nbd-c16-coa  (FUJIFILM)

Journal: PLoS ONE

Article Title: Generation of a ceramide synthase 6 mouse lacking the DDRSDIE C-terminal motif

doi: 10.1371/journal.pone.0271675

Figure Lengend Snippet: CerS activity was assayed using 20 μg of protein from homogenates from the indicated tissues, and C16:0-CoA was used as substrate. Data are means ± S.D., n = 4. No significant differences were detected.

Article Snippet: NBD-sphinganine and palmitoyl-CoA (C16-CoA) were from Avanti Polar Lipids (Alabaster, AL).

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes

doi: 10.1038/s41598-021-81949-3

Figure Lengend Snippet: ACOT activities of reconstituted ABCD1. ( A ) Thioesterase activity of reconstituted ABCD1. Proteoliposomes containing ABCD1 (1.84 μg) or negative control liposomes containing non-specific protein (1.62 μg) were incubated with 2 μM NBD-C16-CoA for 10, 20, and 30 min at 37 °C. The “no incubation (0 min)” was run as the reaction was stopped before the addition of NBD-C16-CoA. Each reaction was subjected to TLC to separate NBD-C16-CoA and NBD-C16. ( B ) The ACOT activity was determined after the amount of NBD-C16 was quantified using the image analysis software Image J, and the ACOT activity of ABCD1 (filled circle) and non-specific protein (open circle) are shown. Error bars indicate the standard error (n = 3). ( C ) Acylation of ABCD1 with NBD-C16-CoA. ABCD1-liposomes were incubated with NBD-C16-CoA or NBD-C16 for 30 min at 37 °C and then subjected to SDS-PAGE. The gel was fixed with methanol and stained with CBB (left panel) after the detection of NBD fluorescence (right panel). The arrow head indicates ABCD1.

Article Snippet: NBD-C16-CoA was incubated with Nuclease P1 (FUJIFILM Wako, Osaka, Japan) in 50 mM Tris–HCl pH 8.0 and 1 mM DTT at 37 °C for 3 h, then subjected to TLC.

Techniques: Activity Assay, Negative Control, Incubation, Software, SDS Page, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes

doi: 10.1038/s41598-021-81949-3

Figure Lengend Snippet: Amino acid residues responsible for the ACOT activity of ABCD1 ( A ) Proteoliposomes containing ABCD1 (2.70 μg) were incubated with NBD-C16-CoA in the presence or absence of each compound (1 mM) for 30 min at 37 °C. Aliquots were subjected to TLC as shown in Fig. A. Relative ACOT activities were evaluated by quantifying the intensities of NBD-C16 using the image analysis software Image J (upper graph). ACOT activity without any reagents (0.27 nmol/min/mg) has been normalized to 1. The aliquots were also subjected to SDS-PAGE and the acylation of ABCD1 with NBD-C16-CoA was analyzed (lower panel). Error bars indicate the standard error (n = 3). The arrow head indicates ABCD1. PMSF: phenylmethylsulfonyl fluoride, DFP: diisopropylfluorophosphate, BNPP: bis-(4-nitrophenyl)phosphate, p CMB: p -chloromercuribenzoic acid, DEPC: diethyl pyrocarbonate. ( B ) ACOT activity was measured in the presence of various concentrations of p CMB for 30 min at 37 °C. Aliquots were subjected to TLC as shown in Fig. B. Relative ACOT activities were evaluated by quantifying the intensities of NBD-C16 using the image analysis software Image J. ACOT activity without p CMB (0.29 nmol/min/mg) has been normalized to 1. ( C ) Stability of covalent linkage between ABCD1 and NBD-C16. ABCD1-liposomes were incubated with NBD-C16-CoA for 30 min at 37 °C and then subjected to SDS-PAGE. The gels were fixed and then divided. Each gel was incubated with 1 M Tris–HCl pH 7.0, 1 M Hydroxylamine pH 7.0, 0.1 N KOH or 0.1 N HCl for 18 h at room temperature, respectively. The gels were stained with CBB after the detection of NBD fluorescence. The arrow head indicates ABCD1.

Article Snippet: NBD-C16-CoA was incubated with Nuclease P1 (FUJIFILM Wako, Osaka, Japan) in 50 mM Tris–HCl pH 8.0 and 1 mM DTT at 37 °C for 3 h, then subjected to TLC.

