The Mole Quantity and Turnover Number of Rpe65 in the Generation of 11–Cis Retinal in the Living Mouse Eye

Abstract

Abstract: : Purpose: In common strains of mice to (1) determine the mole quantity and turnover number of Rpe65 in the synthesis of 11–cis retinal; (2) assess whether the quantity and functionality of Rpe65 is dependent on the Leu450Met polymorphism; (3) test the hypothesis that the rate of rhodopsin regeneration is limited by the quantity of Rpe65. Methods: We bred mice with 5 combinations of the Rpe65 L450/M450 polymorphism: Balb/c (L/L); C57BL/6N (M/M); Balb/c × C57BL/6N (L/M); Balb/c × Rpe65^–/– (L/–); C57BL/6N × Rpe65^–/– (M/–). In mice of each genotype we measured the quantity of Rpe65 per eye, the level of Rpe65 message (relative to that in Balb/c), and the initial rate of rhodopsin regeneration after a nearly complete bleach of rhodopsin. The latter rate provides an estimate of the maximum rate of 11–cis retinal synthesis in vivo. Results: The quantity of Rpe65 per eye ranged from 2.9 pmol (Balb/c) to 0.16 pmol (C57BL/6N × Rpe65^–/–). The amount of Rpe65 mRNA differed little among L/L, M/M & L/M mice, but was ∼ 40% lower in L/– and M/– mice; the protein/message ratio was similar in these latter mice. The initial rate of rhodopsin regeneration ranged from 0.36 to 2.2 % min^–1 and is describable as a Michaelis saturation function of Rpe65 with K_m = 0.76 pmol/eye, V_max = 2.8 % min^–1. In Balb/c mice, which have the highest regeneration rate (2.2% min^–1), the production of 11–cis retinal is nearly saturated, and thus not limited by the quantity of Rpe65. Given a total rhodopsin mass of ∼ 600 pmol/eye, for mice with Rpe65 levels well below the K_m, the initial rate of regeneration per mol Rpe65 is (V_max × 600)/ K_m = 22 min^–1, and is independent of the L/M polymorphism. Conclusions: (1) In animals with low expression, Rpe65 limits the initial rate of rhodopsin regeneration, presumably by limiting the rate of delivery of retinyl esters to the isomerohydrolase. (2) The apparent retinyl ester turnover per Rpe65 molecule under the conditions of our experiments is ∼ 20 min^–1, independent of the L/M variant expressed. (3) In animals such as Balb/c mice, which express it at high levels, Rpe65 does not limit the rate of regeneration, and thus, of 11–cis retinal synthesis.

Keywords: retinoids/retinoid binding proteins • photoreceptors • retinal pigment epithelium