TG biosynthetic process from endoplasmic reticulum membranes to lipid bodies in the fungus.
对产油真菌拉曼被孢霉变种角孢被孢霉（Mortierella ramanniana var. angulispora）的亚细胞组分中的三酰甘油（TG）生物合成酶进行了表征。当将该真菌的膜或脂质体组分与[14C]油酰辅酶A一起孵育而不添加外源酰基受体时，放射性主要掺入TG中，这表明二酰甘油酰基转移酶（DGAT）利用内源性二酰甘油将[14C]油酰辅酶A掺入TG中。添加3-磷酸甘油或溶血磷脂酸会增加膜组分中放射性标记的磷脂酸（PA），这反映了3-磷酸甘油酰基转移酶（GPAT）和溶血磷脂酸酰基转移酶（LPAAT）的存在。添加3-磷酸甘油时，溶血磷脂酸中未出现标记积累，这表明GPAT在顺序酰化中起限速作用。在脂质体组分中，添加溶血磷脂酸同样会增加放射性标记的PA，而添加3-磷酸甘油导致放射性标记的PA增加幅度小得多。对GPAT、LPAAT、磷脂酸磷酸酶（PAP）和DGAT的定量测定基本上证实了从[1-14C]油酰辅酶A掺入实验中获得的结果；LPAAT在膜和脂质体组分中的活性最高，GPAT在脂质体组分中显著较低，而DGAT在脂质体组分中高得多。膜组分中的GPAT和LPAAT对油酰辅酶A作为底物的偏好性远高于棕榈酰辅酶A。结果表明，TG生物合成酶具有不同的亚细胞分布，在脂质体组分中的富集顺序为：GPAT＜LPAAT≈PAP＜DGAT。这可能反映了该真菌中从内质网膜到脂质体的TG生物合成过程。
