Myszka D G, Swenson R P
Department of Biochemistry, Ohio State University, Columbus 43210.
J Biol Chem. 1991 Nov 5;266(31):20725-31.
Two different photoaffinity analogs of 4-hydroxy coumarin, 3-(p-azidobenzyl)-4-hydroxycoumarin (AzBHC) and 3-(4-azido-5-iodosalicylamido)-4-hydroxycoumarin (AzISAHC), are being used in the identification of warfarin-binding proteins present in mammalian tissue (Myszka, D. G., and Swenson, R. P. (1990) Biochem. Biophys. Res. Commun. 172, 415-422; Myszka, D. G., and Swenson, R. P. (1991) J. Biol. Chem. 266, 4789-4797). In this study, [14C]AzBHC, but not [125I]AzISAHC, was observed to specifically label a 15,000-dalton protein present in both the microsomal and cytosolic fractions of rat liver. Pretreatment of the crude protein samples with warfarin or dicoumarol completely protected the 15-kDa protein from modification by [14C]AzBHC, indicating that this photoaffinity reagent is specifically labeling a coumarin-binding protein. 4-Hydroxycoumarin itself and AzISAHC were unable to block the incorporation of this photoaffinity probe. The 15-kDa protein was isolated by two-dimensional electrophoresis and subjected to amino-terminal sequence analysis. The first 20 amino acid residues analyzed were found to be identical with the amino-terminal sequence of rat liver fatty acid-binding protein (L-FABP) (Gordon J. I., Alpers, D. H., Ockner, R. K., and Strauss, A. W. (1983) J. Biol. Chem. 258, 3356-3363). Photoaffinity labeling and protection experiments carried out on purified preparations of L-FABP paralleled the labeling results obtained in the microsomes and cytosol, confirming that L-FABP is capable of specifically binding AzBHC, warfarin, and dicoumarol. Oleic acid, an established ligand for L-FABP, can compete with the binding of the photoaffinity probe; however, it was less effective in protecting the protein than warfarin. The specificity of labeling of crude liver fractions by warfarin photoaffinity analogs reported here as well as the high concentration of FABP in liver tissue together suggest that this protein may represent a major hepatic receptor responsible for the uptake and/or transport of various oral 4-hydroxycoumarin-based anticoagulant drugs.
4-羟基香豆素的两种不同的光亲和类似物，3-(对叠氮苄基)-4-羟基香豆素(AzBHC)和3-(4-叠氮-5-碘水杨酰胺基)-4-羟基香豆素(AzISAHC)，正被用于鉴定哺乳动物组织中存在的华法林结合蛋白(Myszka, D. G., and Swenson, R. P. (1990) Biochem. Biophys. Res. Commun. 172, 415 - 422; Myszka, D. G., and Swenson, R. P. (1991) J. Biol. Chem. 266, 4789 - 4797)。在本研究中，观察到[14C]AzBHC，而非[125I]AzISAHC，能特异性标记大鼠肝脏微粒体和胞质溶胶部分中存在的一种15000道尔顿的蛋白质。用华法林或双香豆素预处理粗蛋白样品，可完全保护15 kDa蛋白不被[14C]AzBHC修饰，这表明这种光亲和试剂特异性标记了一种香豆素结合蛋白。4-羟基香豆素本身和AzISAHC无法阻断这种光亲和探针的掺入。通过二维电泳分离出15 kDa的蛋白，并进行氨基末端序列分析。分析的前20个氨基酸残基与大鼠肝脏脂肪酸结合蛋白(L-FABP)的氨基末端序列相同(Gordon J. I., Alpers, D. H., Ockner, R. K., and Strauss, A. W. (1983) J. Biol. Chem. 258, 3356 - 3363)。对纯化的L-FABP制剂进行的光亲和标记和保护实验与在微粒体和胞质溶胶中获得的标记结果相似，证实L-FABP能够特异性结合AzBHC、华法林和双香豆素。油酸是L-FABP的一种既定配体，它可以与光亲和探针的结合竞争；然而，它在保护蛋白质方面不如华法林有效。本文报道的华法林光亲和类似物对粗肝部分标记的特异性以及肝组织中FABP的高浓度共同表明，这种蛋白可能代表负责摄取和/或转运各种口服4-羟基香豆素类抗凝药物的主要肝脏受体。
