Erlanson-Albertsson C
Biochim Biophys Acta. 1980 Mar 21;617(3):371-82. doi: 10.1016/0005-2760(80)90003-x.
The binding between colipase and two triacylglycerol substrates, tributyrin and Intralipid, in the presence of bile salts have been determined quantitatively by a method based on equilibrium partition in an aqueous two-phase system. In the model proposed the triacylglycerol, in the form of spherical droplets covered with bile salt, is assumed to have a certain number of independent binding sites at the surface for colipase. The binding of colipase to tributyrin at pH 7.0 in the presence of 4 mM sodium taurodeoxycholate and 150 mM NaCl had a dissociation constant Kd = 3.3 . 10(-7) M; the concentration of binding sites was 1.2 . 10(-6) M in a 102 mM tributyrin emulsion. When tributyrin was dispersed in 1 mM and 12 mM sodium taurodeoxycholate the dissociation constant was somewhat higher, 6.3 . 10(-7) M and 6.0 . 10(-7) M, respectively. Thus the binding strength was optimal at 4 mM sodium taurodeoxycholate. At the same time the concentration of binding sites decreased from 4.1 . 10(-6) M for 1 mM sodium taurodeoxycholate to 1.4 . 10(-6) M for 12 mM sodium taurodeoxycholate. This indicated that at higher bile salt concentration the bile salt acted as non-competitive inhibitors on the binding of colipase to the substrate, thus binding to other sites than colipase to the substrate. The binding of colipase to Intralipid, an emulsion of a long-chain triacylglycerol stabilized with phosphatidylcholine and glycerol, was more complex with indications of several different binding sites with different affinity. The majority of these had a dissociation constant Kd = 1.2 . 10(-6) M in the presence of 4 mM sodium taurodeoxycholate and 150 mM. With each droplet having a diameter of 10(-4) cm, the number of binding sites on each droplet was determined to 1.96 . 10(5) and the average area available for each colipase molecule to 1600 A at saturation. Colipase on denaturation has a surface of 1320 A.
通过一种基于水相两相系统中平衡分配的方法，定量测定了在胆盐存在下，共脂肪酶与两种三酰甘油底物（三丁酸甘油酯和英脱利匹特）之间的结合情况。在所提出的模型中，呈球形液滴形式且覆盖有胆盐的三酰甘油，被假定在其表面具有一定数量的共脂肪酶独立结合位点。在4 mM牛磺脱氧胆酸钠和150 mM氯化钠存在的情况下，pH 7.0时共脂肪酶与三丁酸甘油酯的结合解离常数Kd = 3.3×10⁻⁷ M；在102 mM三丁酸甘油酯乳液中，结合位点的浓度为1.2×10⁻⁶ M。当三丁酸甘油酯分散在1 mM和12 mM牛磺脱氧胆酸钠中时，解离常数略高，分别为6.3×10⁻⁷ M和6.0×10⁻⁷ M。因此，在4 mM牛磺脱氧胆酸钠时结合强度最佳。同时，结合位点的浓度从1 mM牛磺脱氧胆酸钠时的4.1×10⁻⁶ M降至12 mM牛磺脱氧胆酸钠时的1.4×10⁻⁶ M。这表明在较高胆盐浓度下，胆盐对共脂肪酶与底物的结合起非竞争性抑制剂的作用，即与共脂肪酶以外的其他位点结合到底物上。共脂肪酶与英脱利匹特（一种用磷脂酰胆碱和甘油稳定的长链三酰甘油乳液）的结合更为复杂，显示出有几个具有不同亲和力的不同结合位点。在4 mM牛磺脱氧胆酸钠和150 mM存在的情况下，其中大多数的解离常数Kd = 1.2×10⁻⁶ M。每个液滴直径为10⁻⁴ cm，测定出每个液滴上的结合位点数为1.96×10⁵ 个，饱和时每个共脂肪酶分子可利用的平均面积为1600 Å。变性的共脂肪酶表面积为1320 Å。
