Abstract
The lipid/protein stoichiometries of a naturally crystalline biological membrane, the purple membrane (PM) of Halobacterium salinarum, have been obtained by a combination of (31)P- and (1)H-NMR analyses of the lipid extract. In total, 10 lipid molecules per retinal were found to be present in the PM lipid extract: 2-3 molecules of phosphatidylglycerophosphate methyl ester (PGP-Me), 3 of glycolipid sulfate, 1 of phosphatidylglycerol, 1 of archaeal glycocardiolipin (GlyC), 2 of squalene plus minor amounts of phosphatidylglycerosulfate (PGS) and bisphosphatidylglycerol (archaeal cardiolipin) (BPG) and a negligible amount of vitamin MK8. The novel data of the present study are necessary to identify the lipids in the electron density map, and to shed light on the structural relationships of the lipid and protein components of the PM.
Highlights
The lipid/protein stoichiometries of a naturally crystalline biological membrane, the purple membrane (PM) of Halobacterium salinarum, have been obtained by a combination of 31P- and 1H-NMR analyses of the lipid extract
TLC and electro-spray ionization mass spectrometry (ESI-MS) analyses have been used to gain preliminary insights into the lipid composition of the lipid extract of the PM isolated from Halobacterium salinarum cells
These data suggest that the novel cardiolipins are specific components of the PM and, at the same time, raise doubts about the specificity of the location of S-TGD-1, which had been previously considered located in the PM [3, 8]
Summary
The mixture was shaken and centrifuged; 6.25 ml each of chloroform and water were added to the combined supernatant extracts to obtain a two-phase system. The lipids of PM were separated by TLC in solvent A (chloroform-methanol-90% acetic acid, 65:4:35, v/v/v). Each component was further purified by re-chromatography in neutral solvent B (chloroform-methanol-water, 65:25:4, v/v/v) and recovered from silica as just described. 1 –5 mg of individual purified phospholipids or of total lipid extract was dissolved in 0.8 ml of deuterated chloroform. To this solution 0.4 ml of methanol reagent (containing Cs/EDTA) was added, and the mixture stirred gently. Inverse-detected 1H-31P-correlated 2D NMR spectra were obtained by using the standard gradient-enhanced pulse sequence INVIETGPSI, using a 7 Hz coupling constant for magnetization transfer (18 – 20)
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