Naoki Kamo

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Acetabularia rhodopsins are the first microbial rhodopsins discovered in a marine plant organism, Acetabularia acetabulum. Previously, we expressed Acetabularia rhodopsin II (ARII) by a cell-free system from one of two opsin genes in A. acetabulum cDNA and showed that ARII is a light-driven proton pump [Wada, T., et al. (2011) J. Mol. Biol.411, 986–998]. In this study, the photochemistry of ARII was examined using the flash-photolysis technique, and data were analyzed using a sequential irreversible model. Five photochemically defined intermediates (P_i) were sufficient to simulate the data. Noticeably, both P₃ and P₄ contain an equilibrium mixture of M, N, and O. Using a transparent indium tin oxide electrode, the photoinduced proton transfer was measured over a wide pH range. Analysis of the pH-dependent proton transfer allowed estimation of the pK_a values of some amino acid residues. The estimated values were 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values were assigned as the pK_a of Asp81 (Asp85^BR) in the dark, Asp92 (Asp96^BR) at N, Glu199 (Glu204^BR) at M, Glu199 in the dark, an undetermined proton-releasing residue at the release, and the pH to start denaturation, respectively. Following this analysis, the proton transfer of ARII is discussed.

Japan's largest platform for academic e-journals: J-STAGE is a full text database for reviewed academic papers published by Japanese societies

Mycophenolic acid (MPA), an immunosuppressant, is excreted as its glucuronized form, MPAG. In humans, MPAG is mostly excreted into urine, whereas more than 80% of the dose is excreted into bile in rats. The aim of this study was to clarify the cause of the species difference. We investigated whether MPAG is a substrate of human organic anion transporters (hOATs), and we compared the affinities of multi-drug resistance-associated protein 2 (MRP2) for MPAG in rats and humans. The inhibitory effects of MPAG on the uptake of typical substrates via hOAT1 and hOAT3 were determined using HeLa cells heterologously expressing hOAT1 and Xenopus laevis oocytes heterologously expressing hOAT3. MPAG transport activity via hOAT1 and hOAT3 was determined by the two-microelectrode voltage-clamp technique using Xenopus laevis oocytes expressing hOAT1 and hOAT3. The affinities of MPAG for hMRP2 and rMrp2 were determined by the inhibitory effects of MPAG on p-aminohippuric acid (a typical substrate) uptake using membrane vesicles expressing hMRP2 or rMrp2. MPAG inhibited the uptake of PAH via hOAT1 and hOAT3, and calculated IC50 values were 222.6+/-26.6 microM and 41.5+/-11.5 microM, respectively. However, MPAG was not transported by hOAT1 and hOAT3. MPAG strongly inhibited the uptake of PAH via both rMrp2 and hMRP2. However, the magnitudes of inhibitory effects were different. The calculated IC50 values were 286.2+/-157.3 microM and 1036.8+/-330.5 microM, respectively. MPAG is not a substrate but is an inhibitor of hOAT1 and hOAT3. The affinity of rMRP2 to MPAG was about 3.6 times as high as that of hMRP2. Therefore, the difference of affinity between hMRP2 and rMrp2 is a possible mechanism of the difference of excretion ratio of MPAG between rats and human.