Clarke A R, Holbrook J J
Biochim Biophys Acta. 1985 Mar 1;827(3):358-68. doi: 10.1016/0167-4838(85)90220-1.
The binding of lipoprotein lipase to a fluorescently labelled apolipoprotein C-II in free solution has been followed by measuring fluorescence anisotropy. The formation of a weak, binary complex in which a single apolipoprotein C-II molecule associates non-cooperatively with each subunit of the dimeric enzyme was observed. The dissociation constant for this complex in 0.05 M NaCl is 0.2 X 10(-6) M and it is weakened markedly by raising the salt concentration and by the binding of heparin to the enzyme. The assembly of the same protein-protein complex on the surface of glycerol trioleate globules has been monitored by steady-state and pre-steady-state kinetics. In these circumstances the lipoprotein lipase-apolipoprotein C-II interaction is much tighter (Kd = (7-10) X 10(-9) M) and is insensitive to salt and heparin. The mechanism of activation of the enzyme at low concentrations of apolipoprotein C-II is described by a kinetic model in which apolipoprotein C-II binds preferentially to the form of the enzyme which is associated with the triacylglycerol substrate. This preference leads to a stabilization of the enzyme-substrate complex, thus reducing the apparent Ks.
通过测量荧光各向异性，跟踪了游离溶液中脂蛋白脂肪酶与荧光标记的载脂蛋白C-II的结合。观察到形成了一种弱的二元复合物，其中单个载脂蛋白C-II分子与二聚体酶的每个亚基非协同结合。该复合物在0.05 M NaCl中的解离常数为0.2×10⁻⁶ M，提高盐浓度以及肝素与酶的结合会使其显著减弱。通过稳态和预稳态动力学监测了相同蛋白质-蛋白质复合物在三油酸甘油酯球表面的组装。在这些情况下，脂蛋白脂肪酶-载脂蛋白C-II相互作用紧密得多（Kd = (7 - 10)×10⁻⁹ M），并且对盐和肝素不敏感。低浓度载脂蛋白C-II激活该酶的机制由一个动力学模型描述，其中载脂蛋白C-II优先结合与三酰甘油底物相关的酶形式。这种偏好导致酶-底物复合物的稳定，从而降低了表观Ks。
