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Phospholipase D activity in the intestinal mitochondria: activation by oxygen free radicals
M Madesh1, S A Ibrahim, K A Balasubramanian
1Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College & Hospital, Vellore, India.
Abstract:
A prominent feature of cell damage caused by oxidative stress is morphological and functional changes in the mitochondria. The present study looked at the effect of free radical exposure on intestinal mitochondrial lipids. Free radical exposure did not alter neutral lipids, but among the phospholipids, phosphatidylethanolamine (PE) content was decreased on exposure to superoxide anion, generated by xanthine-xanthine oxidase or menadione with a concomitant increase in the level of phosphatidic acid (PA), suggesting activation of phospholipase D (PLD). This enzyme did not show transphosphatidylation activity in the presence of ethanol or butanol, and the product formed was phosphatidic acid (PA). This was confirmed by separation of reaction products by HPLC. This alteration in mitochondrial phospholipid was abolished by the presence of superoxide dismutase. Exposure to H2O2 did not have any significant effect. Activation of PLD by free radicals was further confirmed by quantitation of ethanolamine released from PE. Absence of any change in the content of lysophospholipid or diglyceride following exposure of mitochondria to superoxide ruled out the involvement of phospholipase A2 or C in the altered lipid composition. Moreover, inclusion of phospholipase A2 inhibitors, chlorpromazine, or p-bromophenacyl bromide did not prevent the generation of PA on exposure to free radicals. These findings suggest that superoxide anion stimulates intestinal mitochondrial PLD resulting in PE degradation and PA formation. These alterations in mitochondrial lipids may play a role in causing the functional alteration seen in oxidative stress.
Insights
Superoxide radicals damage intestinal mitochondria by altering phospholipids, specifically decreasing phosphatidylethanolamine (PE) and increasing phosphatidic acid (PA) via phospholipase D (PLD) activation. This damage was prevented by superoxide dismutase.
Area of Science:
- Mitochondrial biochemistry
- Oxidative stress research
- Lipid metabolism
Background:
- Oxidative stress induces mitochondrial damage, affecting cell function.
- Mitochondria are key targets of oxidative damage.
- Understanding lipid alterations in mitochondria is crucial for cell health.
Purpose of the Study:
- To investigate the impact of free radical exposure on intestinal mitochondrial lipids.
- To identify specific lipid changes and the enzymes involved.
- To elucidate the role of superoxide anion in mitochondrial lipid modification.
Main Methods:
- Exposure of intestinal mitochondria to superoxide anion (generated by xanthine-xanthine oxidase or menadione) and hydrogen peroxide (H2O2).
- Analysis of neutral lipids and phospholipids, including phosphatidylethanolamine (PE) and phosphatidic acid (PA).
- Enzyme activity assays for phospholipase D (PLD), phospholipase A2 (PLA2), and phospholipase C (PLC), including inhibitor studies and HPLC analysis.
Main Results:
- Superoxide anion exposure decreased PE and increased PA, suggesting PLD activation.
- PLD activity was confirmed by transphosphatidylation and ethanolamine release.
- Changes were abolished by superoxide dismutase, but H2O2 had no significant effect.
- Absence of lysophospholipid/diglyceride changes ruled out PLA2/PLC involvement.
Conclusions:
- Superoxide anion stimulates intestinal mitochondrial PLD, leading to PE degradation and PA formation.
- These mitochondrial lipid alterations may contribute to functional changes observed in oxidative stress.
- Targeting PLD or mitigating superoxide damage could protect mitochondrial function.