Jacobson P B, Marshall L A, Sung A, Jacobs R S
Department of Biological Sciences, University of California, Santa Barbara 93106.
Biochem Pharmacol. 1990 May 15;39(10):1557-64. doi: 10.1016/0006-2952(90)90521-l.
The marine natural product, manoalide (MLD), was investigated to determine if this drug inhibited purified human synovial fluid phospholipase A2 (HSF-PLA2). Utilizing classical Michaelis-Menten kinetics, apparent Km and Vmax values for HSF-PLA2 of 1.34 mM and 0.47 mumol [3H]palmitic acid released/min/mg protein were obtained using dipalmitoylphosphatidylcholine (DPPC) as the substrate, and 38.0 microM and 18.8 mumol [3H]arachidonic acid released/min/mg protein with Escherichia coli as a natural substrate. These kinetic parameters were utilized subsequently to evaluate the inhibitory effects of manoalide on HSF-PLA2. Inhibition of HSF-PLA2 by MLD was concentration and time dependent with IC50 values of 0.2 and 0.02 microM for DPPC and E. coli respectively. Dialysis studies and examination of DPPC or E. coli hydrolysis versus enzyme concentration indicate that MLD is an irreversible inhibitor of HSF-PLA2. Substrate specificity was also examined in the absence and presence of MLD using dipalmitoylphosphatidylethanolamine (DPPE) as a substrate. MLD inhibited the hydrolysis of DPPE (greater than 90% inhibition at 2 microM), and preliminary results indicate that DPPC was more readily hydrolyzed than DPPE under the substrate conditions of the assay. While the cellular source of secreted HSF-PLA2 is unknown, these studies indicate that MLD can inactivate secreted phospholipase A2 isolated from patients with inflammatory joint disease.
对海洋天然产物 manoalide（MLD）进行了研究，以确定该药物是否能抑制纯化的人滑液磷脂酶 A2（HSF-PLA2）。利用经典的米氏动力学，以二棕榈酰磷脂酰胆碱（DPPC）为底物时，HSF-PLA2 的表观 Km 和 Vmax 值分别为 1.34 mM 和 0.47 μmol [3H]棕榈酸释放量/分钟/毫克蛋白质；以大肠杆菌为天然底物时，分别为 38.0 μM 和 18.8 μmol [3H]花生四烯酸释放量/分钟/毫克蛋白质。随后利用这些动力学参数评估 manoalide 对 HSF-PLA2 的抑制作用。MLD 对 HSF-PLA2 的抑制作用呈浓度和时间依赖性，对 DPPC 和大肠杆菌的 IC50 值分别为 0.2 和 0.02 μM。透析研究以及 DPPC 或大肠杆菌水解与酶浓度关系的检查表明，MLD 是 HSF-PLA2 的不可逆抑制剂。还在有和没有 MLD 的情况下，以二棕榈酰磷脂酰乙醇胺（DPPE）为底物研究了底物特异性。MLD 抑制了 DPPE 的水解（在 2 μM 时抑制率大于 90%），初步结果表明在该测定的底物条件下，DPPC 比 DPPE 更容易被水解。虽然分泌型 HSF-PLA2 的细胞来源尚不清楚，但这些研究表明 MLD 可以使从炎症性关节疾病患者中分离出的分泌型磷脂酶 A2 失活。
