Detection of choline and phosphatidic acid (PA) catalyzed by phospholipase D (PLD) using MALDI-QIT-TOF/MS with 9-aminoacridine matrix

Abstract

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.

PRMT8 as a phospholipase regulates Purkinje cell dendritic arborization and motor coordination

“…We previously reported a MALDI-QIT-TOF/MS method that directly detects DPPC-derived choline and dipalmitoylphosphatidic acid (DPPA) catalyzed by PLD ( 44 ). Because DPPC was significantly increased in the cerebellum of prmt8 −/− mice, we investigated whether PRMT8, but not the PRMT8 K107R mutant, directly hydrolyzed DPPC to produce choline and DPPA.…”

“…S6, B to D). In addition, we also assessed the PC hydrolysis activity of PRMT8 using the headgroup-release assay with [choline-methyl- 3 H]DPPC ([ 3 H]DPPC) as a substrate ( 44 , 45 ). As expected, wild-type PRMT8, but not the K107R mutant, generated [ 3 H]choline as a water-soluble product (fig.…”

Activity and Function of the PRMT8 Protein Arginine Methyltransferase in Neurons

“…Indeed, aberrant reduction of acetylcholine and choline levels, as well as increased PC levels, are detected in the cerebellum of Prmt8 −/− mice [10]. Using MALDI-QIT-TOF/MS, Kim et al further identify lysine-107 on PRMT8 as the essential amino acid residue for its phospholipase activity in vitro, and its PC hydrolysis activity promotes neurite branching in PC12 cells upon treatment with nerve growth factor (NGF) [10,41].…”