Fig. 5

11cROL-oxidase activity of ZCRDH and various mammalian RDH candidates for the cone 11cROL-oxidase and substrate kinetics for ZCRDH and RDH12 HEK293T cells were transfected with non-recombinant pcDNA3.1 or the same plasmid containing the coding regions for the indicated proteins. All clones were made with a C-terminal FLAG tag. Assays were carried out using homogenate of the transfected cells as an enzyme source with 2 μM 11cROL and 1 mM NADP+ cofactor for 1 min at 37°C with gentle agitation. (A) 11cROL-oxidase activities (11cRAL synthesis): zf-ZCRDH, zebrafish ZCRDH; hum-RDH11, human RDH11; hum RDH12, human RDH12; hum-RDH13, human RDH13; and mus-RDH14, mouse RDH14. (B) 11cROL-oxidase activities: zf-ZCRDH-L, zebrafish ZCRDH-like; zf-RDH12, predicted zebrafish RDH12; zf-RDH12-L, predicted zebrafish RDH12-like; carp ZCRDH, carp ZCRDH; and carp RDH13-L, carp RDH13-like. (C) Immunoblots containing equal protein amounts of HEK293T cell homogenates transfected with the indicated plasmids and reacted with an anti-FLAG antibody. Note the similar intensities of immunoreactive bands containing the different proteins. The same blot was re-probed with an antibody against β-tubulin as a protein loading control, shown below the anti-FLAG immunoblot. (D and E) (D) Michaelis-Menton analysis of 11cROL oxidation of ZCRDH and (E) human RDH12 using the indicated 11cROL concentrations and 1 mM NADP+. The assay conditions for kinetic analysis are as described in this figure. Calculated KM and Vmax values are shown. Error bars show standard deviation of the mean for three replicates (n = 3). See also Figures S1 and S3 and Tables S1 and S2 .