Clarke A R, Luscombe M, Holbrook J J
Biochim Biophys Acta. 1983 Sep 14;747(1-2):130-7. doi: 10.1016/0167-4838(83)90131-0.
The interactions of bovine milk lipoprotein lipase (triacylglycero-protein acylhydrolase, EC 3.1.1.34) with the glycosaminoglycans heparin and heparan sulphate were investigated using the technique of fluorescence polarization spectroscopy. The type of complex formed with the enzyme depends on the chain length of the heparin. In 0.05 M NaCl and when the heparin was in molar excess, one heparin chain of Mr 10000-18400 formed a very stable complex with the dimeric protein molecule (the 1:1 complex). With excess protein, weaker interactions produced complexes with higher molecular weights. These two classes of complex were also detected with shorter heparins (Mr 6600-8000), although in these circumstances the more stable complex possessed a heparin:protein dimer ratio of 2:1. In higher salt (0.2 M NaCl) and lower heparin concentrations (less than 6 . 10(-8) M) the weaker class of compound was undetectable and Kd values of 4 . 10(-8) M and 6 . 10(-9) M were assigned to the 2:1 and 1:1 complexes, respectively. Heparan sulphate of Mr 17000 could only form one class of complex. This had a 1:1 stoichiometry and with Kd values of 3 . 10(-8) M and 1.6 . 10(-7) M at 0.05 and 0.2 M NaCl, respectively. The results could be explained if there is a distinct binding region for glycosaminoglycans on each subunit of the dimeric enzyme and a single heparin chain of Mr greater than 10000 can satisfy both sites to form a 1:1 complex. Smaller heparin chains are unable to span the sites and, in order to occupy them, two chains must interact with each enzyme molecule.
利用荧光偏振光谱技术研究了牛乳脂蛋白脂肪酶（三酰甘油 - 蛋白质酰基水解酶，EC 3.1.1.34）与糖胺聚糖肝素和硫酸乙酰肝素的相互作用。与该酶形成的复合物类型取决于肝素的链长。在0.05 M NaCl中且肝素摩尔过量时，一条相对分子质量为10000 - 18400的肝素链与二聚体蛋白分子形成非常稳定的复合物（1:1复合物）。当蛋白过量时，较弱的相互作用产生了分子量更高的复合物。用较短的肝素（相对分子质量6600 - 8000）也检测到了这两类复合物，不过在这种情况下，更稳定的复合物中肝素与蛋白二聚体的比例为2:1。在较高盐浓度（0.2 M NaCl）和较低肝素浓度（小于6×10⁻⁸ M）下，较弱的一类复合物无法检测到，2:1和1:1复合物的解离常数（Kd）值分别为4×10⁻⁸ M和6×10⁻⁹ M。相对分子质量为17000的硫酸乙酰肝素只能形成一类复合物。其化学计量比为1:1，在0.05 M和0.2 M NaCl时的Kd值分别为3×10⁻⁸ M和1.6×10⁻⁷ M。如果在二聚体酶的每个亚基上存在一个糖胺聚糖的独特结合区域，且一条相对分子质量大于10000的肝素链能够同时占据这两个位点以形成1:1复合物，那么这些结果就能得到解释。较短的肝素链无法跨越这些位点，为了占据它们，两条链必须与每个酶分子相互作用。
