Journal: Journal of Lipid Research
Article Title: Identification and characterization of LPLAT7 as an sn -1-specific lysophospholipid acyltransferase
doi: 10.1016/j.jlr.2022.100271
Figure Lengend Snippet: Biochemical characterization of LPLAT7. A, B, Preference of LPLAT7 for sn -2 lysophospholipids. A: LPLAT activities of LPLAT7 against LPC. Oleoyl (C18:1) LPCs (both sn -2-rich and sn -1-rich, 20 μM) and stearoyl (C18:0)-CoA (4 μM) were used as acyl acceptors and an acyl donor, respectively. Membrane fractions from HEK293A cells transfected with a human LPLAT7 expression vector or an empty vector (Mock) were used as enzyme sources. B: Kinetic analyses of LPTAT7 activities. Substrate concentration dependence of LPLAT7 was determined with the indicated concentrations of LPCs (both sn -2-rich and sn -1-rich) in the presence of C18:0-CoA (4 μM). C, D, Determination of the glycerol sn positions into which LPLAT7 introduces a fatty acid. C: LPLAT activities of LPLAT7 against LPC using a set of labeled LPCs and a labeled-acyl-CoA. Palmitoyl (C16:0)- d 31 LPCs (both sn -2-rich and sn -1-rich, 10 μM) and 13 C 18 18:0-CoA (2 μM) were used as acyl acceptors and an acyl donor, respectively. D: The ratio of two labeled fatty acids at the sn -1 position of the LPLAT7 reaction products. The reaction products of LPLAT7 in (C) were subjected to a PLA 2 reaction. The amount of the resulting two sn -1-acyl LPCs (1– 13 C 18 18:0-2-hydroxy-GPC and 1-16:0- d 31 -2-hydroxy-GPC) was determined by LC-MS/MS. E: LPLAT activities of LPLAT7 toward acyl acceptors with different head groups. Various sn -2-rich C18:1 lyso-PLs (LPC, LPE, LPS, LPG and LPA) and soy LPI, each 20 μM and C18:0-CoA (4 μM) were used. F: Kinetic analyses of LPTAT7 activities. Substrate concentration dependence of LPLAT7 was determined with the indicated concentrations of LPCs or LPEs in the presence of C18:0-CoA (4 μM). G: LPLAT activities of LPLAT7 toward the three acyl donors, determined using C16:0-CoA, C18:1-CoA, or C18:0-CoA (4 μM) with sn -2-rich C18:1 LPC (20 μM). H: Kinetic analyses of LPTAT7 activities. Substrate concentration dependence of LPLAT7 was determined with the indicated concentrations of acyl-CoAs (C18:0-, C18:1- and C16:0-CoAs) in the presence of sn -2-rich C18:1 LPC (20 μM). I: LPLAT activities of LPLAT7 toward various acyl acceptors with different fatty acids. Several LPCs (C16:0, C18:1, linoleoyl (C18:2), arachidonoyl (C20:4), and docosahexaenoyl (C22:6), each 20 μM) and C18:0-CoA (4 μM) were used.
Article Snippet: 13 C 16 Palmitoyl ( 13 C 16 16:0) coenzyme A and 13 C 18 stearoyl ( 13 C 18 18:0) coenzyme A were purchased from Taiyo Nippon Sanso (Tokyo, Japan).
Techniques: Membrane, Transfection, Expressing, Plasmid Preparation, Concentration Assay, Labeling, Liquid Chromatography with Mass Spectroscopy
Proteintech is a verified supplier 
Fig. 1 A)) as acyl acceptors, 
Fig. 1 A) as an acyl acceptor and stearoyl-CoA (C18:0-CoA, A: HeLa, B: HEK293A) and palmitoyl-CoA (C16:0-CoA, C: HeLa, D: HEK293A) as acyl donors. Data are shown as the mean ± SD of three data points. The data are representative of two independent experiments with similar results. " width="100%" height="100%">
