retinaldehyde and sodium-borohydride

Retinoids in the eggs of four teleosts, chum salmon (Oncorhynchus keta), black porgy (Acanthopagrus schlegeli), marbled flounder (Pleuronectes yokohamae), and stingfish (Inimicus japonicus), were analyzed by high performance liquid chromatography. Retinal (RAL1) or both RAL1 and 3,4-didehydroretinal (RAL2) were major or exclusive retinoids in the eggs of every species examined. In O. keta eggs, both RAL1 and RAL2 were present at the ratio of approximately 3:4, whereas RAL1 was the only retinal in the eggs of the other three marine species. RAL1 was the exclusive retinoid in the eggs of P. yokohamae and I. japonicus, whose eggs lack lipid bodies. In the eggs of O. keta and A. schlegeli, which have lipid bodies, retinylesters were also detected, and retinals composed 69% and 93%, respectively, of total retinoids. In O. keta eggs, retinals were present mostly in the aqueous part and were bound to a protein homologous to lipovitellin 1, an amphibian yolk protein, and retinylesters were located in lipids. These results indicate that retinals are the essential mode of retinoid storage in eggs of teleosts and they are the precursors of functional retinoids, such as retinoic acid and visual pigment chromophores. Retinylesters are additional retinoids that accompany lipid accumulation.

Topics: Animals; Borohydrides; Chromatography, Gel; Chromatography, High Pressure Liquid; Egg Proteins; Electrophoresis, Polyacrylamide Gel; Female; Fishes; Molecular Weight; Ovum; Protein Denaturation; Retinaldehyde; Retinoids; Species Specificity

The ligand-binding property of a cytoplasmic membrane-bound protein from bovine retinal pigment epithelium (RPE) has been demonstrated. The putative RPE-retinal G protein coupled receptor (RGR) covalently binds both all-trans- and 11-cis-retinal after reduction by sodium borohydride. The 32-kDa receptor binds all-trans-retinal preferentially, rather than the 11-cis isomer. The amino acid sequence of the opsin-related protein in humans is 86% identical to that of bovine RGR, and a lysine residue, analogous to the retinaldehyde attachment site of rhodopsin, is conserved in the seventh transmembrane domain of RGR in both species. The human gene that encodes the novel retinaldehyde receptor spans 14.8 kb and is split into seven exons. The structure of the gene is distinct from that of the visual pigment genes. These findings support the notion that the rgr gene represents the earliest independent branch of the vertebrate opsin gene family. A second form of human RGR in retina is predicted by alternative splicing of its precursor mRNA. This RGR variant results from the alternative use of an internal acceptor splice site in the second intron of the human gene, and it contains an insertion of four amino acids in the connecting loop between the second and thrid transmembrane domains. Since RGR binds all-trans-retinal preferentially, one of its functions may be to catalyze isomerization of the chromophore by a retinochrome-like mechanism.

Topics: Amino Acid Sequence; Animals; Base Sequence; Borohydrides; Carrier Proteins; Cattle; DNA, Complementary; Enzyme-Linked Immunosorbent Assay; Eye Proteins; Gene Expression Regulation; GTP-Binding Proteins; Humans; Microsomes; Molecular Sequence Data; Nucleic Acid Hybridization; Pigment Epithelium of Eye; Precipitin Tests; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Restriction Mapping; Retinaldehyde; Rhodopsin; RNA, Messenger; Rod Opsins; Sequence Homology, Amino Acid; Stereoisomerism

When retinochrome absorbs light, it bleaches to m-retinochrome, which may act as a direct supplier of 11-cis-retinal to protein opsin to form rhodopsin. The present experiments were aimed at further elucidating the molecular state of m-retinochrome. Retinochrome and m-retinochome were mixed each with sodium borohydride, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resultant fluorescence bands were examined. The reduced retinochrome showed only one band due to N-retinyl protein, whereas the reduced m-retinochrome had two bands. When extracted with successive volumes of n-hexane, m-retinochrome released the 11-cis-retinal chromophore in a time-course consisting of fast and slow phases. These findings indicated that m-retinochrome exists in two forms, with loose and tight coupling of the chromophore to the protein moiety. Those forms were usually balanced in a molar ratio of about 1:2, and the proportion of the tight form of m-retinochrome was increased in the presence of excess 11-cis-retinal. Based upon these findings, the role of m-retinochrome in the visual cells is discussed.

