Menashe M, Romero G, Biltonen R L, Lichtenberg D
J Biol Chem. 1986 Apr 25;261(12):5328-33.
The hydrolysis of small unilamellar vesicles made of dipalmitoylphosphatidylcoline by pancreatic phospholipase A2 has been studied under various conditions of temperature and enzyme and substrate concentration using the following three different experimental protocols. When the enzyme was added to the substrate vesicles after being separately adjusted to the temperature of the experiments hydrolysis occurred instantaneously only in the temperature range where the lipid is known to exist in its gel phase, while above the transition range no hydrolysis occurred. Within the transition range, the time course of hydrolysis was characterized by initial very slow rate of hydrolysis (latency phase) followed by an abrupt increase in the rate after a time tau, which is a complex function of temperature and enzyme to substrate ratio. When an enzyme-substrate mixture was first preincubated below Tm and then temperature jumped to a temperature above or within the transition range, the latency phase was markedly shortened. When the temperature jump was to the transition range, this effect is observed even if Ca2+ is absent in the preincubation mixture. However, instantaneous hydrolysis was observed upon temperature jumping the mixture to a temperature high above Tm only if Ca2+ was present in the preincubation medium. In temperature-scanning experiments, hydrolysis was followed while changing the temperature of the enzyme-substrate mixture continuously. Heating an enzyme-substrate mixture from room temperature resulted in an abrupt onset of hydrolysis when the transition range was approached. These results lead us to conclude that two distinctly different steps precede rapid hydrolysis of dipalmitoylphosphatidylcholine small unilamellar vesicles by pancreatic phospholipase A2: a Ca2+-independent binding of the enzyme to the substrate vesicles, which for chemically pure bilayers occurs best in the gel phase. This step is followed by a Ca2+-dependent activation of the initially formed enzyme-substrate complex. The latter step only occurs under conditions where the bilayer possesses packing irregularities and probably involves a reorganization of the enzyme-substrate complex. At least one of these two steps appears to involve enzyme-enzyme interaction.
利用以下三种不同的实验方案，在不同温度、酶浓度和底物浓度条件下，研究了胰腺磷脂酶A2对由二棕榈酰磷脂酰胆碱制成的小单层囊泡的水解作用。当酶和底物囊泡分别调节至实验温度后，将酶加入底物囊泡中，水解仅在已知脂质以凝胶相存在的温度范围内瞬间发生，而在转变温度范围以上则不发生水解。在转变温度范围内，水解的时间进程表现为初始水解速率非常缓慢（延迟期），随后在时间τ后速率突然增加，τ是温度和酶与底物比例的复杂函数。当酶 - 底物混合物首先在低于熔点温度（Tm）下预孵育，然后温度跃升至转变温度范围以上或以内时，延迟期明显缩短。当温度跃升至转变温度范围时，即使预孵育混合物中不存在Ca2+，也会观察到这种效应。然而，只有当预孵育介质中存在Ca2+时，将混合物温度跃升至远高于Tm的温度时才会观察到瞬间水解。在温度扫描实验中，在连续改变酶 - 底物混合物温度的同时跟踪水解情况。从室温加热酶 - 底物混合物，当接近转变温度范围时会突然开始水解。这些结果使我们得出结论，胰腺磷脂酶A2对二棕榈酰磷脂酰胆碱小单层囊泡进行快速水解之前有两个明显不同的步骤：酶与底物囊泡的Ca2+非依赖性结合，对于化学纯的双层膜，这种结合在凝胶相中最佳。此步骤之后是最初形成的酶 - 底物复合物的Ca2+依赖性激活。后一步骤仅在双层具有堆积不规则性的条件下发生，并且可能涉及酶 - 底物复合物的重组。这两个步骤中至少有一个似乎涉及酶 - 酶相互作用。
