Mustonen P, Lehtonen J Y, Kinnunen P K
Institute of Biomedicine, Department of Medical Chemistry, University of Helsinki, Finland.
Biochemistry. 1998 Sep 1;37(35):12051-7. doi: 10.1021/bi980430q.
Binding of quinacrine to phospholipids and porcine pancreatic phospholipase A2 (PLA2) was investigated using fluorescence resonance energy transfer, Langmuir films, assay for the enzymatic activity, and molecular modeling. No significant binding of this drug to the zwitterionic phosphatidylcholine was observed whereas a high affinity for acidic phospholipids was revealed by quenching of pyrene-labeled phospholipid analogues. Partial reversal of this binding was observed due to the addition of 4 mM CaCl2. Quinacrine efficiently and independently of the lipid surface pressure penetrated into monolayers of phosphatidylglycerol while only a weak penetration into phosphatidylcholine films was evident. Quinacrine also bound to eosin-labeled PLA2, and the addition of 4 mM CaCl2 reversed this interaction almost completely. In the presence of acidic phospholipids both the drug and the enzyme were attached to the lipid surface. Studies on the influence of quinacrine on the activity of PLA2 toward pyrene-labeled phospholipid analogues revealed that the hydrolysis of phosphatidylcholine was progressively reduced as a function of increasing [quinacrine]. At low [CaCl2] and low quinacrine:lipid molar ratios (<1:5) quinacrine enhanced slightly the rate of hydrolysis of acidic phospholipids whereas at higher drug:lipid molar ratios (>1:2) an inhibition was observed. In the presence of 1 mM CaCl2 quinacrine inhibited PLA2-catalyzed hydrolysis of phosphatidylglycerol only when the drug:lipid molar ratio exceeded 1:1. The presence of 4 mM CaCl2 abolished nearly completely the inhibition with all the substrate analogues used. Our data suggest that the inhibition of PLA2 by quinacrine is due to its binding to the enzyme. This is supported also by molecular modeling which suggested a binding site for quinacrine close to the active site and Ca2+ binding site of the enzyme. Importantly, our data indicate that quinacrine binds avidly to acidic phospholipids and their presence may influence the drug-enzyme interaction and the inhibition of the enzyme action. Accordingly, presence of quinacrine may interfere also with other processes that require the presence of acidic lipids and/or Ca2+, such as the function of the nicotinic acetylcholine receptor.
利用荧光共振能量转移、朗缪尔膜、酶活性测定和分子建模等方法，研究了喹吖因与磷脂及猪胰磷脂酶A2（PLA2）的结合情况。未观察到该药物与两性离子磷脂酰胆碱有明显结合，而芘标记的磷脂类似物的猝灭显示其对酸性磷脂具有高亲和力。由于加入4 mM氯化钙，观察到这种结合有部分逆转。喹吖因高效且独立于脂质表面压力渗透到磷脂酰甘油单层中，而对磷脂酰胆碱膜的渗透则很微弱。喹吖因也与曙红标记的PLA2结合，加入4 mM氯化钙几乎完全逆转了这种相互作用。在酸性磷脂存在的情况下，药物和酶都附着在脂质表面。关于喹吖因对PLA2作用于芘标记的磷脂类似物活性影响的研究表明，随着[喹吖因]的增加，磷脂酰胆碱的水解逐渐减少。在低[氯化钙]和低喹吖因:脂质摩尔比（<1:5）时，喹吖因略微提高了酸性磷脂的水解速率，而在较高的药物:脂质摩尔比（>1:2）时，则观察到抑制作用。在存在1 mM氯化钙的情况下，只有当药物:脂质摩尔比超过1:1时，喹吖因才抑制PLA2催化的磷脂酰甘油水解。4 mM氯化钙的存在几乎完全消除了对所有所用底物类似物的抑制作用。我们的数据表明，喹吖因对PLA2的抑制作用是由于其与酶的结合。分子建模也支持这一点，该模型表明喹吖因的结合位点靠近酶的活性位点和钙离子结合位点。重要的是，我们的数据表明喹吖因与酸性磷脂紧密结合，其存在可能影响药物 - 酶相互作用以及酶作用的抑制。因此，喹吖因的存在也可能干扰其他需要酸性脂质和/或钙离子存在的过程，如烟碱型乙酰胆碱受体的功能。
