Journal of Agricultural and Food Chemistry Figure 3. Comparison of the signals obtained from cytoplasmic and mitochondrial photoproteins. hT1R2/hT1R3-expressing cells were transiently transfected with each apophotoprotein expression plasmid and loaded with 10 μM coelenterazine. Each point indicates the mean ( SEM (n = 6). mt-, mitochondrial. luminescence-based assay with aequorin (Figure 1D) and the ﬂuo-4 assay (Figure 1C). The EC50 values measured in both assays were almost equal, that is, in the aequorin assay, EC50 was measured at 0.9 mM; in the ﬂuo-4 assay, EC50 was measured at 1.7 mM, although the signal/background ratio obtained from the luminescence-based assay was lower than that in the ﬂuores- cence-based assay; that is, in the aequorin assay, signal/back- ground = 2.8, and in the ﬂuo-4 assay, signal/background = 12.0. This result indicates that the luminescence-based assay using a photoprotein is equally suitable to the conventional ﬂuores- cence-based taste evaluation system for detecting the response of the sweet taste receptor-expressing cells. Evaluation of the Luminescence-Based Assay System in the Presence of an Artificial Fluorescent Substance. To investigate whether the luminescence-based assay could be applied to fluorescent samples, the luminescent signals from the photoprotein-transfected cells were measured in response to aspartame in the presence of a fluorescent substance, which was excited and detected at the wavelengths used in the fluo-4 measurements (Ex. 485 nm/Em. 525 nm). We ﬁrst selected BCECF, which is widely used as a ﬂuorescent pH indicator, as a model ﬂuorescent substance.27 When the ﬂuo- 4-loaded cells were treated with 10 40 nM BCECF alone and the ﬂuorescent intensity was monitored, a robust and dose- dependent rectangular ﬂuorescence response was detected im- mediately after the BCECF solution was added (Figure 2A). Because the BCECF-derived ﬂuorescence intensity remained approximately constant, it could be clearly distinguished from the response mediated by the sweet taste receptor, whose signal gradually increased after the addition of sweetener and declined with time (Figures 1A and 2B, 5 mM aspartame alone). In the ﬂuorescence-based assay, the strong BCECF-derived ﬂuores- cence intensity interfered with detection of the cellular response to aspartame, and we failed to detect a dose response relation in aspartame when more than 10 nM BCECF was added (Figure 2B,C). In contrast, when the aequorin-expressing cells were treated with 10 40 nM BCECF and the luminescence intensity was monitored, the addition of BCECF did not result in nonspeciﬁc or dose-dependent luminescent signals. In addition, the time response curves were nearly the same as the curve obtained after assay buﬀer application alone (Figure 2D), although BCECF was ARTICLE Table 1. Comparison of Measured EC50 Values and Signal/ Background Ratios from Cytoplasmic and Mitochondrial Photoproteins Using hT1R2/hT1R3-Expressing Cellsa photoprotein EC50 (mM) signal/background aequorin 1.2 4.9 mt-aequorin 1.5 11.6 clytin b mt-clytin 1.4 5.1 obelin 0.7 3.3 mt-obelin 1.2 2.6 clytin-II 0.8 6.6 mt-clytin-II 1.0 14.4 a The measured EC50 values and signal/background ratios were calcu- lated by ﬁtting the data to the Hill's equation. Signal/background = maximum signal/minimum signal. b , value not determined. added at a high enough concentration to produce robust signals in the ﬂuo-4 assay. Moreover, the cellular response to aspartame was precisely detected in the presence of BCECF using the aequorin assay, even when 40 nM BCECF was added to 5 mM aspartame (Figure 2E). The luminescence intensity of the aspartame-treated cells exhibited a dose dependence, and the EC50 values were approximately equal to each other, regardless of the presence of any of the BCECF concentrations tested (Figure 2F). Consequently, we conﬁrmed that the luminescence- based assay using aequorin could be used as an appropriate evaluation system for the human sweet taste receptor in the presence of an artiﬁcial ﬂuorescent substance. Selection of Photoproteins for Use in the Luminescence- Based Assay of Sweet Taste Receptor-Expressing Cells. It is well-known that the luminescence intensity varies according to photoprotein type under the same experimental conditions. To optimize our luminescence-based assay using Ca2+-sensitive photoproteins, we evaluated the luminescent properties of four photoproteins: aequorin, obelin, clytin, and clytin-II (Figure 3). When human sweet taste receptor-expressing cells were transfected with expression plasmids encoding each apophoto- protein, that is, apoaequorin, apoobelin, apoclytin, and apoclytin- II, and then loaded with coelenterazine to form the active photoproteins, the luminescence intensities of the four photo- proteins in response to aspartame could be detected in all cases except when clytin was used (Figure 3, closed symbols). The dose-dependent response to aspartame was detected using each indicator, and their EC50 values were all approximately 1 mM (Table 1). Among these photoproteins, only clytin-II produced a stronger luminescent signal intensity than aequorin (approximately three times higher). We also evaluated the cellular response to aspartame using mitochondrial photoproteins because localization of photopro- teins to the mitochondria has been reported to enhance lumines- cence in many GPCR assays.20,23,28 The luminescence intensities produced by aequorin, clytin, and clytin-II were increased upon mitochondrial targeting without changes being observed in the EC50 values (Figure 3, open symbols; Table 1). Among the photoproteins, mitochondrial clytin-II yielded the highest sig- nals, approximately ﬁve times higher than those produced by nonmitochondrially targeted aequorin, which was expressed in the cytoplasm (Figure 3). Mitochondrial clytin-II and mitochon- drial aequorin generated the highest signal/background ratios, which were nearly equivalent to the ratio obtained using the 12134 dx.doi.org/10.1021/jf2029835 |J. Agric. Food Chem. 2011, 59, 12131–12138