L-a-lysophosphatidylcholine

For cell permeabilization various methods can be applied. Treatment with L-a-lysophosphatidylcholine (LPC) has proven to be a cheap, easy, and versatile way to perforate cells. If soluble cytoplasmic proteins need to be labeled other reagents generating smaller holes, such as Staphylococcus aureus a-toxin, can be used. If only the plasma membrane needs to be permeabilized the LPC concentration should be adjusted to obtain 95% trypan blue-stainable cells. 

Lecithins and related phospholipids usually contain a saturated fatty acid in the C-1 position but an rmsaturated acid, which may contain from one to four double bonds, at C-2. Arachidonic acid is often present here. Hydrolysis of the ester linkage at C-2 yields a l-acyl-3-phosphoglycerol, better known as a lysophosphatidylcholine. The name comes from the powerful detergent action of these substances which leads to lysis of cells. Some snake venoms contain phospholipases that form lysophosphatidylcholine. Lysophosphatidic acid (l-acyl-glycerol-3-phosphate) is both an intermediate in phospholipid biosynthesis (Chapter 21) and also a signaling molecule released into the bloodstream by activated platelets. ... 

All of the above SMases are C type SMases that produce Cer and choline phosphate an SMase D (which produces Cer 1-phosphate and choline) is found in the venom of brown recluse spider, Corynebacterium pseudotuberculosis (which commonly infects sheep). Vibrio damsela (an aquatic bacterium that causes wound infections in humans), and the human pathogen Arcanobacterium haemolyticum. The venom SMase D produces much of the tissue damage caused by these organisms (A.P Truett, 1993) by sustained activation of inflammation (S.H. Farsky, 2005). Interestingly, SMase also acts as a lysophosphatidylcholine phosphodiesterase to produce lysophosphatidic acid, another inflammatory mediator (L.A. van Meeteren, 2004). 

Prepare 10 ml of permeabilization buffer NIM warmed to room temperature containing 0.5 mg/ml L-a-lysolecithin (a-lysophosphatidylcholine cat. L 4129 Sigma Chemical Co.), 1.0 ngjml leupeptin (cat. 1034 626 Boehringer-Mannheim Canada, Laval, QC), and 100 /xAf phenylmethylsulfonyl fluoride (PMSF, cat. 837 091 Sigma Chemical Co). Note Lysolecithin is poorly soluble in cold NIM. 

Arroyo-Caro, J.M., Chileh, T., Kazachkov, M., Zou, J., Lopez-Alonso, D., Garcia-Maroto, F., 2013b. Molecular characterization of a lysophosphatidylcholine acyltransferase gene belonging to the MBOAT family in Ricinus communis L. Lipids 48, 663-674. 

Huang, Y.H., Schafer-Elinder, L., Wu, R., Claesson, H.E., and Frostegard J., 1999b, Lysophosphatidylcholine (LPC) induces proinflammatory cytokines by a plateletactivating factor (PAE) receptor-dependent mechanism, Clin. Exp. Immunol. 116 326-331. 

This enzyme [EC 2.3.1.23], also called lysolecithin acyl-transferase and lysophosphatidylcholine acyltransferase, catalyzes the reaction of an acyl-CoA derivative with 1-acyl-5 n-glycero-3-phosphocholine to yield coenzyme A and l,2-diacyl-5 n-glycero-3-phosphocholine. The enzyme preferentially acts on unsaturated acyl-CoA derivatives, but l-acyl-5 n-glycero-3-phosphoinositol can also act as the acceptor. 

Gomez-Munoz A., O Brien L., Hundal R., and Steinbrecher U. P. (1999). Lysophosphatidylcholine stimulates phospholipase D activity in mouse peritoneal macrophages. J. Lipid Res. 40 ... 

Fig. 8.1 A schematic diagram illustrating the involvement of NF-k I in gpl20, ROS, NO, PG, IL-1/3 and TNF-a-mediated neurotoxicity. NMDA-R, N-Methyl-D-aspartate receptor, cPLA2, cytosolic phospholipase A2 lyso-PtdCho, lysophosphatidylcholine AA, arachidonic acid cAMP, cyclic adenosine monophosphate PKA, protein kinase A TNF-a, tumor necrosis factor-a TNF-a-R, TNF-a-receptor IL-1/8, interleukin-1 /3 IL-l/i-R, IL-1/8-receptor, IL-6, interleukin-6 MARK, mitogen-activated protein kinase NO, nitric oxide PG, prostaglandins EP-R, prostaglandin receptors NF-kB, nuclear factor-icB NF-kB-RE, nuclear factor-/cB-response element I/cB, inhibitory subunit of NF-icB HIV-1, human immunodeficiency virus type 1 gpl20, HIV-1 coat glycoprotein COX-2, cyclooxygenase-2 iNOS, inducible nitric oxide synthase SPLA2, secretory phospholipase A2 SOD, superoxide dismutase MMP, matrix metalloproteinase and VCAM-1, vascular adhesion molecule-1...

