Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) (EC 3.1.4.3) and phosphatidylethanolamine (PE)-specific PLC (PE-PLC) (EC 3.1.4.62), which generate diacylglycerol (DG) and are tricyclodecan-9-yl-xanthogenate (D609)-sensitive, were detected in detergent-insoluble fractions of mammalian tissues approximately 70 and 35 years ago, respectively. However, the genes and proteins involved in PC-PLC and PE-PLC activities remain unknown. In a recent study, we observed that mammalian sphingomyelin synthase (SMS) 1 and SMS-related protein display PC-PLC and PE-PLC activities in vitro. In the present study, we showed that human SMS2, which is located in detergent-insoluble fractions of the plasma membrane, also possesses PC-PLC activity (approximately 41% of SMS activity), PE-PLC activity (approximately 4%), ceramide phosphoethanolamine synthase (CPES) activity (approximately 46%), and SMS activity in the presence of phospholipid-detergent mixed micelles. Moreover, purified SMS2 reconstituted in detergent-free proteoliposomes (near-native environments) showed PC-PLC, PE-PLC, and CPES activities. Notably, in the presence of approximately 2 mol% ceramide and 4 mol% PC (1:2 ratio), PC-PLC activity was almost equal to SMS activity. SMS2 as PC/PE-PLC showed substrate selectivity for saturated fatty acid– and/or monounsaturated fatty acid–containing PC and PE species. The PC-PLC/SMS inhibitor D609 inhibited all enzyme activities (SMS, PC-PLC, PE-PLC, and CPES) of SMS2. Moreover, Zn2+ strongly inhibited all the enzymatic activities of SMS2. Interestingly, diacylglycerol inhibited the SMS activity of SMS2 (feedback control). These results indicate that mammalian SMS2 has unique enzymatic properties and is a candidate for a long-sought mammalian PC/PE-PLC.

中文翻译：

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鞘磷脂合酶 2 的多种活性产生含饱和脂肪酸和/或单不饱和脂肪酸的甘油二酯


磷脂酰胆碱 （PC） 特异性磷脂酶 C （PC-PLC） （EC 3.1.4.3） 和磷脂酰乙醇胺 （PE） 特异性 PLC （PE-PLC） （EC 3.1.4.62） 产生甘油二酯 （DG） 并且对三环十二烷-9-基黄原酸酯 （D609） 敏感，分别在大约 70 年前和 35 年前在哺乳动物组织的去污剂不溶性组分中检测到。然而，参与 PC-PLC 和 PE-PLC 活性的基因和蛋白质仍然未知。在最近的一项研究中，我们观察到哺乳动物鞘磷脂合酶 （SMS） 1 和 SMS 相关蛋白在 体外显示 PC-PLC 和 PE-PLC 活性。在本研究中，我们表明位于质膜的去污剂不溶性部分中的人类 SMS2 也具有 PC-PLC 活性（约 SMS 活性的 41%）、PE-PLC 活性（约 4%）、神经酰胺磷酸乙醇胺合酶 （CPES） 活性（约 46%）和在磷脂-洗涤剂混合胶束存在下的 SMS 活性。此外，在不含洗涤剂的蛋白脂质体（接近天然环境）中重构的纯化 SMS2 显示出 PC-PLC、PE-PLC 和 CPES 活性。值得注意的是，在大约 2 mol% 神经酰胺和 4 mol% PC（比例为 1：2）存在下，PC-PLC 活性几乎等于 SMS 活性。SMS2 作为 PC/PE-PLC 显示出对含有饱和脂肪酸和/或单不饱和脂肪酸的 PC 和 PE 物种的底物选择性。PC-PLC/SMS 抑制剂 D609 抑制 SMS2 的所有酶活性 （SMS、PC-PLC、PE-PLC 和 CPES）。此外，Zn2+ 强烈抑制 SMS2 的所有酶活性。有趣的是，甘油二酯抑制了 SMS2 的 SMS 活性 （反馈控制）。这些结果表明，哺乳动物 SMS2 具有独特的酶学特性，是长期寻找的哺乳动物 PC/PE-PLC 的候选者。