Transmembrane movement of phosphatidylglycerol and diacylglycerol sulfhydryl analogs

Abstract

Transmembrane movement of phospholipids is a fundamental step in the process of biological membrane assembly and intracellular lipid sorting. To facilitate study of transmembrane movement, we have synthesized analogues of phosphatidylglycerol and diacylglycerol in which a sulfhydryl group replaces a hydroxyl group in the polar head group. A rapid, continuous assay for the movement of phospholipids across single-walled lipid vesicles was developed that exploits the reactivity of these analogues toward 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a nonpenetrating, colorimetric, sulfhydryl reagent. In the reaction of DTNB with vesicles containing phosphatidylthioglycerol, a phosphatidylglycerol analogue, two kinetic phases were seen, which represent the reaction of DTNB with phosphatidylthioglycerol in the outer and inner leaflets of the bilayer. Analysis of the slow second phase indicated that the half-time for phosphatidylthioglycerol transbilayer movement was in excess of 8 days. In a similar experiment using dioleoylthioglycerol, a diacylglycerol analogue, the reaction was complete within 15 s. The large difference in translocation rates between these two lipids indicates that the primary barrier to transmembrane movement is the polar head group and implies that phospholipid translocation events in biological membranes may not be unlike those for molecules similar to the polar head groups alone.

Phospholipid-Dependence of Plant UDP-Glucose Sterol β-d-Glucosyl Transferase

“…2). These results are in good agreement with the size of vesicles obtained by other authors using Green and Bell's method (9,10,30).…”

“…After 10 min of incubation at 4°C, the mixtures (500 IuL) were chromatographed through a Sephadex G-50 column (0.75 x 4 cm) equilibrated with buffer 2 containing 3 mM fmercaptoethanol. The liposomes eluting in the void volume (570 ,L) and devoid of pCMBS were tested for their enzymatic activity in the presence or in the absence of 0.2% Triton X-100: to 170 ,L of these liposomes were added 10 (24). Sterols were extracted with hexane and analyzed by GLC (1).…”

Sphingolipid transport in eukaryotic cells

“…2). This predicts that they translocate across cellular membranes spontaneously within seconds, as had been reported for diacylglycerol [216]. The rate of translocation across a phosphatidylcholine bilayer has been measured for diacylglycerol and Cer carrying a hydrophobic C 5 -DMB moiety as the sn-2-acyl and N-acyl, respectively.…”

Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement

“…with no ionized residues) will diffuse very rapidly through the membrane, as no polar groups are present. This has been shown for diacylglycerol, which diffuses with a characteristic time of less than 1 min in artificial and biological membranes (Allan et al, 1978;Ganong and Bell, 1984). A priori, this should be true for cholesterol since related molecules such as bile salts equilibrate in a bilayer with characteristic times well below 1 s (Cabral et al, 1987).…”