Techniques: Activity Assay, Incubation, Software, SDS Page, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes

doi: 10.1038/s41598-021-81949-3

Figure Lengend Snippet: Substrate specificity of ABCD1-catalyzed ACOT activity. ( A ) Proteoliposomes containing ABCD1 (3.04 μg) were incubated with NBD-C16-CoA or NBD-C16-3′-dephosphoCoA (NBD-C16-dePiCoA) for 30 min at 37 °C. The aliquots were subjected to TLC. ( B ) Aliquots were also subjected to SDS-PAGE. The gel was fixed with methanol and stained with CBB (left panel) after the detection of NBD fluorescence (right panel). The arrow head indicates ABCD1. ( C ) Proteoliposomes containing ABCD1 (3.16 μg) were incubated with NBD-C16-CoA or NBD-C6-CoA for 30 min at 37 °C. The aliquots were subjected to TLC. ( D ) Aliquots were also subjected to SDS-PAGE. The gel was fixed with methanol and stained with CBB (left panel) after the detection of NBD fluorescence (right panel). The arrow head indicates ABCD1.

Article Snippet: NBD-C16-CoA was incubated with Nuclease P1 (FUJIFILM Wako, Osaka, Japan) in 50 mM Tris–HCl pH 8.0 and 1 mM DTT at 37 °C for 3 h, then subjected to TLC.

Techniques: Activity Assay, Incubation, SDS Page, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes

doi: 10.1038/s41598-021-81949-3

Figure Lengend Snippet: Relationship between ATPase activity and the ACOT activity of ABCD1 ( A ) Effect of ATP on ABCD1-catalyzed ACOT activity. ABCD1-liposomes were incubated with NBD-C16-CoA in the presence of various concentration of ATP for 30 min at 37 °C. The aliquots were subjected to TLC as shown in Fig. . The relative ACOT activities were evaluated by quantifying the intensities of NBD-C16 using the image analysis software Image J. Error bars indicate the standard error (n = 3). ( B ) His-ABCD1(a.a.1–431) and His-ABCD1(K513A) were prepared using the same procedure as the purification of His-ABCD1 and then reconstituted into liposomes. Proteoliposomes containing ABCD1wild type, ABCD1(K513A) or ABCD1(1–431) (2.68 μg, 3.21 μg or 3.71 μg, respectively) were incubated with NBD-C16-CoA for 30 min at 37 °C. The aliquots were subjected to TLC to separate hydrolyzed NBD-C16 and NBD-C16-CoA. ( C ) Relative ACOT activities of ABCD1(a.a.1–431) and ABCD1(K513A) were evaluated. The intensities of NBD-C16 were normalized by the signal intensities of each of the proteins obtained by immunoblot analysis using the image analysis software Image J. Error bars indicate the standard error (n = 3). Wild type activity (0.33 nmol/min/mg) has been normalized to 1. ( D ) Aliquots were also subjected to SDS-PAGE. The gel was fixed with methanol and stained with CBB (left panel) after the detection of NBD fluorescence (right panel). The arrow heads indicate the wild type, a.a.1–431 or K513A.

Article Snippet: NBD-C16-CoA was incubated with Nuclease P1 (FUJIFILM Wako, Osaka, Japan) in 50 mM Tris–HCl pH 8.0 and 1 mM DTT at 37 °C for 3 h, then subjected to TLC.

Techniques: Activity Assay, Incubation, Concentration Assay, Software, Purification, Western Blot, SDS Page, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes

