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Optical-rotatory-dispersion studies of compounds related to cholesterol in liposomes and the membranes of erythrocyte 'ghosts'.

ABSTRACT: 1. Steroid molecules containing the alpha,beta-unsaturated oxo group in various positions were incorporated with egg phosphatidylcholine into liposomes and into human erythrocyte membranes. 2. The liposomes formed contained 0.3-0.94mol of steroid/mol of phospholipid and the steroids replaced 19-76% of the erythrocyte membrane sterol. 3. The optical rotatory dispersion (o.r.d.) spectra of the steroids in these structures were compared with those obtained in solvents of different polarity. 4. The o.r.d. spectra of cholesta-4,6-dien-3-one and 3-hydroxycholest-3-en-2-one in liposomes resembled those obtained with polar solvents such as ethanol or triethyl phosphate-water (1:1, v/v). 5. The o.r.d. spectra of 3-hydroxycholest-7-en-6-one and 3-hydroxycholest-5-en-7-one in liposomes resembled those obtained with moderately polar solvents such as dioxan. 6. The o.r.d. spectrum of 3-hydroxycholest-8(14)-en-15-one in liposomes resembled those obtained with non-polar solvents such as cyclohexane. 7. 3-Hydroxycholest-3-en-2-one did not exchange with erythrocyte membrane cholesterol, but the other steroids did do so and the o.r.d. spectra of the membranes containing them closely resembled those obtained with liposomes. 8. From the results, the position of sterol molecules with respect to the phospholipid molecules in liposomes and membranes of human erythrocyte ;ghosts' can be deduced.

SUBMITTER: Green JR

PROVIDER: S-EPMC1165789 | biostudies-other | 1973 Sep

REPOSITORIES: biostudies-other

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Project description:1. The optical rotatory dispersion (ORD) of five homodinucleotides, ApAp(3'), CpCp(3'), GpGp(3'), IpIp(3') and UpUp(3') (where A, C, G, I and U represent adenosine, cytidine, guanosine, inosine and uridine respectively, and p to the left of a nucleoside symbol indicates a 5'-phosphate and to the right it indicates a 3'-phosphate), were measured as a function of pH, ionic strength and Mg(2+) concentration. 2. The ORD titrations of ApAp(3') and CpCp(3'), which were made by measuring the ORD curves at closely spaced pH intervals, exhibit a maximum at approx. pH5.0 and 5.7 for ApAp(3') and CpCp(3') respectively in the profile of the magnitude of the first Cotton effect versus pH. The results indicate that the conformational rigidity of these dinucleotides depends on the ionization state of a 3'-terminal phosphate group. 3. ApAp(3') was shown to exist as an approximately 1:1 equilibrium mixture of the two major ionic species represented by Ap((-1))Ap((-1)) and Ap((-1))Ap((-2)) at pH6.16, whereas at pH7.5 it exists exclusively as a form of Ap((-1))Ap((-2)). 4. To ascertain the effects of the presence of a terminal phosphate group and of the ionization of the secondary phosphate on the conformation of adenylate dimer, we measured the ORD of ApA, ApAp(3')CH(3) and ApAp(2'). The rotatory power of the first Cotton effect in the above series of dinucleotides decreased at 20 degrees in the order ApA> ApAp(3')CH(3) approximately ApAp(3')((-1))> ApAp(2') at pH7> ApAp(3') at pH7. 5. The pH-rotation profiles were also obtained for ApAp(2'), CpCp(2') and UpUp(3'), but no corresponding maximum was observed. Although simple nearest-neighbour calculations based on the ORD data of IpIp(3') and 5'-IMP account for the observed ORD spectrum of polyinosinic acid at low salt concentration, there were large discrepancies between calculated and experimental results of the polyguanylic acid ORD even at low ionic strength. 6. The extent to which the amplitude of the Cotton effects of IpIp(3') increases with salt concentration, especially by the addition of Mg(2+), was much greater than that observed for ApAp(3'). The implication of such salt effects on the ORD is considered.

Project description:Estrogen components of some hormone replacement formulations have been implicated in the initiation of breast cancer. Some of these formulations contain equine estrogens such as equilin and equilenin that are metabolized to the genotoxic catechol 4-hydroxyequilenin (4-OHEN). Auto-oxidation generates the o-quinone form that reacts with dC and dA in oligodeoxynucleotides to form unusual stable cyclic bulky adducts, with four different stereoisomers identified for each base adduct. The dC and dA adducts have the same unsaturated bicyclo[3.3.1]nonane type linkage site with identical stereochemical characteristics. Stereochemical effects may play an important part in the biological consequences of the formation of 4-OHEN-DNA adducts, and the assignment of the absolute configurations of the stereoisomeric 4-OHEN-dC and -dA adducts is therefore needed to understand structure-function relationships. We used density functional theory (DFT) to compute the specific optical rotations and electronic circular dichroism (ECD) spectra of the four 4-OHEN-C stereoisomers, and the results were compared with experimentally measured optical rotatory dispersion (ORD) and ECD spectra. The predicted ORD curves for the four stereoisomeric base adducts reproduced the shapes and signs of experimental spectra in the transparent spectral region. The stereochemistry of the C3' atom was determined by comparison of the calculated and experimental ORD and ECD spectra, and the stereochemistry of C2' was determined by mass spectrometric methods. Combining the ORD and mass spectrometry data, the absolute configurations of the four 4-OHEN-C and the stereochemically identical -dC adducts have been identified. The molecular architecture of the linkage site at the 4-OHEN-C/A and 4-OHEN-dC/dA is identical, and it is shown that the deoxyribose group does not substantially contribute to the optical activities. The absolute configurations of the 4-OHEN-dA adducts were thus deduced by comparing the experimental ORD with computed ORD values of 4-OHEN-A adducts.

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Optical-rotatory-dispersion studies of compounds related to cholesterol in liposomes and the membranes of erythrocyte 'ghosts'.

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