Detection of choline and phosphatidic acid (PA) catalyzed by phospholipase D (PLD) using MALDI-QIT-TOF/MS with 9-aminoacridine matrix
| Autor: | Akiyoshi Fukamizu, Motoki Matsui, Kyung-Eui Park, Hiroaki Daitoku, Jun-Dal Kim, Yusuke Nagashima, Gwi Gun Park, Koichiro Kako |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Phosphatidic Acids
Applied Microbiology and Biotechnology Biochemistry Analytical Chemistry Choline chemistry.chemical_compound 9-Aminoacridine Limit of Detection Phosphatidylcholine medicine Phospholipase D Phospholipase D activity Humans Molecular Biology Enzyme Assays Chromatography Chemistry PLD2 Hydrolysis Organic Chemistry General Medicine Phosphatidic acid Aminacrine HEK293 Cells Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Biocatalysis lipids (amino acids peptides and proteins) Acetylcholine Biotechnology medicine.drug |
| Zdroj: | Bioscience, biotechnology, and biochemistry. 78(6) |
| ISSN: | 1347-6947 |
| Popis: | Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant phospholipids of plasma membrane, resulting in the production of choline and phosphatidic acid (PA). Choline is a precursor of the neurotransmitter acetylcholine, whereas PA functions as an intracellular lipid mediator of diverse biological functions. For assessing PLD activity in vitro, PLD-derived choline has been often analyzed with radioactive or non-radioactive methods. In this study, we have developed a new method for detecting choline and PA with MALDI-QIT-TOF/MS by using 9-aminoacridine as a matrix. The standard calibration curves showed that choline and PA could be detected with linearity over the range from 0.05 and 1 pmol, respectively. Importantly, this method enables the concomitant detection of choline and PA as a reaction product of PC hydrolysis by PLD2 proteins. Thus, our simple and direct method would be useful to characterize the enzymatic properties of PLD, thereby providing insight into mechanisms of PLD activation. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|