doi: 10.1038/s41598-021-81949-3

Figure Lengend Snippet: Transport of the NBD-C16 derived from NBD-C16-CoA into ABCD1- liposomes. ( A ) Proteoliposomes containing ABCD1 (4.81 μg) or non-specific protein were incubated with NBD-C16-CoA at 37 °C for the indicated periods. After incubation, the remaining NBD-C16-CoA in the outside portion of the liposomes was quenched with sodium dithionite. In some cases, the liposomes were treated with sodium dithionite after the addition of 0.01% Triton X-100. Subsequently, ABCD1-liposomes were precipitated by centrifugation and then resuspended with 80% acetone. NBD-C16 and NBD-C16-CoA were separated by TLC. ( B ) Transport of NBD-C16 into ABCD1-liposomes was tested under various conditions. ABCD1-liposomes were incubated with NBD-C16-CoA in the presence of p CMB (1 mM) or palmitoyl-CoA (20 μM), or in the absence of ATP. NBD-C16-3′-dephosphoCoA (NBD-C16-dePiCoA) and NBD-C6-CoA were also examined as the substrate. Relative NBD-C16 transport activities were evaluated by quantifying the intensities of NBD-C16 using the image analysis software Image J (right graph). Error bars indicate the standard error (n = 3). (C) NBD-C16 transport activity for ABCD1(K513A) was tested by the same procedure as used for the wild type. The relative transport activity ABCD1(K513A) was evaluated as follows. The intensities of NBD-C16 were normalized by the signal intensities of the wild type and K513A obtained by immunoblot analysis using the image analysis software Image J. Error bars indicate the standard error (n = 3).

Article Snippet: NBD-C16-CoA was incubated with Nuclease P1 (FUJIFILM Wako, Osaka, Japan) in 50 mM Tris–HCl pH 8.0 and 1 mM DTT at 37 °C for 3 h, then subjected to TLC.

Techniques: Derivative Assay, Incubation, Centrifugation, Software, Activity Assay, Western Blot

Journal: The Journal of Cell Biology

Article Title: Mdm1 maintains endoplasmic reticulum homeostasis by spatially regulating lipid droplet biogenesis

doi: 10.1083/jcb.201808119

Figure Lengend Snippet: PXA domain of Mdm1 recruits FA-CoA ligase Faa1 to LD budding sites. (A) Left: Light microscopy of GFP-tagged Mdm1 ER-PM in yeast stably expressing mCherry-tagged Faa1 in the presence of 0.2% oleate. Scale bar, 2 µm. LDs (gray) were visualized by using MDH. Arrows indicate co-enrichment of Mdm1 ER-PM , Faa1, and LDs. Scale bar, 2 µm. (B) Line tracing of light microscopy images in (A). (C) Left: Light microscopy of different fragments of Mdm1 ER-PM in yeast stably expressing mCherry-tagged Faa1 in the presence of 0.2% oleate. LDs (gray) were visualized by using MDH. Scale bar, 2 µm. Arrows indicate co-enrichment of Mdm1 ER-PM , Faa1, and LDs. Right: Illustrated representation of the topology of different Mdm1 ER-PM constructs being expressed. (D) Quantification of Mdm1 ER-PM foci associated with Faa1 from images in A and C. Data represent the number of LD-associated foci over the total Mdm1 ER-PM foci. Mean ± SD; n > 50 cells; *, P < 0.01; **, P < 0.005; Student’s t test. (E) Diagram depicting the pathway of neutral lipid synthesis. (F) Quantification of BODIPY-C16 incorporation into neutral lipid (NL) BODIPY-DAG in mdm1 Δ and faa1 Δ relative to WT yeast (1-h incubation). n = 3; **, P < 0.005; Student’s t test. Raw data are shown in Fig. S3G. (G) Quantification of BODIPY-C16 incorporation into neutral lipid (NL) BODIPY-TAG in mdm1 Δ and faa1 Δ relative to WT yeast (1-h incubation). n = 3; **, P < 0.005; Student’s t test. Raw data are shown in Fig. S3 G (high exposure). (H) Quantification of NBD-C16-CoA incorporation into NBD-TAG in mdm1 Δ and faa1 Δ relative to WT yeast (1-h incubation). n = 3; ns, nonsignificant; Student’s t test. Raw data are shown in Fig. S3 H.

Article Snippet: Yeast lysates (100 µg in 50 µl buffer) were mixed with 50 µM of BODIPY-C16 (Invitrogen; D3821) or NBD-C16-CoA (Avanti; 810705) in the presence of 20 µl 1 M Tris-HCl, pH 7.6, 4 µl 1 M MgCl 2 , 10 µl 4 mM 1–2-dioleoyl-sn-glycerol DOG (Sigma-Aldrich; D0138), 10 µl 12.5 mg/ml BSA, and 96 µl water per reaction and incubated at 30°C.

Techniques: Light Microscopy, Stable Transfection, Expressing, Construct, Incubation