Phoborhodopsin, a repellent phototaxis receptor in Halobacterium halobium, exhibits vibrational fine structure, a feature that has not been identified for any other rhodopsin pigment at physiological temperatures. This conclusion follows form analysis of the absorption properties of the pigment in H. halobium membranes containing native retinal and an array of retinal analogues. The absorption spectrum of the native pigment has a maximum at 487 nm with a pronounced shoulder at 460 nm; however, the bandwidth is that expected for a single retinylidene species. Gaussian band-shape simulation with a spacing corresponding to the vibrational frequencies of polyene stretching modes reproduces the structured absorption spectra of native pigment as well as of analogue phoborhodopsin. Absorption shifts produced by a series of dihydroretinal and other retinal analogues strongly indicate that the dominant factor regulating the color of the pigment is planarization of the retinal ring with respect to the polyene chain.

In mammals, retinol is the precursor for retinoids, which affect various aspects of morphogenesis and development. However, B lymphocytes, although retinol-dependent, do not use retinoic acid as mediator. Retinol is metabolized by B lymphocytes and other cell lines to optically active 14-hydroxy-4,14-retro-retinol; it is this compound that mediates the growth control. Thus another second messenger molecule, in addition to retinoic acid and retinal, is derived from retinol.

Retinoids are a large family of natural and synthetic compounds related to vitamin A that have pleiotropic effects on body physiology, reproduction, immunity, and embryonic development. The diverse activities of retinoids are primarily mediated by two families of nuclear retinoic acid receptors, the RARs and RXRs. Retinoic acids are thought to be the only natural ligands for these receptors and are widely assumed to be the active principle of vitamin A. However, during an unbiased, bioactivity-guided fractionation of Xenopus embryos, we were unable to detect significant levels of all-trans or 9-cis retinoic acids. Instead, we found that the major bioactive retinoid in the Xenopus egg and early embryo is 4-oxoretinaldehyde, which is capable of binding to and transactivating RARs. In addition to its inherent activity, 4-oxoretinaldehyde appears to be a metabolic precursor of two other RAR ligands, 4-oxoretinoic acid and 4-oxoretinol. The remarkable increase in activity of retinaldehyde and retinol as a consequence of 4-oxo derivatization suggests that this metabolic step could serve a critical regulatory function during embryogenesis.Retinoic acid (RA) is a vitamin A metabolite that has profound effects on growth, differentiation, and development (1). RA is a potent teratogen in human fetal development (2) and influences limb development and regeneration (for reviews, see refs. 3 and 4). RA is also capable of altering positional identities along the vertebrate anteroposterior axis. Treatment of early Xenopus embryos with RA elicits concentrationdependent anterior truncations, which some have interpreted to represent posterior transformations (5). The majority of the effects of RA are likely to result from alterations in gene expression elicited by the two classes of ligand-dependent transcription factors, RA receptors (RARs) and 9-cis RA receptors (RXRs) (for review, see ref. 6).Our interest in the effects of endogenous retinoids in Xenopus development led us to undertake a search for naturally occurring compounds capable of activating RARs. In preliminary experiments, we were intrigued by the relative paucity of all-trans and 9-cis RAs compared with other RAR agonists in the egg and early embryo, leading us to look for other retinoid derivatives. Previous attempts had focused on separating embryonic extracts by HPLC and matching the retention times of components having absorbance at 350 nm with those of known standard retinoids (5, 7-9). To identify potentially novel compounds, we used HPLC fractionation and purification guided simply by biological activity as measured by transcriptional regulation. This strategy does not presume prior knowledge of endogenous retinoids and has the benefit of being able to identify compounds with unusual fractionation and absorption properties.This approach identified 4-oxoretinaldehyde (4-oxo-RAL) as the major bioactive retinoid in the Xenopus egg and early embryo. In addition to being able to bind to and transactivate RARs, 4-oxo-RAL appears to be a metabolic precursor o...

