The interconversion of the retinenes and vitamins a in vitro

Abstract

The retinene 1 which results from the bleaching of rhodopsin now appears to be vitamin A 1 aldehyde. Morton et al. have given the best evidence for this, and have shown that retinene 1 can be prepared by the mild oxidation of vitamin A 1. A simple procedure is described for performing this process chromatographically on a column of manganese dioxide. In the retina, retinene 1 is converted irreversibly to vitamin A 1 by an enzyme system in which reduced cozymase (reduced Coenzyme I, DPNH 2 serves as coenzyme. The essential process is the transfer of two hydrogen atoms from DPNH 2 to retinene 1, reducing its aldehyde group to the primary alcohol group of vitamin A 1. The enzyme system which performs this reduction can be assembled in solution from the following components: the coenzyme, DPNH 2; as substrate, synthetic retinene 1; and the apoenzyme extracted with dilute salt solutions from homogenized frog or cattle retinas. The apoenzyme is non-dialysable, is precipitated by half-saturated ammonium sulphate, and is destroyed by heating at 100°C within 30 seconds. Its p H optimum lies at about 6.5. In the rods of freshwater fishes, a parallel enzyme system reduces retinene 2 to vitamin A 2. This can be assembled from the following components, all in true solution: the coenzyme, DPNH 2; as substrate, synthetic retinene 2, prepared by the chromatographic oxidation of vitamin A 2 on manganese dioxide; and the apoenzyme extracted with dilute salt solutions from freshwater fish retinas (sunfish, yellow perch). The apoenzyme from frog retinas reduces retinene 2 as effectively as retinene 1. Similarly the fish apoenzyme acts equally well upon both retinenes. One need consider only one apoenzyme, retinene reductase, which together with one coenzyme, DPNH 2, reduces either of the retinenes to the corresponding vitamin A. The retinene reductase system brings a second vitamin into the chemistry of rod vision. It presents the novel phenomenon of one vitamin regenerating another, for the central component of DPNH 2 is nicotinamide, the anti-pellagra factor of the vitamin B complex. Rhodopsin solutions and retinal homogenates rapidly lose their power to reduce the retinenes, through destruction of their DPN by a nucleotidase. Rhodopsin solutions which have lost their activity in this way are re-activated by the addition of new DPNH 2. The coenzyme can also be protected by the presence of free nicotinamide and of α-tocopheryl phosphate. On addition to the enzyme system, the synthetic retinenes rapidly couple with other molecules, and primarily with protein. The normal state of the retinenes in retinas and retinal extracts is a labile equilibrium between the free and the coupled condition. The retinenes couple with a variety of retinal molecules, and migrate freely from one to the other.