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1.
Fig. 3.

Fig. 3. From: Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle.

Isomerization activity is inhibited by the iron chelator 2,2-DP. Extracts of 293-F cell cultures transfected with pVitro2/RPE65+CRALBP and pVitro3/LRAT+RDH5 vectors were treated with 2.5 μM all-trans-retinol for 3.5 h in the presence of 0-2,000 μM 2,2-DP. Retinoids were extracted, saponified, and analyzed by HPLC. Values for 11-cis-retinol synthesis in the presence of chelator are normalized to activity in the presence of ethanol vehicle alone, taken as 100% activity. Results are means and SDs of three independent experiments.

T. Michael Redmond, et al. Proc Natl Acad Sci U S A. 2005 September 20;102(38):13658-13663.
2.
Fig. 2.

Fig. 2. From: Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle.

RPE65, CRALBP, LRAT, and RDH5 are expressed only in transfected 293-F cells. (A) Immunoblot analysis of whole-cell lysates probed with antisera to, from top, RPE65, CRALBP, RDH5, and LRAT. Lane 1, untransfected 293-F cells; lane 2, 293-F cells transfected with an irrelevant plasmid; lane 3, 293-F cells transfected with pVitro2/RPE65+CRALBP and pVitro3/LRAT+RDH5. (B) Single-channel and merged confocal images of 293-F cells transfected with pVitro2/RPE65+CRALBP and labeled to demonstrate coexpression of RPE65 and CRALBP. Immunolabeling: (a) rabbit antibody to RPE565 (green); (b) mouse monoclonal antibody to CRALBP (red); (c) merged triple-labeled image; (d) merged image of untransfected 293 cells labeled with RPE65 and CRALBP antibodies. DAPI labels cell nuclei (blue) in all images.

T. Michael Redmond, et al. Proc Natl Acad Sci U S A. 2005 September 20;102(38):13658-13663.
3.
Fig. 1.

Fig. 1. From: Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle.

Specific transfection of RPE65 and LRAT induces 11-cis-retinol synthesis. (A) 11-cis-retinol synthesis by 293-F cells transfected with RPE65. Synthesis of 11-cis-retinol is seen only in 293-F cells transfected with pVitro2/RPE65+CRALBP and pVitro3/LRAT+RDH5 (red trace), and not when pVitro3/LRAT+RDH5 alone is transfected (blue trace). Traces are representative of multiple analyses. Peak identification: 1, 11-cis-retinol; 2, 13-cis-retinol; 3, all-trans-retinol. (Inset) Absorption spectra of peaks 1, 2, and 3 confirm the identity of the isomers (29). (B) Isomerization of all-trans- to 11-cis-retinol occurs optimally with coexpression of RPE65 and LRAT. Cultures are transfected with six different combinations of pVitro2/RPE65+CRALBP, pVitro2/RPE65, pVitro2/CRALBP, and/or pVitro3/LRAT+RDH5, or no DNA, and incubated with 2.5 μM all-trans-retinol, and retinols are analyzed after 6 h. Data represent means and SDs of three independent sets of experiments.

T. Michael Redmond, et al. Proc Natl Acad Sci U S A. 2005 September 20;102(38):13658-13663.
4.
Fig. 6.

Fig. 6. From: Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle.

Comparison of RPE65 with ACO. (A) Selected residues of RPE65 are plotted to equivalent positions on the carbon backbone of the ACO crystal structure (22) by using the visual molecular dynamics program (Version 1.8.3; www.ks.uiuc.edu/Research/vmd) (42). Residues are iron-coordinating (blue), conserved residues associated with disease (pink), conserved/nonconserved cysteines (yellow), and weak pathogenic/nonpathogenic mutations (red). The iron-coordinating residues are clustered in the proposed substrate binding tunnel. Residues more sensitive to mutation tend to occur in the core. (B) Sequence alignment of RPE65 and ACO showing key iron-coordinating, cysteine, and other residues. The original alignment obtained with DeepView/Swiss-PdbViewer (Version 3.7; http://ca.expasy.org/spdbv) was manually adjusted to maximize gap suppression and alignment with predicted structural features (β-sheets and α-helices). Residues are color-coded as above. *, identity; ·, similarity.

T. Michael Redmond, et al. Proc Natl Acad Sci U S A. 2005 September 20;102(38):13658-13663.
5.
Fig. 4.

Fig. 4. From: Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle.

Effect of mutation of iron-coordinating residues on RPE65 activity and expression. (A) Mutation of iron-coordinating residues abolishes isomerase activity. Synthesis of 11-cis-retinol is normalized to RPE65 immunoreactivity quantified by densitometry. Relative activities of mutants are compared with wild type, expressed as the mean and SD of three determinations. (B) Effect of histidine and glutamate mutations on RPE65 expression. Equivalent volumes of whole-cell lysates of transfections with pVitro2/RPE65+CRALBP and pVitro3/LRAT+RDH5 constructs analyzed by immunoblot using RPE65 (Upper) and CRALBP (Lower) antibodies. The expression level of all mutants except H180A was lower than wild type. Lane 1, untransfected control; lane 2, wild-type RPE65; lane 3, H180A; lane 4, H241A; lane 5, H313A; lane 6, H527A; lane 7, E417A. The lower band in Upper is a nonspecific reactant present in both untransfected and transfected 293-F cells.

T. Michael Redmond, et al. Proc Natl Acad Sci U S A. 2005 September 20;102(38):13658-13663.
6.
Fig. 5.

Fig. 5. From: Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle.

Effect of mutation of “palmitoylation switch” cysteines. (A) Individual conservative changes do not affect 11-cis-retinol synthesis by RPE65, but nonconservative (C330Y) or double (CC329/330SS) mutations abolish activity. Differences between wild type, C231S, C329S, and C330T are not significant. Synthesis of 11-cis-retinol is normalized to RPE65 immunoreactivity quantified by densitometry. Relative activities of mutants are compared with wild type, expressed as the mean and SD of three independent determinations. (B) Effect of cysteine mutations on RPE65 expression. Equivalent volumes of whole-cell lysates of cells transfected with pVitro2/RPE65+CRALBP and pVitro3/LRAT+RDH5 constructs analyzed by immunoblot with primary antibodies to RPE65 (Upper) and CRALBP (Lower). Lane 1, wild-type RPE65; lane 2, C231S; lane 3, C329S; lane 4, C330T; lane 5, C330Y; lane 6, CC329/330SS. The lower band in Upper is a nonspecific reactant present in both untransfected and transfected 293-F cells (see Fig. 4).

T. Michael Redmond, et al. Proc Natl Acad Sci U S A. 2005 September 20;102(38):13658-13663.

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