Topics: Animals; Borohydrides; Decapodiformes; Electrophoresis, Polyacrylamide Gel; Hot Temperature; Hydroxylamines; Retinal Pigments; Retinaldehyde

The previously described chymotryptic fragment of bacteriorhodopsin, C-2 (amino acids 1-71), is cleaved by 70% formic acid to two fragments, A-1 (amino acids 1-36) and A-2 (amino acids 37-71), which have been separated by high pressure liquid chromatography. The fragments A-1 and A-2, separately or together, are not able to replace C-2 in forming a stable bacteriorhodopsin-like complex with the fragment C-1 (amino acids 72-248) and all-trans-retinal. A second set of bacteriorhodopsin fragments, B-1 (amino acids 1-155) and B-2 (amino acids 156-248), have been prepared by sodium borohydride cleavage of bacteriorhodopsin. Following denaturation, fragments B-1 and B-2 reassociate in the presence of retinal to regenerate the native bacteriorhodopsin chromophore (approximately 40%). Fragment B-1 also interacts with fragment C-1 and all-trans-retinal to form a complex with spectral properties and secondary structure similar to those of bacteriorhodopsin. Vesicles prepared from the reconstituted fragment complexes (B-1 + B-2) or (B-1 + C-1), show proton pumping activities comparable to the previously described activity fragments C-1 and C-2.

Topics: Bacteriorhodopsins; Borohydrides; Carotenoids; Circular Dichroism; Halobacterium; Kinetics; Peptide Fragments; Protein Binding; Protein Conformation; Retinaldehyde

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 two 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. (a) 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. (b) The difference spectrum of absorption of metaretinochrome caused by alkalinization shows two minimum peaks at approximately 420 and 470 nm. (c) 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 two fluorescent bands: one at the position that corresponds to the retinochrome protein (mol wt 27,000 +/- 2,000), and another at the front of migration, where no band of 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. The results of this and earlier (Seki et al., 1982) papers are summarized, and it is strongly suggested that 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.

Topics: Absorption; Ammonium Hydroxide; Animals; Borohydrides; Chromatography, High Pressure Liquid; Decapodiformes; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Hydroxides; Membrane Proteins; Photic Stimulation; Retina; Retinal Pigments; Retinaldehyde; Retinoids; Rhodopsin; Spectrophotometry, Ultraviolet; Stereoisomerism

The rate of regeneration of rhodopsin, from 11-cis-retinal and opsin, and bacteriorhodopsin from all-trans-retinal and bacterio-opsin, in the presence or absence of compounds whose structures partially resemble retinal were measured. Some of these compounds severely slowed down the regeneration process, but did not influence the extent of regeneration. In the case of compounds with a carbonyl functional group they were not joined to the active site of the apo-protein via a Schiff's base linkage since after treatment with NaBH4 an active apo-protein remained. The most effective inhibitors of rhodopsin regeneration were molecules whose structure could be superimposed on 9-cis or 11-cis retinal up to carbon atom 11. These C13 and C15 molecules were not distinguished between aldehyde, ketone or alcohol functional groups. The regeneration of bacteriorhodopsin was not inhibited by retinal analogues with short side chains. The most effective inhibitors were the all-trans C17-aldehyde (beta-ionylideneacetaldehyde) or C18-ketone (beta-ionylidenepent-3-ene-2-one) which, compared to retinal, lack two or three carbon atoms from the end of the poylene chain. The inhibition was very dependent upon the presence of the all-trans isomer and required aldehyde or ketone as functional group nitriles and alcohols were less effective. However, similarly to retinol, the all-trans C17 and C18 alcohols underwent a bathochromic shift and showed fine-structured spectra when mixed with bacterio-opsin.

Topics: Animals; Bacteriorhodopsins; Borohydrides; Carotenoids; Eye Proteins; Halobacterium; Retinal Pigments; Retinaldehyde; Rhodopsin; Rod Cell Outer Segment; Rod Opsins; Structure-Activity Relationship; Vitamin A