Specific inhibition of rat brain phospholipase D by lysophospholipids

Abstract

Although the importance of phospholipase D (PLD) in signal transduction in mammalian cells is well documented, the negative regulation of PLD is poorly understood. This is primarily due to a lack of known specific inhibitors of PLD. We herein report that the activity of partially purified rat brain PLD is inhibited by certain lysophospholipids, such as lysophosphatidylinositol, lysophosphatidylglycerol, and lysophosphatidylserine in a highly specific manner. Inhibition of PLD by lysophospholipids was dosedependent: the concentration of lysophosphatidylinositol required for half-maximal inhibition was about 3 m . An analysis of the enzyme-kinetics suggested that lysophospholipids act as non-competitive inhibitors of PLD activity. As expected, PLD activity was stimulated by ADP-ribosylation factor (Arf) and phosphatidylinositol 4,5-bisphosphate (PIP 2 ). The inhibition of PLD by lysophospholipids, however, was not affected by the presence or absence of Arf or by an increase in PIP 2 concentration. A protein-binding assay suggested that lysophospholipids bind directly to PLD. These results indicate that the observed inhibition of PLD by lysophospholipids is due to their direct interaction rather than to an interaction between lysophospholipids and either Arf or PIP 2 . The present study suggests that certain lysophospholipids are specific inhibitors of rat brain PLD in a cell-free system and may provide the new opportunities to investigate mechanisms by which PLD is regulated by lysophospholipids, presumably liberated by phospholipase A 2 activation, in mammalian cells. -Ryu, S. B. and J. P. Palta. Specific inhibition of rat brain phospholipase D by lysophospholipids.

Synthesis and Evaluation of Fluorogenic Substrates for Phospholipase D and Phospholipase C

“…A longer incubation time with lysoDDPB mixed micelles (Supporting Information, Figure S3) only amplified fluorescence from Genzyme PLD(P). These results support the idea that lysophospholipids are substrates for scPLD 14 but are not substrates for HKD PLD. ,

1 Fluorescence evolution (λ Ex /λ Em = 500/530 nm) during (a) 3 min incubation of DDPB mixed micelles with PLD from various sources (blue ⧫ = peanut PLD; red ▪ = cabbage PLD; yellow ▴ = Strep. PMF PLD; brown × = Genzyme PLD(P); ○ = scPLD), (b) 60 min incubation of DDPB mixed micelles with PLD and PLA 2 from various sources (blue ⧫ = peanut PLD; red ▪ = cabbage PLD; brown ▴ = Strep.

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“…These results support the idea that lysophospholipids are substrates for scPLD 14 but are not substrates for HKD PLD. 29,30 Next, mixed micelles of the PC analogues were tried as substrates for PC-PLC, from Bacillus cereus and Clostridium perfringens, and PI-PLC, from B. cereus (Figure 1c). Over 3 min, DDPB gave an overwhelmingly large fluorescence response with B. cereus PC-PLC and virtually none with B. cereus PI-PLC or C. perfringens PC-PLC.…”

Genetic overlap and causality between blood metabolites and migraine

“…First, as LPEs and anandamide are both formed from membrane phosphatidylethanolamines, higher levels of LPE(20:4) may limit the resource required to produce anandamide. Second, LPEs inhibit phospholipase D activity 47 that produces anandamide in neurons. These findings and our analyses highlight a causal role for LPEs, specifically LPE(20:4), indicating a potential therapeutic target for migraine.…”