Banner C D, Göttlicher M, Widmark E, Sjövall J, Rafter J J, Gustafsson J A
Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Sweden.
J Lipid Res. 1993 Sep;34(9):1583-91.
Using a novel combination of analytical chemical and molecular biological techniques, lipophilic components of human plasma separated according to their physico-chemical properties were screened for their ability to activate the rat peroxisome proliferator-activated receptor (rPPAR). Activation of an rPPAR/glucocorticoid receptor chimera stably expressed in CHO cells by fractions in the initial screening guided further subfractionation. Characterization of an active subfraction by gas chromatography alone and in combination with mass spectrometry (GC-MS), indicated the presence of free fatty acids. Individual active components in this mixture were isolated by a final fractionation using high performance liquid chromatography (HPLC). GC-MS analyses of HPLC fractions able to activate the chimeric receptor identified palmitic acid, oleic acid, linoleic acid, and arachidonic acid as endogenous activators of rPPAR. No other activators were identified. This approach is able to specifically extract and identify endogenous activators of PPAR from a complex biological extract and as such may be valuable in the identification of activators of other orphan receptors in the steroid hormone receptor superfamily.
运用分析化学和分子生物学技术的新型组合，根据物理化学性质分离出的人血浆亲脂性成分，被筛选其激活大鼠过氧化物酶体增殖物激活受体（rPPAR）的能力。在初步筛选中，通过各组分对稳定表达于CHO细胞中的rPPAR/糖皮质激素受体嵌合体的激活作用，指导进一步的亚分级分离。单独使用气相色谱以及与质谱联用（GC-MS）对活性亚组分进行表征，表明存在游离脂肪酸。通过使用高效液相色谱（HPLC）进行最终分级分离，分离出该混合物中的各个活性成分。对能够激活嵌合受体的HPLC级分进行GC-MS分析，确定棕榈酸、油酸、亚油酸和花生四烯酸为rPPAR的内源性激活剂。未鉴定出其他激活剂。这种方法能够从复杂的生物提取物中特异性地提取和鉴定PPAR的内源性激活剂，因此在鉴定类固醇激素受体超家族中其他孤儿受体的激活剂方面可能具有价值。
