Metaretinochrome in membranes as an effective donor of 11-cis retinal for the synthesis of squid rhodopsin.

Abstract

Aporetinochrome, which is a protein moiety of retinochrome without chromophore retinal, is found in the membrane containing retinochrome. All of the prosthetic retinal of retinochrome in membranes, which is all-trans retinal, is bound to the chromophoric site on the protein moiety, with protonated Schiff bases showing an absorption band with the maximum at 495 nm. On exposure to light, retinochrome is converted to metaretinochrome at room temperature. The prosthetic retinals of metaretinochrome in membranes, which are 11-cis retinals, are in 2 states: retinals bound to the chromophoric site with protonated Schiff bases, and the free retinals, which are separated from the protein moiety. These states are suggested from the following observations. The ratio of the absorbance at 470 nm of metaretinochrome to that at 495 nm of the parental retinochrome differs because of differences in samples and is higher in the purer preparations. The difference spectrum of absorption of metaretinochrome caused by alkalinization shows 2 minimum peaks at .apprx. 420 and 470 nm. The rate of bleaching of metaretinochrome in membranes with dilute NH2OH is much faster than that of retinochrome, and the absorption band in the near-UV region is more susceptible to NH2OH than the visible absorption band. The state of the prosthetic retinals in metaretinochrome was confirmed directly by the reaction of metaretinochrome in membranes with NaBH4. After treatment with NaBH4, the sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern shows 2 fluorescent bands: one at the position that corresponds to the retinochrome protein (MW 27,000 .+-. 2000) and another at the front of migration, where no band protein is observed. Retinoids extracted from the NaBH4-treated metaretinochrome in membranes and analyzed with high-pressure liquid chromatography show a main peak of 11-cis retinol. Metaretinochrome in the squid retina may play the role of 11-cis retinal donor for opsin and contribute to the synthesis of the squid rhodopsin.