Biozentrum of the Martin-Luther-University Halle-Wittenberg, Weinbergweg 22, 06120 Halle, Germany.
Amino Acids. 2013 Feb;44(2):373-81. doi: 10.1007/s00726-012-1340-9. Epub 2012 Jun 19.
Mechanism and substrate specificity of the proton-coupled amino acid transporter 2 (PAT2, SLC36A2) have been studied so far only in heterologous expression systems such as HeLa cells and Xenopus laevis oocytes. In this study, we describe the identification of the first cell line that expresses PAT2. We cultured 3T3-L1 cells for up to 2 weeks and differentiated the cells into adipocytes in supplemented media containing 2 μM rosiglitazone. During the 14 day differentiation period the uptake of the prototype PAT2 substrate L-[(3)H]proline increased ~5-fold. The macro- and microscopically apparent differentiation of 3T3-L1 cells coincided with their H(+) gradient-stimulated uptake of L-[(3)H]proline. Uptake was rapid, independent of a Na(+) gradient but stimulated by an inwardly directed H(+) gradient with maximal uptake occurring at pH 6.0. L-Proline uptake was found to be mediated by a transport system with a Michaelis constant (K(t)) of 130 ± 10 μM and a maximal transport velocity of 4.9 ± 0.2 nmol × 5 min(-1 )mg of protein(-1). Glycine, L-alanine, and L-tryptophan strongly inhibited L-proline uptake indicating that these amino acids also interact with the transport system. It is concluded that 3T3-L1 adipocytes express the H(+)-amino acid cotransport system PAT2.
迄今为止，质子偶联氨基酸转运体 2（PAT2，SLC36A2）的作用机制和底物特异性仅在异源表达系统（如 HeLa 细胞和非洲爪蟾卵母细胞）中进行了研究。在本研究中，我们描述了鉴定出的第一个表达 PAT2 的细胞系。我们培养 3T3-L1 细胞长达 2 周，并在含有 2 μM 罗格列酮的补充培养基中将细胞分化为脂肪细胞。在 14 天的分化期间，原型 PAT2 底物 L-[3H]脯氨酸的摄取增加了约 5 倍。3T3-L1 细胞的宏观和微观可见分化与它们在 H+梯度刺激下摄取 L-[3H]脯氨酸的情况相吻合。摄取过程迅速，与 Na+梯度无关，但被内向 H+梯度刺激，最大摄取发生在 pH 6.0。发现 L-脯氨酸摄取是由一种转运系统介导的，其米氏常数（K(t)）为 130±10 μM，最大转运速度为 4.9±0.2 nmol×5 min-1mg 蛋白-1。甘氨酸、L-丙氨酸和 L-色氨酸强烈抑制 L-脯氨酸摄取，表明这些氨基酸也与转运系统相互作用。结论是，3T3-L1 脂肪细胞表达 H+-氨基酸共转运系统 PAT2。
