Article Figures & Data
Figures
- FIG. 1.
Evaluation of Mms16 as an anchor molecule for protein assembly onto BMP membrane. (A) SDS-PAGE of BMP membrane proteins. Forty micrograms of protein was applied to the gel. Standard molecular mass makers (in kilodaltons) were used. An arrowhead indicates Mms16. (B) Expression of HA-tagged Mms16 on BMPs. Fluorescent intensity (expressed in arbitrary units [a.u.]) was determined by immunoassay using rhodamine-conjugated anti-HA antibody when 50 μg of BMPs was used in 100 μl of PBST. WT, BMPs from wild-type AMB-1; 16HA, BMPs from AMB-1 transformant harboring pUM16HA; HA16, BMPs from AMB-1 transformant harboring pUMHA16.
- FIG. 2.
Efficiencies of anchoring Mms16 and MagA onto BMP membrane and cell membrane. Luminescent intensity of BMPs (50 μg) (A) and cell membrane (1 μg) (B) was determined with a luminescence reader (Lucy-2) after addition of luciferin solution (100 μl). (C) Schematic diagram of each plasmid. Solid, shaded, and white blocks indicate genes encoding luciferase, Mms16, and MagA, respectively. Shaded and white triangles indicate the promoters of mms16 and magA, respectively. The numbers 1 to 4 indicate the following: 1, pUML (promoter pmms16 and no anchor); 2, pUM16L (promoter pmms16 and anchor Mms16); 3, pUMML (promoter pmms16 and anchor MagA); 4, pKML (promoter pmagA and anchor MagA). Plasmids 1, 2, and 3 were derived from pUMG (27), and plasmid 4 was derived from pRK415 (24).
- FIG. 3.
Expression analysis of D1R on BMPs by antibody and antagonist binding. (A) Schematic diagram for preparation of recombinant BMPs and assembly of D1R. In step a, a plasmid (pUM16D1) containing a gene fusion for Mms16 and D1R expression was transformed into wild-type AMB-1. In step b, the AMB-1 transformant harboring pUM16D1 was disrupted to release recombinant BMPs. In step c, recombinant BMPs were magnetically separated and purified by stringent washing. D1R was assembled on recombinant BMPs. (B) Expression analysis of D1R was performed with anti-D1R antibody by a method derived from enzyme-linked immunosorbent assay of BMPs extracted from AMB-1 (bar 1) and BMPs extracted from an AMB-1 transformant harboring pUM16D1 (bar 2). Luminescence intensity was determined with the secondary ALP-conjugated antibody and Lumiphos 530. (C) Binding of [3H]SCH23390 to the surfaces of the BMPs shown in bars 1 and 2. A sample of 100 μg of BMPs was used for each assay. Results represent the average with standard deviations from more than three experiments.
- FIG. 4.
Specific saturation binding of a radiolabeled D1R-specific antagonist [3H]SCH23390 to recombinant BMPs extracted from AMB-1 transformants harboring pUM16D1.
- FIG. 5.
Fluorescence competition binding analysis of dopamine to D1R on BMP surfaces. Recombinant BMPs were incubated with 10−7 M BODIPY-labeled SCH23390. Concentrations of dopamine, a D1R agonist, are indicated.
