PubMed · 13241537
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1955. [Bilamid-choline].. https://pubmed.ncbi.nlm.nih.gov/13241537/
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For the purpose of developing highly sensitive and convenient determination of plasmalogens, the high-performance liquid chromatography (HPLC) method using radioactive iodine ((125)I) was investigated. Radioactive triiodide (1-) ion ((125)I(3)(-)), which is an actual iodine form capable of reacting with vinyl ether bond ([bond]CH(2)[bond]O[bond]CH[double bond]CH[bond]) of plasmalogens, could be safely and efficiently produced by oxidizing a commercial radioactive sodium iodine (Na(125)I) with hydrogen peroxide (H(2)O(2)) under acid condition (pH 5.5-6.0), which is called iodine-125 reagent. I(3)(-) specifically reacted with plasmalogens at the molar ratio of 1:1 in methanol, and 1 or 2 mol of plasmalogens was involved in the binding with iodine per iodine atom, resulting in the formation of stable iodine-binding phospholipids. The HPLC system with Diol column and acetonitrile/water as a mobile phase was available for separating iodine-binding phospholipids from nonbinding free iodine and for separately eluting iodine-binding phospholipids derived from choline and ethanolamine plasmalogens. Using iodine-125 reagent (1.85 MBq/ml), plasmalogens were detectable at high sensitivity of 10,000-15,000 cpm/nmol, which is more than 1000-fold higher sensitivity than the classical determination with nonradioactive iodine. Plasmalogen concentrations in human plasma were measured with the HPLC system and determined as, on average, 129.1+/-31.3 microM (n=8) in a 1.2 content ratio of choline to ethanolamine plasmalogens, a concentration that nearly agrees with the value reported previously.
Phosphatidylcholine (PtdCho) is the most abundant phospholipid in numerous eukaryotes and is generally thought to be essential for membrane structure and cellular function. We designed a specific test of this idea by using genetic and biochemical manipulation of yeast. Yeast mutants (pem1 pem2Delta) lacking the phosphatidylethanolamine (PtdEtn) methyltransferase enzymes require choline for growth and cannot make N-methylated phospholipids. When these strains are grown on a glucose carbon source supplemented with 20 mm propanolamine (Prn), the PtdCho level declines precipitously to the limits of detection (<0.6%), and the hexagonal phase-forming, primary amine-containing lipids, PtdEtn and PtdPrn, constitute approximately 60% of the total phospholipid content of the cell. When the lipids were analyzed by mass spectrometry, there was no compensatory shift in unsaturation of the PtdEtn and PtdPrn toward more bilayer-forming species. Thus the majority of the cellular amino phospholipids remained hexagonal phase-forming. The pem1 pem2Delta cells will also grow without choline, in the presence of Prn, on nonfermentable carbon sources (requiring functional mitochondria) and accumulate nearly 70% of their phospholipid as hexagonal phase-forming types. These data provide compelling evidence that the functions of PtdCho and N-methylated lipids in membranes are nonessential in Saccharomyces cerevisiae.
The purpose of this work was to develop a simple procedure for the creation of a specific biorecognition layer for histidine-tagged (His-tagged) molecules. Such a layer was prepared by the spontaneous fusion of vesicles containing readily available plain (DOPC) and iminodiacetic acid (DOGS-NTA) phospholipids on a silica surface resulting in the formation of an NTA-containing supported lipid bilayer. The frequency surface acoustic waveguide device which supports Love waves was used to follow the real-time formation of the biorecognition layer. The mole percent of the DOGS-NTA phospholipids in the supported bilayer was optimized by following the kinetics of the fusion for the different NTA-containing lipids. Fluorescently labeled lipids were used with observations of the fluorescence recovery after photobleaching to confirm the presence of lipid bilayers. After saturating all NTA-molecules with Ni(2+), the binding of a His-tagged protein fragment within the concentration range of 0.04 and 0.4 mM to a 5 mol % DOGS-NTA/DOPC was detected; binding curves were used to calculate the apparent association constant k(on) = 2.56 x 10(4) M(-)(1) s(-)(1), dissociation constant k(off) = 1.3 x 10(-)(3) s(-)(1), and equilibrium constant k(eq) = 1.97 x 10(7) M(-)(1). The described method could find significant applications as a generic technique for preparing biorecognition layers for His-tagged proteins. In addition, the acoustic waveguide device, which provides high sensitivity together with flexibility in terms of the substrate material used, is shown to be an attractive alternative to direct optical biosensors.