We consider the problem of color regulation in visual pigments for both bovine rhodopsin (lambda max = 500 nm) and octopus rhodopsin (lambda max = 475 nm). Both pigments have 11-cis-retinal (lambda max = 379 nm, in ethanol) as their chromophore. These rhodopsins were bleached in their native membranes, and the opsins were regenerated with natural and artificial chromophores. Both bovine and octopus opsins were regenerated with the 9-cis- and 11-cis-retinal isomers, but the octopus opsin was additionally regenerated with the 13-cis and all-trans isomers. Titration of the octopus opsin with 11-cis-retinal gave an extinction coefficient for octopus rhodopsin of 27,000 +/- 3000 M-1 cm-1 at 475 nm. The absorption maxima of bovine artificial pigments formed by regenerating opsin with the 11-cis dihydro series of chromophores support a color regulation model for bovine rhodopsin in which the chromophore-binding site of the protein has two negative charges: one directly hydrogen bonded to the Schiff base nitrogen and another near carbon-13. Formation of octopus artificial pigments with both all-trans and 11-cis dihydro chromophores leads to a similar model for octopus rhodopsin and metarhodopsin: there are two negative charges in the chromophore-binding site, one directly hydrogen bonded to the Schiff base nitrogen and a second near carbon-13. The interaction of this second charge with the chromophore in octopus rhodopsin is weaker than in bovine, while in metarhodopsin it is as strong as in bovine.

All-trans-retinoic acid (at-RA) induces cell differentiation in a wide variety of cell types, including F9 embryonic teratocarcinoma cells, and can influence axial pattern formation during embryonic development. We now identify a novel retinoid synthetic pathway in differentiating F9 cells that results in the intracellular production of 4-oxoretinol (4-oxo-ROL) from retinol (vitamin A). Approximately 10-15% of the total retinol in the culture is metabolized to 4-hydroxyretinol and 4-oxo-ROL by the at-RA-treated, differentiating F9 cells over an 18-hr period, but no detectable metabolism of all-trans-retinol to at-RA or 9-cis-retinoic acid is observed in these cells. Remarkably, we show that 4-oxo-ROL can bind and activate transcription of the retinoic acid receptors whereas all-rans-retinol shows neither activity. Low doses of 4-oxo-ROL (e.g., 10-9 or 10-1o M) can activate the retinoic acid receptors even though, unlike at-RA, 4-oxo-ROL does not contain an acid moiety at the carbon 15 position. 4-oxo-ROL does not bind or transcriptionally activate the retinoid X receptors. Treatment of F9 cells with 4-oxo-ROL induces differentiation without conversion to the acid and 4-oxo-ROL is active in causing axial truncation when administered toXenopus embryos at the blastula stage. Thus, 4-oxo-ROL is a natural, biologically active retinoid that is present in differentiated F9 cells. Our data suggest that 4-oxo-ROL may be a novel signaling molecule and regulator of cell differentiation.Retinoids are derivatives of retinol (vitamin A) that are required for the appropriate functioning of a wide number of cell types involved in processes such as cell differentiation, axial pattern formation in embryogenesis, reproduction, and vision (for reviews, see refs. 1-3). Retinoids also exhibit cancer preventive actions and are used to treat some types of cancers (for review, see ref.2). The biological and transcriptional effects of retinoids are mediated by their interactions with the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) (for review, see ref. 4). RARs and RXRs can heterodimerize and bind response elements in a variety of target genes (for reviews, see refs. 1 and 4). The ligands for these receptors are generally thought to be acid derivatives of retinol; all-trans-retinoic acid (at-RA) has been shown to bind to the RARs with high affinity and 9-cis-retinoic acid (9-cis-RA) binds and activates the RXRs as well the RARs (for reviews, see refs. 4 and 5). The N-terminal portion of the ligand binding domain (E domain) is essential for the recognition of at-RA by various RARs (6).Much recent research in the vitamin A field has focused on at-RA and its isomers. Retinol has been reported to be metabolized to at-RA in a large number of different cultured cells and in various tissues from developing embryos (3, 5). However, the complexity of the biological actions of vitamin A suggests that retinol derivatives other than at-RA mediate some of these actions. Consistent with this idea, two nonacidic retinol d...