A mass spectrometry-based method for the assay of ceramide synthase substrate specificity.

Anal Biochem

Department of Biochemistry and Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE 68588, USA. Electronic address:

Published: June 2015

The acyl composition of sphingolipids is determined by the specificity of the enzyme ceramide synthase (EC 2.3.1.24). Ceramide contains a long-chain base (LCB) linked to a variety of fatty acids to produce a lipid class with potentially hundreds of structural variants. An optimized procedure for the assay of ceramide synthase in yeast microsomes is reported that uses mass spectrometry to detect any possible LCB and fatty acid combination synthesized from unlabeled substrates provided in the reaction. The assay requires the delivery of substrates with bovine serum albumin for maximum activity within defined limits of substrate concentration and specific methods to stop the reaction and extract the lipid that avoid the non-enzymatic synthesis of ceramide. The activity of ceramide synthase in yeast microsomes is demonstrated with the four natural LCBs found in yeast along with six saturated and two unsaturated fatty acyl-coenzyme As from 16 to 26 carbons in length. The procedure allows for the determination of substrate specificity and kinetic parameters toward natural substrates for ceramide synthase from potentially any organism.

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ab.2015.02.016DOI Listing

Publication Analysis

Top Keywords

ceramide synthase
20
assay ceramide
8
substrate specificity
8
synthase yeast
8
yeast microsomes
8
ceramide
7
synthase
5
mass spectrometry-based
4
spectrometry-based method
4
method assay
4

Similar Publications

Plant sphingolipids are lipophilic membrane components essential for different cellular functions but they also act as signaling molecules in various aspects of plant development. However, the interaction between plant sphingolipids and abscission remains largely uncharacterized. Here, the possible role of sphingolipids in regulating fruit abscission was examined in the abscission zone (AZ) of olive fruit.

View Article and Find Full Text PDF

Bisphenol A exacerbates colorectal cancer progression through enhancing ceramide synthesis.

Toxicology

January 2025

Department of Occupational Health and Environmental Health, School of Public Health, Anhui Medical University, Hefei, Anhui 230032, PR China. Electronic address:

Bisphenol A (BPA) is a typical environmental endocrine disruptor which have been broadly confirmed to be associated with malignant tumors, including colorectal cancer (CRC). Lipid metabolism reprogramming performed important biological effects in cancer progression. While the role of lipid metabolism in CRC progression upon BPA exposure remain elusive.

View Article and Find Full Text PDF

Protective effects and bioinformatic analysis of narciclasine on vascular aging via cross-talk between inflammation and metabolism through inhibiting skeletal muscle-specific ceramide synthase 1.

Mech Ageing Dev

December 2024

School of Medicine, Nankai University, 94 Weijin Road, Tianjin 30071, China; Institude of Chinese PLA Geriatric Medicine, The Second Medical Centre, Chinese PLA General Hospital & National Clinical Research Center for Geriatric Diseases, Beijing 100853, China. Electronic address:

Objective: The senescence of smooth muscle is one of the independent risk factors in atherosclerosis progression in which the vascular inflammation plays an important role on vascular dysfunction. This study is designed to explore the novel vascular aging biomarkers and screen the potential molecular interventional targets through bioinformatic analysis.

Results: Transcriptional analysis was conducted based on the GSE16487 open access database, which included 15 human vascular tissue samples from two groups: young group (≤ 60 years old, n = 8) and aged group (≥ 75 years old, n = 7).

View Article and Find Full Text PDF

produces the mycotoxin fumonisin B (FB), which disrupts sphingolipid biosynthesis by inhibiting ceramide synthase and affects the health of plants, animals, and humans. The means by which protects itself from its own mycotoxin are not completely understood. Some fumonisin () cluster genes do not contribute to the biosynthesis of the compound, but their function has remained enigmatic.

View Article and Find Full Text PDF

This study investigated the protective effects of a ceramides derivates from the peach (PF3) on photoaging by UV-irradiated hairless mice. Mice were randomly divided into seven groups: AIN93G without UVB exposure (normal control, NC), AIN93G with UVB exposure (control, C), AIN93G supplemented 100 mg/kg body weight (BW) of L-ascorbic acid with UVB exposure (AA), AIN93G supplemented 100 mg/kg BW of arbutin with UVB exposure (Arbutin), AIN93G supplemented 10 mg/kg BW of PF3 with UVB exposure (10PF3), AIN93G supplemented 20 mg/kg BW of PF3 with UVB exposure (20PF3), and AIN93G supplemented 40 mg/kg BW of PF3 with UVB exposure (40PF3). The study examined the impact of PF3 on skin hydration, wrinkle formation, and melanogenesis using enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (real-time PCR), and Western blot analysis.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!