Spin-labeled lipid analogs

Lipid vesicles containing phosphatidyl choline (95%) and the spin-labeled analog (5%) were prepared by sonication... 

Fig. 2. General structural features of fluorescent and spin-labeled lipid analogs. The fluorescent lipids usually contain a short-chain fatty acid, amino-caproic acid, that is derivatized with 4-nitrobenzo-2-oxa-1,3-diazole (NBD), or valeric acid that is derivatized with a boron dipyrromethene difluoride (BODIPY) moiety. For fluorescent phospholipids, X can be hydrogen or the esterified forms of choline, ethanolamine, serine, or inositol. R>r fluorescent sphingolipids, Y can be hydrogen or the esterified fonns of phosphocholine or mono- or oligosaccharides typical of glycosphingolipids. The spin-labeled lipids modified in the fatty acid portion contain a 4-doxylpentanoyl fatty acid in the sn-2 position. Those modified in the polar head group contain a tempocholine moiety in place of choline. The X substituent for the acyl spin-labeled lipids can be hydrogen or the esterified forms of choline, ethanolamine, or serine.

Paramagnetic analogs of phospholipids have also been used to investigate lipid transport phenomena in model membrane systems (R.D. Komberg, 1971) and in biological membranes. Representative structures are shown in Fig. 2. Several of these spin-labeled lipid analogs that are modified in the fatty acid chain can be readily and reversibly transferred... 

Direct experiments to examine the transbilayer movement of phospholipids (R.D. Kornberg, 1971) made use of spin-labeled analogs of PC in which the choline moiety was replaced with the tempocholine probe,, A -dimethyl-A -(l -oxyI-2, 2, 6, 6 -tetramethyl-4 -piperidyl)-ethanolamine (Fig. 2). These workers found that only the electron spin resonance signal generated by molecules in the outer leaflet of unilamellar liposomes could be rapidly quenched by ascorbate. The electron spin resonance signal from lipid molecules initially residing at the inner leaflet of liposomes was accessible to ascorbate with a r,/2 of >6.5 h, indicating slow transbilayer lipid movement (Fig. 3). 

Additional studies utilized spin-labeled analogs of PC, PE, PS, and SM, and the t for the translocation of these lipid analogs from the cytosolic face to the lumenal face of the microsomes was calculated to be 20 min. The transport process did not require ATP and the translocation of each class of lipid showed identical sensitivity to inhibition by N-ethylmaleimide. Furthermore, different species of lipid showed transport kinetics that were consistent with mutual competition for a single transporter. These results indicate that the ER has a relatively non-specific, ATP-independent transporter that is capable of translocating multiple species of lipid across the bilayer. Several attempts have been made to reconstitute the protein components of the ER necessary for ATP-independent transbilayer lipid movement within liposomes (J. Backer, 1987 S. Gummadi, 2002). These approaches have met with only limited success and thus far no specific proteins or genes have been directly implicated in the process. 

Lipid vesicles containing phosphatidyl choline (95%) and the spin-labeled analog (5%) were prepared by sonication and purified by gel-filtration chromatography. The outside diameter of these liposomes was about 25 nm. The amplitude of the paramagnetic resonance spectrum decreased to 35% of its initial value within a few minutes of the addition of ascorbate. There was no detectable change in the spectrum within a few minutes after the addition of a second aliquot of ascorbate. However, the amplitude of the residual spectrum decayed exponentially with a half-time of 6.5 hours. How would you interpret these changes in the amplitude of the paramagnetic spectrum ... 

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