Frolov A, Cho T H, Murphy E J, Schroeder F
Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station 77843-4466, USA.
Biochemistry. 1997 May 27;36(21):6545-55. doi: 10.1021/bi970205t.
Although native rat liver fatty acid binding protein (L-FABP) is composed of isoforms differing in isoelectric point, their comparative structure and function are unknown. These properties of apo- and holo-L-FABP isoforms were resolved by circular dichroism, time-resolved fluorescence spectroscopy, and binding/displacement of fluorescent ligands. Both apo-isoforms had similar hydrodynamic radii of 18.5 A, but apo-isoform I had a greater alpha-helical content and exhibited a longer Tyr lifetime, indicative of secondary and tertiary structural differences from isoform II. Isoforms I and II both had two fatty acid or fatty acyl CoA binding sites. Ligand binding decreased the isoform hydrodynamic radii by 3-4 A and increased Tyr rotational motions in a more restricted range. Fatty acyl CoAs were more effective than fatty acids in altering the isoform structures. Scatchard analysis showed that both isoforms bound cis- parinaric acid with high affinity (Kd values 41 and 60 nM, respectively) and bound trans-parinaric acid with 2- and 7-fold, respectively, higher affinity than for cis-parinaric acid. In contrast, isoform I had higher affinity for cis- and trans-parinaroyl CoAs (Kd values of 33 and 14 nM) than did isoform II (Kd values of 110 and 97 nM), thereby resulting in biphasic plots of parinaroyl-CoA binding to native L-FABP. Finally, displacement studies indicated that each isoform displayed distinct specificities for fatty acid/fatty acyl CoA chain length and unsaturation. Thus, rat L-FABP isoforms differ markedly in both structure and ligand binding function.
尽管天然大鼠肝脏脂肪酸结合蛋白（L-FABP）由等电点不同的同工型组成，但其比较结构和功能尚不清楚。通过圆二色性、时间分辨荧光光谱以及荧光配体的结合/置换，解析了脱辅基和全蛋白L-FABP同工型的这些特性。两种脱辅基同工型的流体动力学半径相似，均为18.5 Å，但脱辅基同工型I具有更高的α-螺旋含量，且酪氨酸寿命更长，这表明其与同工型II在二级和三级结构上存在差异。同工型I和II均有两个脂肪酸或脂肪酰基辅酶A结合位点。配体结合使同工型流体动力学半径减小3 - 4 Å，并在更受限的范围内增加了酪氨酸的旋转运动。脂肪酰基辅酶A在改变同工型结构方面比脂肪酸更有效。Scatchard分析表明，两种同工型均以高亲和力结合顺式十八碳四烯酸（Kd值分别为41和60 nM），且结合反式十八碳四烯酸的亲和力分别比顺式十八碳四烯酸高2倍和7倍。相比之下，同工型I对顺式和反式十八碳四烯酰基辅酶A的亲和力（Kd值分别为33和14 nM）高于同工型II（Kd值分别为110和97 nM），从而导致十八碳四烯酰基辅酶A与天然L-FABP结合的双相图。最后，置换研究表明，每种同工型对脂肪酸/脂肪酰基辅酶A链长和不饱和度表现出不同的特异性。因此，大鼠L-FABP同工型在结构和配体结合功能上均存在显著差异。
