Metal ion stimulation of phospholipase D-like activity of isolated rat intestinal mitochondria
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M Madesh1, K A Balasubramanian
1Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College & Hospital, Vellore, India.
Abstract:
Presence of phospholipase D-like (PLD) activity in the intestinal mitochondria was identified using endogenous phospholipids as substrate. The enzyme had a pH optimum of 6.5, did not show trans-phosphatidylation 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 high-performance liquid chromatography and analysis of composition of the PA formed which gave phosphate/fatty acid ratio of 1:2 PLD-like activity was further confirmed by the formation of ethanolamine and choline as products of enzyme action. This activity was stimulated by various metal ions; when stimulated by Mg2+ and Ba2+, it hydrolyzed both phosphatidylcholine and phosphatidylethanolamine, and when stimulated by Ca2+, it preferentially hydrolyzed phosphatidylethanolamine. There was no requirement for sodium oleate for the PLD-like activity in mitochondria. These results suggest that intestinal mitochondria have an active PLD-like enzyme which differs in certain properties from phospholipase D from other tissues.
Insights
Intestinal mitochondria possess phospholipase D-like (PLD) activity, producing phosphatidic acid. This enzyme
Area of Science:
- Biochemistry
- Cell Biology
Background:
- Mitochondria play crucial roles in cellular metabolism and signaling.
- Phospholipase D (PLD) enzymes are involved in phospholipid metabolism and signal transduction.
Purpose of the Study:
- To investigate the presence and characteristics of phospholipase D-like (PLD) activity in intestinal mitochondria.
- To determine the substrate specificity and optimal conditions for this mitochondrial PLD-like activity.
Main Methods:
- Incubation of intestinal mitochondria with endogenous phospholipids.
- Analysis of reaction products using high-performance liquid chromatography (HPLC).
- Characterization of enzyme activity based on pH optimum, trans-phosphatidylation, metal ion stimulation, and substrate preference.
Main Results:
- PLD-like activity was identified in intestinal mitochondria, with a pH optimum of 6.5.
- The enzyme produced phosphatidic acid (PA) and did not exhibit trans-phosphatidylation activity with short-chain alcohols.
- Activity was modulated by metal ions (Mg2+, Ba2+, Ca2+), with differential hydrolysis of phosphatidylcholine and phosphatidylethanolamine.
Conclusions:
- Intestinal mitochondria possess a distinct PLD-like enzyme with unique properties compared to PLD from other tissues.
- This finding suggests a potential role for mitochondrial PLD-like activity in intestinal phospholipid metabolism and function.