Spectrum and purity of bovine rhodopsin

The consumption of trans fatty acid (TFA) is linked to the elevation of LDL cholesterol and is considered to be a major health risk factor for coronary heart disease. Despite several decades of extensive research on this subject, the underlying mechanism of how TFA modulates serum cholesterol levels remains elusive. In this study, we examined the molecular interaction of TFAderived phospholipid with cholesterol and the membrane receptor rhodopsin in model membranes. Rhodopsin is a prototypical member of the G-protein coupled receptor family. It has a wellcharacterized structure and function and serves as a model membrane receptor in this study. Phospholipid-cholesterol affinity was quantified by measuring cholesterol partition coefficients. Phospholipid-receptor interactions were probed by measuring the level of rhodopsin activation. Our study shows that phospholipid derived from TFA had a higher membrane cholesterol affinity than their cis analogues. TFA phospholipid membranes also exhibited a higher acyl chain packing order, which was indicated by the lower acyl chain packing free volume as determined by DPH fluorescence and the higher transition temperature for rhodopsin thermal denaturation. The level of rhodopsin activation was diminished in TFA phospholipids. Since membrane cholesterol level and membrane receptors are involved in the regulation of cholesterol homeostasis, the combination of higher cholesterol content and reduced receptor activation associated with the presence of TFAphospholipid could be factors contributing to the elevation of LDL cholesterol.Trans fatty acid (TFA), 1 the stereoisomer of the naturally occurring cis fatty acid (CFA), is considered to be a major health risk factor for coronary heart disease. TFA and CFA are stereoisomers that only differ in the geometry of the CdC double bond. TFA adopts a more linear configuration similar to that of saturated fatty acid, while CFA adopts a bent configuration (illustrated in Figure 1). The linear configuration of TFA allows stronger intermolecular chain-chain interaction, resulting in a higher transition temperature for TFA chain melting (1). TFA is mainly produced by partial hydrogenation of unsaturated oils and is widely found in a variety of foods, including margarines, vegetable shortening, frying oils, etc.(2). The average American consumes more than 5 g of TFA daily, which is equivalent to ∼7% of total fat consumption (3). It is widely documentedȈthat TFA is incorporated in serum lipids, lipoproteins, and adipose tissues in the form of triglyceride, phospholipids, and cholesterol esters (4-10), although the efficiency of incorporation varied among different tissues or lipid forms. The dietary intake of TFA is associated with the elevation of LDL cholesterol (11-15) and increased risk for coronary heart disease (16-18). However, the cause for LDL cholesterol elevation by TFA, or in a more general scope, the cause for LDL cholesterol regulation by fatty acid, is not well-understood. In the past several decades, extensive studies...

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“…The rhodopsin concentration was determined by absorbance at 500 nm using an extinction coefficient of 40 000 M cm -1 (37).…”

Section: Miscellaneous Assaysmentioning

confidence: 99%

Trans Fatty Acid Derived Phospholipids Show Increased Membrane Cholesterol and Reduced Receptor Activation as Compared to Their Cis Analogs

2005

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“…Theories to explain the depolarization of fluorescence due to energy transfer are not as well-developed (10 (11). The purity (ratio) of the rhodopsin was typically 1.7-1.8, with my best samples having a purity of 1.65.…”

mentioning

confidence: 99%

Energy Transfer in Rhodopsin, N -Retinyl-Opsin, and Rod Outer Segments

Ebrey

1971

Proc. Natl. Acad. Sci. U.S.A.

