Walton P A, Possmayer F
Department of Biochemistry, University of Western Ontario, London, Canada.
Biochem J. 1989 Jul 15;261(2):673-8. doi: 10.1042/bj2610673.
Lung contains both Mg2+-dependent and Mg2+-independent phosphatidate phosphohydrolase activities. Addition of Triton X-100 (0.5%) or chlorpromazine (1 mM) leads to a marked increase in the total phosphatidate phosphohydrolase activity in rat lung microsomes (microsomal fractions), but a decrease in the Mg2+-dependent activity. These observations suggest that the Mg2+-independent activity is stimulated, whereas the Mg2+-dependent activity is inhibited. However, the possibility exists that Triton X-100 could stimulate the Mg2+-dependent enzymic activity in an Mg2+-independent manner. In addition, the positively charged amphiphilic drug could be replacing the enzyme's requirement for Mg2+. These two possibilities were examined by using subcellular fractions in which the Mg2+-dependent phosphatidate phosphohydrolase had been abolished by heat treatment at 55 degrees C for 15 min. Heat treatment does not affect the microsomal Mg2+-independent phosphohydrolase to any great extent. Since the 6-8-fold stimulations due to Triton X-100 and chlorpromazine are retained after heat treatment of this fraction, the Mg2+-independent activity must be involved. Addition of Triton X-100 and chlorpromazine to cytosol virtually abolishes the Mg2+-dependent phosphatidate phosphohydrolase activity and decreases the Mg2+-independent activity by half. Heat treatment also abolishes the Mg2+-dependent activity and decreases the Mg2+-independent activity by over half. The Mg2+-independent phosphatidate phosphohydrolase activity remaining after heat treatment was not affected by Triton X-100 or chlorpromazine. These studies demonstrate that Triton X-100 and chlorpromazine specifically stimulate the heat-stable Mg2+-independent phosphatidate phosphohydrolase activity in rat lung microsomes. In contrast, the heat-labile Mg2+-independent phosphatidate phosphohydrolase activities in cytosol are inhibited by these reagents. Triton X-100 and chlorpromazine inhibit the Mg2+-dependent phosphatidate phosphohydrolase activities in both rat lung microsomes and cytosol. These results are consistent with the view that a single Mg2+-dependent phosphatidate phosphohydrolase present in both microsomes and cytosol is specifically involved in glycerolipid metabolism.
肺中同时存在镁离子依赖性和非镁离子依赖性的磷脂酸磷酸水解酶活性。添加曲拉通X-100（0.5%）或氯丙嗪（1 mM）会导致大鼠肺微粒体（微粒体组分）中总磷脂酸磷酸水解酶活性显著增加，但镁离子依赖性活性降低。这些观察结果表明，非镁离子依赖性活性受到刺激，而镁离子依赖性活性受到抑制。然而，存在曲拉通X-100可能以非镁离子依赖性方式刺激镁离子依赖性酶活性的可能性。此外，带正电荷的两亲性药物可能替代了酶对镁离子的需求。通过使用亚细胞组分来检验这两种可能性，其中镁离子依赖性磷脂酸磷酸水解酶已在55℃下热处理15分钟而被消除。热处理在很大程度上不影响微粒体的非镁离子依赖性磷酸水解酶。由于该组分经热处理后，曲拉通X-100和氯丙嗪引起的6至8倍刺激仍然存在，所以必定涉及非镁离子依赖性活性。向胞质溶胶中添加曲拉通X-100和氯丙嗪实际上消除了镁离子依赖性磷脂酸磷酸水解酶活性，并使非镁离子依赖性活性降低一半。热处理也消除了镁离子依赖性活性，并使非镁离子依赖性活性降低超过一半。热处理后剩余的非镁离子依赖性磷脂酸磷酸水解酶活性不受曲拉通X-100或氯丙嗪的影响。这些研究表明，曲拉通X-100和氯丙嗪特异性地刺激大鼠肺微粒体中热稳定的非镁离子依赖性磷脂酸磷酸水解酶活性。相反，胞质溶胶中热不稳定的非镁离子依赖性磷脂酸磷酸水解酶活性受到这些试剂的抑制。曲拉通X-100和氯丙嗪抑制大鼠肺微粒体和胞质溶胶中的镁离子依赖性磷脂酸磷酸水解酶活性。这些结果与以下观点一致，即微粒体和胞质溶胶中存在的单一镁离子依赖性磷脂酸磷酸水解酶特异性地参与甘油脂质代谢。
