Matsumoto Yusaku, Sugimori Daisuke
Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan.
Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan.
J Biosci Bioeng. 2015 Oct;120(4):372-9. doi: 10.1016/j.jbiosc.2015.02.020. Epub 2015 Apr 17.
The substrate recognition mechanism of phospholipase D and enzymatic measurement of choline plasmalogen were investigated. A phospholipase D (PLD684) from Streptomyces sp. strain NA684 was purified 184-fold from the culture supernatant with 23.7% recovery. Maximum activity for l-α-lysophosphatidylcholine (LPC) hydrolysis was found at pH 5.0 and 80°C. The hydrolytic activity was remarkably affected by the concentration of Triton X-100 in the reaction mixture. In the presence of 0.05-0.5% and 0.1-0.2% (wt/vol) Triton X-100, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and choline plasmalogen were efficiently hydrolyzed by PLD684, respectively. Hydrolysis of LPC and choline lysoplasmalogen did not require Triton X-100; rather, the hydrolytic activity was inhibited by more than 0.05% (wt/vol) Triton X-100. The enzyme preferred mixed micelle substrates to liposomal substrates and hydrolyzed 98.4% of mixed micelle POPC in 1 h. Kinetic analysis showed that the rate-limiting steps of hydrolysis of mixed micelle POPC and emulsified LPC by PLD684 were the bulk step and the surface step, respectively. These results suggest that PLD684 has at least two substrate recognition mechanisms to recognize various phospholipids that have considerably different physical properties derived from their head and tail groups. Understanding of how PLD684 recognizes substrate forms will be useful for elucidating roles of lipolytic proteins in nature. Moreover, we report an enzymatic measurement of choline plasmalogen using PLD684 and phospholipase B. This is the first enzymatic method for measuring choline plasmalogen.
研究了磷脂酶D的底物识别机制以及胆碱缩醛磷脂的酶法测定。从链霉菌属菌株NA684中纯化出一种磷脂酶D（PLD684），其在培养上清液中的回收率为23.7%，纯化倍数为184倍。发现l-α-溶血磷脂酰胆碱（LPC）水解的最大活性出现在pH 5.0和80°C条件下。水解活性受到反应混合物中Triton X-100浓度的显著影响。在存在0.05 - 0.5%和0.1 - 0.2%（重量/体积）Triton X-100的情况下，PLD684分别能有效水解1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（POPC）和胆碱缩醛磷脂。LPC和胆碱溶血缩醛磷脂的水解不需要Triton X-100；相反，超过0.05%（重量/体积）的Triton X-100会抑制水解活性。该酶对混合胶束底物的偏好高于脂质体底物，1小时内可水解98.4%的混合胶束POPC。动力学分析表明，PLD684水解混合胶束POPC和乳化LPC的限速步骤分别是本体步骤和表面步骤。这些结果表明，PLD684至少有两种底物识别机制来识别各种具有因头部和尾部基团而有显著不同物理性质的磷脂。了解PLD684如何识别底物形式将有助于阐明自然界中脂解蛋白的作用。此外，我们报道了一种使用PLD684和磷脂酶B对胆碱缩醛磷脂进行酶法测定的方法。这是第一种测定胆碱缩醛磷脂的酶法。