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N-retinyl, the chromophore of bleached and reduced rhodopsin, N-retinyl-opsin, was used as a covalently attached fluorescence probe to examine the structure of N-retinyl-opsin and the rod outer segment. The efficiency of energy transfer from the protein part of N-retinyl-opsin to the chromophore is 12 4 5%. It is argued that this implies that the N-retinyl-opsin molecule is asymmetrical. Kropf has estimated the efficiency of energy transfer from the protein to the chromophore in native rhodopsin to be about 50%. This difference of efficiencies seems to imply a large movement of the chromophore away from the tryptophans of the opsin after rhodopsin is bleached.From excitation spectrum measurements, it has been found that light absorbed by the protein of the rod outer segments has more action in sensitizing the fluorescence of the chromophore than does light absorbed by the protein part of pure N-retinyl-opsin. Thus, some other tryptophans or tyrosines in either another N-retinylopsin molecule or another protein must be close enough (about 28 A) to the chromophore to transfer energy to it. Measurements of the polarization of the fluorescence of the chromophore suggest, however, that the chromophores of neighboring N-retinyl-opsin molecules are more than 20 A apart. Moreover, these neighboring chromophores do not transfer energy to each other, tending to rule out any clustering of chromophores of different Nretinyl-opsin molecules and suggesting that rhodopsin chromophores do not transfer energy to each other.The mechanism of visual excitation can not be understood without a fuller understanding of the structure and function of the proteins in the membranes of the photoreceptors, particularly rhodopsin. The significance of rhodopsin goes beyond the immediate problems of visual excitation. Of the best-known membrane proteins and membrane structures, rhodopsin and the rod outer segments (r.o.s.) are among the most suitable for extensive study. Structurally, rhodopsin is quite important since it constitutes about 50% of the protein of the bovine r.o.s. (unpublished data). Functionally, it is important since it is the pigment that absorbs the light used in vision. Rhodopsin can be isolated in fairly large quantities in a pure form, and the purity of rhodopsin and r.o.s. samples can be assayed (1). Much is known already about the photochemistry of rhodopsin; it is almost certain that the first step in visual excitation is the isomerization of the chromophore of rhodopsin, retinal, from the li-cis to the all-trans form (2).Because rhodopsin is arranged in a systematic array in the r.o.s., much work utilizing the techniques of both electron microscopy and x-ray diffraction has been possible (3, 4). Because the rod outer segment is an electrically active membrane (5), a number of electrophysiological findings may Abbreviation: r.o.s., rod outer segments. I would like to present evidence here on the structure of the native rhodopsin molecule, the bleached rhodopsin molecule, and the r.o.s. This evidence is based on...

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“…The cattle rhodopsin extracted in the detergent was purified by a modification of the method by Shichi [15] and Shichi et al [14]. The extract was placed on an ECTEOLAcellulose column equilibrated with 0.3% Ammonyx LO (pH 7.2) and rhodopsin eluted with 0.3% Ammonyx LO (pH 7.2).…”

Section: Preparation O F Ros and Rhodopsinmentioning

confidence: 99%

Patterns of Experimental Allergic Uveitis Induced by Rhodopsin and Retinal Rod Outer Segments

Marak¹,

Shichi²,

Rao³

et al. 1980

Ophthalmic Res

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We have observed that both washed outer segments and purified rhodopsin produce primarily a posterior uveitis with little or no anterior segment inflammation if an effort is made to remove all contaminating soluble retinal antigen. The differences in the immunopathologic responses to rhodopsin compared to soluble retinal antigen may be explained by the solubility difference between these antigens. With the soluble retinal antigen there may be anterior segment inflammation and diffuse tissue necrosis. The allergic response to rhodopsin appears to be confined to the posterior segment with tissue necrosis in severe cases restricted to the anatomic distribution of rhodopsin in the outer retina.

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Spectrum and purity of bovine rhodopsin

Cited by 57 publications

References 22 publications

Trans Fatty Acid Derived Phospholipids Show Increased Membrane Cholesterol and Reduced Receptor Activation as Compared to Their Cis Analogs

Trans Fatty Acid Derived Phospholipids Show Increased Membrane Cholesterol and Reduced Receptor Activation as Compared to Their Cis Analogs

Energy Transfer in Rhodopsin, N -Retinyl-Opsin, and Rod Outer Segments

Patterns of Experimental Allergic Uveitis Induced by Rhodopsin and Retinal Rod Outer Segments

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