Journal: The Journal of Biological Chemistry
Article Title: Acid ceramidase ASAH1 is a key regulator of epidermal ceramide levels and composition
doi: 10.1016/j.jbc.2026.111178
Figure Lengend Snippet: Enzyme kinetic parameters of ASAH1. HEK 293T cells were transfected with either the pCE-puro 3× FLAG-4 vector or the pCE-puro ASAH1-3× FLAG plasmid. Culture supernatant was collected 48 h after transfection as the enzyme source. The indicated ceramide species with different classes—C24:0 NS ( A ), C24:0 NP ( B ), C24:0 NH ( C ), and C24:0 AS ( D )—were incorporated into liposomes at final concentrations of 0.4, 0.8, 1.6, and 3.2 μM and incubated with the culture supernatant at 37 °C for 2 h. After incubation, lipids were extracted, and the resulting long-chain bases (ceramide degradation products) were quantified via LC–MS/MS. The data obtained were used to generate Lineweaver–Burk plots by plotting the inverse of reaction velocity (1/ V ) against the inverse of substrate concentration (1/[S]). Values presented are mean ± SD (n = 3). Values of the calculated kinetic parameters ( K M , V max , and k cat / K M ) are also shown. HEK, human embryonic kidney cell line.
Article Snippet: A mixture of 60 μl of 5 mg/ml 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (16:0/18:1 phosphatidylcholine; Avanti Research), dissolved in chloroform/methanol (1:1, v/v), and 40 μl of each ceramide species at 100 μM— N -palmitoyl- d - erythro -sphingosine (C16:0 NS; Avanti Research), N -lignoceroyl- d - erythro -sphingosine (C24:0 NS; Avanti Research), N -nervonoyl- d - erythro -sphingosine (C24:1 NS; Avanti Research), N -lignoceroyl- d - erythro -dihydrosphingosine (C24:0 NdS; Avanti Research), N -lignoceroyl- d - ribo -phytosphingosine (C24:0 NP; Avanti Research), N -lignoceroyl-6-( R )-hydroxysphingosine (C24:0 NH; Avanti Research), N -(2'-( R )-hydroxylignoceroyl)- d - erythro -sphingosine (C24:0 AS; Avanti Research), N -(2'-( R )-hydroxylignoceroyl)- d - ribo -phytosphingosine (C24:0 AP; Avanti Research), N -(30-linoleoyloxy-triacontanoyl)- d - erythro -sphingosine (C30:0 EOS; Cayman Chemical), and N -ω-hydroxytriacontanoyl- d - erythro -sphingosine (C30:0 OS; Cayman Chemical)—was combined and dried.
Techniques: Transfection, Plasmid Preparation, Liposomes, Incubation, Liquid Chromatography with Mass Spectroscopy, Concentration Assay
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![Enzyme kinetic parameters of ASAH1. HEK 293T cells were transfected with either the pCE-puro 3× FLAG-4 vector or the pCE-puro ASAH1-3× FLAG plasmid. Culture supernatant was collected 48 h after transfection as the enzyme source. The indicated ceramide species with different <t>classes—C24:0</t> NS ( A ), C24:0 NP ( B ), C24:0 NH ( C ), and C24:0 AS ( D )—were incorporated into liposomes at final concentrations of 0.4, 0.8, 1.6, and 3.2 μM and incubated with the culture supernatant at 37 °C for 2 h. After incubation, lipids were extracted, and the resulting long-chain bases (ceramide degradation products) were quantified via LC–MS/MS. The data obtained were used to generate Lineweaver–Burk plots by plotting the inverse of reaction velocity (1/ V ) against the inverse of substrate concentration (1/[S]). Values presented are mean ± SD (n = 3). Values of the calculated kinetic parameters ( K M , V max , and k cat / K M ) are also shown. HEK, human embryonic kidney cell line.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7855/pmc12907855/pmc12907855__gr8.jpg)