Richardson, J. L., Farraj, N. F., and Ilium, L. (1992), Enhanced vaginal absorption of insulin in sheep using lysophosphatidylcholine and a bioadhesive microsphere delivery system, Int. J. Pharm., 88, 319-325. 

R)- and (S)-enantiomers of novel lysophosphatidylcholine analogues (59) and (60) have been synthesized from commercially available L- and D-serine as starting materials by a short and efficient method (Scheme 10). These newly designed and prepared lyso PC analogues exhibited much enhanced hyphal transition inhibitory activity against Candida species as compared to the natural lyso PC. ... 

The group VIB (Vly) enzyme is under detailed investigation and initial results indicate that the protein is famesylated (C.M. Jenkins, 2003) and has unusual properties distinct from the group VIA enzyme (M. Murakami, 2005) including hydrolysis of the sn-l fatty acid of phosphatidylcholine leading to the formation of 2-arachidonoyl lysophosphatidylcholine (W. Yan, 2005), a potential precursor for a variety of lipid mediators. 

Thukkani, A. K., J. McHowat, F. F. Hsu, M. L. Brennan, S. L. Hazen, and D. A. Ford. 2003b. Identification of alpha-chloro fatty aldehydes and unsaturated lysophosphatidylcholine molecular species in human atherosclerotic lesions. Circulation 108(25) 3128-33. 

Obtain the following phospholipid standards from Matreya a polar lipid standard containing equal amounts of lysophosphatidylcholine, phosphatidylcholine, phosphatidyl ethanolamine, and cholesterol a sphingomyelin standard containing equal amounts of sphingomyelin, sulfatides, and three cerebrosides individual phosphatidyl inositol and phosphatidyl serine standards. Each standard is supplied in 25-mg amounts dissolved in 1 ml of chloroform-methanol (2 1). Add 4 ml of chloroform-methanol (2 1) to each standard to achieve a final concentration of 5 J.g/ J-l. 

Similar to plasmanylcholine and plasmenylcholine species (see above), due to the lack of a-hydrogen in both the sn-l and sn-2 moieties, further dissociation from [lVH-Alk-59]+ to form [M4-Alk-183]+ and [M-i-Alk-(Alk-l-182)]+ is significantly reduced [1]. Accordingly, the presence of a prominent [M-l-Alk-59]+ ion relative to [M-l-Alk-183] and [M-l-Alk-(Alk-l-182)] ions can be used to discriminate ether subclasses from lysophosphatidylcholine. 

Figure 7.6 Representative product-ion ESI-MS spectra of lysophosphatidylcholine species in both positive- and negative-ion modes after low-energy CID. Product-ion ESI-MS analyses of lithiated (a), protonated (b), and chloride-adducted (c) l-palmitoyl-5H-3-phosphocholine were performed on a Thermo Fisher TSQ Vantage mass spectrometer. Collision activation was performed at collision energy of 22 (a), 21 (b), and 15 (c) eV, and gas pressure of 1 mTorr.

Stymne, S. and Stobart, A.K. 1984a. Evidence for the reversibility of the acyl-CoA Lysophosphatidylcholine acyltransferase in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons and rat liver. Biochem. J. 223 305-314. 

The transfer of phosphatidylcholine from monolayer to vesicles catalyzed by phosphatidylcholine transfer protein shows remarlcable differences for the positional isomers (23). The PC transfer protein acts as a specific carrier of PC between membrane interfaces. This protein has a recognition site for the phosphorylcholine headgroup and binding sites for the sn-1 and sn-2 fatty acyl chain. Lysophosphatidylcholine is not transferred. It was found that the protein transferred C10 0/C18 l PC twice as fast as C18 l/C10 0 PC. Similar differences in rate were observed for C12 0/C18 l PC and C18 l/C12 0 PC but not for isomers carrying myristic acid (Table I). 

---