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The biosynthesis of sphingomyelin and the role of phosphatidylcholine as its precursor in baby hamster kidney-21F cellsRuff, Blair A. January 1981 (has links)
The last step in sphingomyelin's de novo biosyn-thetic pathway was investigated in Baby Hamster Kidney (BHK-21F) cells in tissue culture. Three types of pulse-chase experiments were done to try to identify the precursor for sphingomyelin's phosphocholine moiety. First, [Me- ³H]-choline was used to monitor the movement of the choline moiety in all the possible phosphocholine donors: ie. phosphocholine, CDP-choline, phosphatidylcholine, lysophospha-tidylcholine, and glycerophosphocholine. Radioactivity was observed in phosphocholine before appearing in phosphatidylcholine, glycerophosphocholine, and sphingomyelin. Specific radioactivities of phosphatidylcholine and sphingomyelin revealed a peculiar pattern, if representative of a precursor-product relationship between these two phospholipids. Their specific radioactivities became equal at 22 hours of chase and remained quite similar for the next 24 hours. The other two types of pulse-chase experiments both utilized prelabeled BHK phosphatidyl[Me- ³H]choline in phospholipid vesicles as their 'pulse' source. Phospholipid Exchange Protein(PLEP)-mediated exchange and polyethylene glycol/phytohemagglutinin (PEG/PHA)-mediated fusion between phospholipid vesicles and BHK cells were used to introduce the labeled phosphatidylcholine. In addition, in the 'fusion' experiments, other labeled compounds (glycerophosphocholine and sphingomyelin) were substituted for labeled phosphatidylcholine. PLEP-
mediated exchange of labeled phosphatidylcholine did not result in enough transfer of radioactivity into the cell to adequately monitor individual cell phospholipids or any transfer of label - ie. from phosphatidylcholine to sphingomyelin. However, PEG/PHA-mediated fusion of vesicles and cells did result in enough radioactivity showing up in the cells. When labeled phosphatidylcholine was used, the radioactivity ratio between it and sphingomyelin averaged around 16, depending on the length of the chase (2-52 hours)m The use of either labeled glycerophosphocholine or sphingomyelin resulted in a ratio of about 1.8. One 'cold-trap1 experiment was done by including a large amount of unlabeled glycerophosphocholine with labeled phosphatidylcholine in the vesicle preparation. The resultant radioactivity in sphingomyelin was 50% less than previously, but phosphatidylcholine's had remained the same. The evidence seems to indicate a reversible precursor-product relationship between phosphatidylcholine and sphingomyelin, but does not clearly show whether or not any other intermediate (such as glycerophosphocholine) is also involved. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate
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Sphingomyelins Prevent Propagation of Lipid Peroxidation—LC-MS/MS Evaluation of Inhibition MechanismsColiva, Giulia, Lange, Mike, Colombo, Simone, Chervet, Jean-Pierre, Domingues, M. Rosario, Fedorova, Maria 20 April 2023 (has links)
Free radical driven lipid peroxidation is a chain reaction which can lead to oxidative degradation of biological membranes. Propagation vs. termination rates of peroxidation in biological membranes are determined by a variety of factors including fatty acyl chain composition, presence of antioxidants, as well as biophysical properties of mono- or bilayers. Sphingomyelins (SMs), a class of sphingophospholipids, were previously described to inhibit lipid oxidation most probably via the formation of H-bond network within membranes. To address the “antioxidant” potential of SMs, we performed LC-MS/MS analysis of model SM/glycerophosphatidylcholine (PC) liposomes with different SM fraction after induction of radical driven lipid peroxidation. Increasing SM fraction led to a strong suppression of lipid peroxidation. Electrochemical oxidation of non-liposomal SMs eliminated the observed effect, indicating the importance of membrane structure for inhibition of peroxidation propagation. High resolution MS analysis of lipid peroxidation products (LPPs) observed in in vitro oxidized SM/PC liposomes allowed to identify and relatively quantify SM- and PC-derived LPPs. Moreover, mapping quantified LPPs to the known pathways of lipid peroxidation allowed to demonstrate significant decrease in mono-hydroxy(epoxy) LPPs relative to mono-keto derivatives in SM-rich liposomes. The results presented here illustrate an important property of SMs in biological membranes, acting as “biophysical antioxidant”. Furthermore, a ratio between mono-keto/mono-hydroxy(epoxy) oxidized species can be used as a marker of lipid peroxidation propagation in the presence of different antioxidants.
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