Purple membrane: surface charge density and the multiple effect of pH and cations.
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Biomedical subjects
Publications and source records attributed to Y Koutalos.
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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.
Phosphorus-31 nuclear magnetic resonance (31P-NMR) spectra were obtained from living toad retinae and toad retinal extracts at 4 degrees C. Several phosphorus metabolites--nucleoside di- and triphosphates (NTP), phosphocreatine, phosphodiesters, inorganic phosphate, and phosphomonoesters--were identified from the spectra of whole retinae. The intracellular pH was determined to be 7.27 +/- 0.06 at 4 degrees C and the intracellular MgNTP/NTP ratio was at least 0.77. These results are consistent with those reported by other techniques, and they show that 31P-NMR spectroscopy can be used for noninvasively and quantitatively studying the metabolism of living toad retinae, and for monitoring its changes over time.
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