Sun Yong-Xin, Tsuboi Kazuhito, Okamoto Yasuo, Tonai Takeharu, Murakami Makoto, Kudo Ichiro, Ueda Natsuo
Department of Biochemistry, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan.
Biochem J. 2004 Jun 15;380(Pt 3):749-56. doi: 10.1042/BJ20040031.
Anandamide (an endocannabinoid) and other bioactive long-chain NAEs (N-acylethanolamines) are formed by direct release from N-acyl-PE (N-acyl-phosphatidylethanolamine) by a PLD (phospholipase D). However, the possible presence of a two-step pathway from N-acyl-PE has also been suggested previously, which comprises (1) the hydrolysis of N-acyl-PE to N-acyl-lysoPE by PLA1/PLA2 enzyme(s) and (2) the release of NAEs from N-acyllysoPE by lysoPLD (lysophospholipase D) enzyme(s). In the present study we report for the first time the characterization of enzymes responsible for this pathway. The PLA1/PLA2 activity for N-palmitoyl-PE was found in various rat tissues, with the highest activity in the stomach. This stomach enzyme was identified as group IB sPLA2 (secretory PLA2), and its product was determined as N-acyl-1-acyl-lysoPE. Recombinant group IB, IIA and V of sPLA2s were also active with N-palmitoyl-PE, whereas group X sPLA2 and cytosolic PLA2a were inactive. In addition, we found wide distribution of lysoPLD activity generating N-palmitoylethanolamine from N-palmitoyl-lysoPE in rat tissues, with higher activities in the brain and testis. Based on several lines of enzymological evidence, the lysoPLD enzyme could be distinct from the known N-acyl-PE-hydrolysing PLD. sPLA2-IB dose dependently enhanced the production of N-palmitoylethanolamine from N-palmitoyl-PE in the brain homogenate showing the lysoPLD activity. N-Arachidonoyl-PE and N-arachidonoyl-lysoPE as anandamide precursors were also good substrates of sPLA2-IB and the lysoPLD respectively. These results suggest that the sequential actions of PLA2 and lysoPLD may constitute another biosynthetic pathway for NAEs, including anandamide.
花生四烯乙醇胺（一种内源性大麻素）和其他生物活性长链N-酰基乙醇胺（N-acylethanolamines，NAEs）是由磷脂酶D（PLD）直接从N-酰基磷脂酰乙醇胺（N-acyl-PE）释放形成的。然而，此前也有人提出可能存在一条从N-酰基磷脂酰乙醇胺开始的两步途径，该途径包括：（1）通过磷脂酶A1/磷脂酶A2（PLA1/PLA2）将N-酰基磷脂酰乙醇胺水解为N-酰基溶血磷脂酰乙醇胺（N-acyl-lysoPE）；（2）通过溶血磷脂酶D（lysoPLD）从N-酰基溶血磷脂酰乙醇胺释放NAEs。在本研究中，我们首次报道了负责该途径的酶的特性。在大鼠的各种组织中均发现了针对N-棕榈酰磷脂酰乙醇胺的PLA1/PLA2活性，其中胃中的活性最高。这种胃酶被鉴定为IB组分泌型磷脂酶A2（sPLA2），其产物被确定为N-酰基-1-酰基溶血磷脂酰乙醇胺。重组的IB组、IIA组和V组sPLA2对N-棕榈酰磷脂酰乙醇胺也有活性，而X组sPLA2和胞质型磷脂酶A2α则无活性。此外，我们发现大鼠组织中存在广泛的溶血磷脂酶D活性，可从N-棕榈酰溶血磷脂酰乙醇胺生成N-棕榈酰乙醇胺，在脑和睾丸中的活性较高。基于多条酶学证据，溶血磷脂酶D可能与已知的水解N-酰基磷脂酰乙醇胺的PLD不同。在显示溶血磷脂酶D活性的脑匀浆中，IB组sPLA2剂量依赖性地增强了从N-棕榈酰磷脂酰乙醇胺生成N-棕榈酰乙醇胺的过程。作为花生四烯乙醇胺前体的N-花生四烯酰磷脂酰乙醇胺和N-花生四烯酰溶血磷脂酰乙醇胺分别也是IB组sPLA2和溶血磷脂酶D的良好底物。这些结果表明，磷脂酶A2和溶血磷脂酶D的相继作用可能构成NAEs（包括花生四烯乙醇胺）的另一条生物合成途径。
