144861-12-1Relevant articles and documents
Scalable Synthesis and Purification of Acetylated Phosphatidyl Choline Headgroup
Subramaniam, Rajesh,Jagadeesan, Ramesh,Mathew, Iswarya,Cen, Yana,Balaz, Stefan
, p. 177 - 181 (2017)
The acetylated headgroup of the most abundant mammalian phospholipid, 1,2-diacetyl-3-sn-phosphatidyl choline (DAcPC), has several important applications in research. For instance, it can be dissolved in the same amount of water as in the fluid PC bilayer, to create a surrogate of a PC headgroup stratum, for studying the solvation of small molecules and the influence of their structure on the process. In contrast to PC derivatives with longer acyl chains, DAcPC does not self-aggregate, rendering the aqueous solution homogeneous and suitable for simplified analyses of interactions of molecules with the headgroups. Several studies have been published where DAcPC was used in a crudely purified form. Here we describe a one-step preparation of DAcPC from commercially available bulk chemicals and purification of the product by crystallization and washing. The process gives a good yield and is easily scalable. The availability of enantiopure, crystalline DAcPC could open the door to more extensive biochemical, pharmacological, and nutritional studies of this interesting chemical.
Oxygenation reactions catalyzed by the F557V mutant of soybean lipoxygenase-1: Evidence for two orientations of substrate binding
Hershelman, Dillon,Kahler, Kirsten M.,Price, Morgan J.,Lu, Iris,Fu,Plumeri, Patricia A.,Karaisz, Fred,Bassett, Natasha F.,Findeis, Peter M.,Clapp, Charles H.
, (2019/09/10)
Plant lipoxygenases oxygenate linoleic acid to produce 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPOD) or 9-hydroperoxy-10E,12Z-octadecadienoic acid (9(S)-HPOD). The manner in which these enzymes bind substrates and the mechanisms by which they control regiospecificity are uncertain. Hornung et al. (Proc. Natl. Acad. Sci. USA 96 (1999) 4192–4197) have identified an important residue, corresponding to phe-557 in soybean lipoxygenase-1 (SBLO-1). These authors proposed that large residues in this position favored binding of linoleate with the carboxylate group near the surface of the enzyme (tail-first binding), resulting in formation of 13(S)-HPOD. They also proposed that smaller residues in this position facilitate binding of linoleate in a head-first manner with its carboxylate group interacting with a conserved arginine residue (arg-707 in SBLO-1), which leads to 9(S)-HPOD. In the present work, we have tested these proposals on SBLO-1. The F557V mutant produced 33% 9-HPOD (S:R = 87:13) from linoleic acid at pH 7.5, compared with 8% for the wild-type enzyme and 12% with the F557V,R707L double mutant. Experiments with 11(S)-deuteriolinoleic acid indicated that the 9(S)-HPOD produced by the F557V mutant involves removal of hydrogen from the pro-R position on C-11 of linoleic acid, as expected if 9(S)-HPOD results from binding in an orientation that is inverted relative to that leading to 13(S)-HPOD. The product distributions obtained by oxygenation of 10Z,13Z-nonadecadienoic acid and arachidonic acid by the F557V mutant support the hypothesis that ω6 oxygenation results from tail-first binding and ω10 oxygenation from head-first binding. The results demonstrate that the regiospecificity of SBLO-1 can be altered by a mutation that facilitates an alternative mode of substrate binding and adds to the body of evidence that 13(S)-HPOD arises from tail-first binding.
