Nishihira J, Ishibashi T, Sakai M, Nishi S, Kumazaki T
Department of Biochemistry, School of Medicine, Hokkaido University, Sapporo, Japan.
Biochem Biophys Res Commun. 1993 Feb 15;190(3):823-31. doi: 10.1006/bbrc.1993.1123.
The association of glutathione S-transferase P (GST-P) with various fatty acids was confirmed by gas-liquid chromatography and mass-spectrometry. Palmitic and stearic acids were the major fatty acids bound to the enzyme in which the molar ratio was 1:0.8 (GST-P:fatty acid). To evaluate the association with respect to the fatty acid carbon chain length and identify the binding site, we prepared a series of fatty acid-linked Sepharoses. GST-P tightly bound the fatty acid-linked Sepharoses (CH3(CH2)nCOOH, n = 4 approximately 16), and the site was determined to be residues 121-156 from the amino terminus by tryptic digestion of GST-P bound to a fatty acid-linked Sepharose. By fatty acid affinity labeling, we identified the binding site as residues 141-188 (Biochem. Biophys. Res. Commun., in press). The overlapping hydrophobic residues (residue 141-156) is expected to be essential for the ligand binding site.
通过气液色谱法和质谱法证实了谷胱甘肽S-转移酶P（GST-P）与各种脂肪酸的结合。棕榈酸和硬脂酸是与该酶结合的主要脂肪酸，其摩尔比为1:0.8（GST-P：脂肪酸）。为了评估与脂肪酸碳链长度相关的结合情况并确定结合位点，我们制备了一系列脂肪酸连接的琼脂糖凝胶。GST-P紧密结合脂肪酸连接的琼脂糖凝胶（CH3(CH2)nCOOH，n = 4至16），通过对与脂肪酸连接的琼脂糖凝胶结合的GST-P进行胰蛋白酶消化，确定该位点为氨基末端的121-156位残基。通过脂肪酸亲和标记，我们确定结合位点为141-188位残基（《生物化学与生物物理研究通讯》，即将发表）。重叠的疏水残基（141-156位残基）预计对配体结合位点至关重要。
