Wu W I, Carman G M
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08903, USA.
Biochemistry. 1996 Mar 26;35(12):3790-6. doi: 10.1021/bi952808f.
Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by phospholipids was examined using purified enzyme and Triton X-100/phospholipid-mixed micelles. Anionic phospholipids activated phosphatidate phosphatase activity whereas zwitterionic phospholipids had a slight inhibitory effect on activity. Cardiolipin (A0.5 = 1.9 mol %), CDP-diacylglycerol (A0.5 = 2.6 mol %), and phosphatidylinositol (A0.5 = 5.5 mol %) were the most potent anionic phospholipid activators. Enzyme activation by cardiolipin (n=2.8), CDP-diacylglycerol (n=2.1), and phosphatidylinositol (n=3.3) followed positive cooperative kinetics. A kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase activation by anionic phospholipids. The dependence of phosphatidate phosphatase on phosphatidate was cooperative (n approximately 2.2) in the absence and presence of phospholipid activators. Cardiolipin, CDP-diacylglycerol, and phosphatidylinositol were mixed competitive activators of phosphatidate phosphatase activity. The major effect of the activators was to cause a decrease in the Km for phosphatidate. Sphinganine, a positively charged sphingoid base, inhibited phosphatidate phosphatase activity and antagonized the activation of the enzyme by cardiolipin and phosphatidylinositol. Sphinganine caused an increase in the cooperativity of cardiolipin activation, but had little effect on the A0.5 value for cardiolipin. On the other hand, sphinganine had little effect on the cooperativity of phosphatidylinositol activation, but caused an increase in the A0.5 value for phosphatidylinositol. The activation constants for cardiolipin, CDP-diacylglycerol, and phosphatidylinositol were within the range of their cellular concentrations. These results suggested that the activation of phosphatidate phosphatase activity by anionic phospholipids may be physiologically relevant.
利用纯化的酶和 Triton X - 100/磷脂混合胶束，研究了磷脂对酿酒酵母膜相关磷脂酸磷酸酶（3 - sn - 磷脂酸磷酸水解酶，EC 3.1.3.4）活性的调节作用。阴离子磷脂激活磷脂酸磷酸酶活性，而两性离子磷脂对活性有轻微抑制作用。心磷脂（半激活浓度A0.5 = 1.9 mol%）、CDP - 二酰甘油（A0.5 = 2.6 mol%）和磷脂酰肌醇（A0.5 = 5.5 mol%）是最有效的阴离子磷脂激活剂。心磷脂（n = 2.8）、CDP - 二酰甘油（n = 2.1）和磷脂酰肌醇（n = 3.3）对酶的激活遵循正协同动力学。进行了动力学分析以确定阴离子磷脂激活磷脂酸磷酸酶的机制。在不存在和存在磷脂激活剂的情况下，磷脂酸磷酸酶对磷脂酸的依赖性都是协同的（n约为2.2）。心磷脂、CDP - 二酰甘油和磷脂酰肌醇是磷脂酸磷酸酶活性的混合竞争性激活剂。激活剂的主要作用是使磷脂酸的米氏常数降低。鞘氨醇，一种带正电荷的鞘氨醇碱，抑制磷脂酸磷酸酶活性，并拮抗心磷脂和磷脂酰肌醇对该酶的激活作用。鞘氨醇导致心磷脂激活的协同性增加，但对心磷脂的A0.5值影响不大。另一方面，鞘氨醇对磷脂酰肌醇激活的协同性影响不大，但导致磷脂酰肌醇的A0.5值增加。心磷脂、CDP - 二酰甘油和磷脂酰肌醇的激活常数在其细胞浓度范围内。这些结果表明，阴离子磷脂对磷脂酸磷酸酶活性的激活可能具有生理相关性。
