Rourke A M, Cha Y, Collins D
Department of Pharmaceutics and Drug Delivery, Amgen, Inc., Thousand Oaks, California 91320, USA.
Biochemistry. 1996 Sep 10;35(36):11913-7. doi: 10.1021/bi960491t.
Recombinant granulocyte colony-stimulating factor (rhG-CSF) interacts with liposomes composed of the anionic phospholipid dioleoylphosphatidylglycerol (DOPG), and this interaction enhances the stability of the protein [Collins, D., & Cha, Y. (1994) Biochemistry 33, 4521-4526]. In the present studies, we have examined the interaction of rhG-CSF with phospholipids other than DOPG. Fluorescence spectroscopy of rhG-CSF with a variety of lipid vesicles demonstrated that rhG-CSF inserts into bilayers of anionic, but not zwitterionic, phospholipids. Isothermal titration calorimetry of the interaction between DMPG and rhG-CSF indicates that the binding is saturable and involves 10 lipids/rhG-CSF. Studies of phosphatidylglycerols with varying alkyl chain lengths determined that the stabilization of rhG-CSF by anionic phospholipids required a certain alkyl chain length; no stabilization was observed with lipids of shorter chain length. Also investigated was the stabilization of other growth factors, which are structurally similar to rhG-CSF, by anionic phospholipids. These proteins include recombinant porcine somatotropin (rpSt), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), recombinant human interleukin 4 (rhIL-4), and recombinant human interleukin 2 (rhIL-2). The helical secondary structure of the proteins was recoverable after heating and cooling in the presence of anionic phospholipids as observed by circular dichroism; the presence of zwitterionic lipids did not induce this effect. Results of these investigations concluded that a group of structurally similar proteins interact preferentially with anionic phospholipids and that the complexation of the growth factors with vesicles composed of anionic phospholipids improves the stability of the proteins under conditions where they normally denature.
重组粒细胞集落刺激因子（rhG-CSF）与由阴离子磷脂二油酰磷脂酰甘油（DOPG）组成的脂质体相互作用，这种相互作用增强了蛋白质的稳定性[柯林斯，D.，& 查，Y.（1994年）《生物化学》33卷，4521 - 4526页]。在本研究中，我们研究了rhG-CSF与DOPG以外的磷脂的相互作用。用多种脂质囊泡对rhG-CSF进行荧光光谱分析表明，rhG-CSF可插入阴离子型而非两性离子型磷脂的双层膜中。对二肉豆蔻酰磷脂酰甘油（DMPG）与rhG-CSF之间相互作用的等温滴定量热法表明，这种结合是可饱和的，且每个rhG-CSF涉及10个脂质分子。对具有不同烷基链长度的磷脂酰甘油的研究确定，阴离子型磷脂对rhG-CSF的稳定作用需要一定的烷基链长度；链长较短的脂质未观察到稳定作用。还研究了阴离子型磷脂对其他与rhG-CSF结构相似的生长因子的稳定作用。这些蛋白质包括重组猪生长激素（rpSt）、重组人粒细胞 - 巨噬细胞集落刺激因子（rhGM-CSF）、重组人白细胞介素4（rhIL-4）和重组人白细胞介素2（rhIL-2）。通过圆二色性观察到，在阴离子型磷脂存在下加热和冷却后，蛋白质的螺旋二级结构可恢复；两性离子脂质的存在不会诱导这种效应。这些研究结果得出结论，一组结构相似的蛋白质优先与阴离子型磷脂相互作用，并且生长因子与由阴离子型磷脂组成的囊泡的络合作用提高了蛋白质在正常变性条件下的稳定性。
