Metal Ion Stimulation of Phospholipase D-Like Activity of Isolated Rat Intestinal Mitochondria M. Madesh and K.A. Balasubramanian* The Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College & Hospital, Vellore 632 004, India ABSTRACT: Presence of phospholipase D-like (PLD) activity Under certain pathological conditions, such as oxidative in the intestinal mitochondria was identified using endogenous stress, increase in cytosolic Ca2+ occurs and this in turn in- phospholipids as substrate. The enzyme had a pH optimum of creases the Ca2+ sequestration by mitochondria (13,14). It has 6.5, did not show trans-phosphatidylation activity in the pres- been proposed that PA may act as a Ca2+ ionophore (15,16) ence of ethanol or butanol, and the product formed was phos- and facilitate the influx of Ca2+ through the plasma mem- phatidic acid (PA). This was confirmed by separation of reac- brane. Although presence of PLD activity has been shown in tion products by high-performance liquid chromatography and liver, skeletal muscle and brain mitochondria, very little is analysis of composition of the PA formed which gave phos- phate/fatty acid ratio of 1:2. PLD-like activity was further con- known on the properties of mitochondrial enzyme (17–19). firmed by the formation of ethanolamine and choline as prod- Since mitochondria are important in calcium homeostasis and ucts of enzyme action. This activity was stimulated by various PA might play a role in this process, the present study looks metal ions; when stimulated by Mg2+ and Ba2+, it hydrolyzed at the possible presence of PLD-like activity and its proper- both phosphatidylcholine and phosphatidylethanolamine, and ties in the intestinal mitochondria. when stimulated by Ca2+, it preferentially hydrolyzed phos- phatidylethanolamine. There was no requirement for sodium oleate for the PLD-like activity in mitochondria. These results MATERIALS AND METHODS suggest that intestinal mitochondria have an active PLD-like en- Various lipid standards, Hepes, fluorescamine, p-bro- zyme which differs in certain properties from phospholipase D mophenacylbromide (pBPB), chlorpromazine (CHLP), and from other tissues. bovine serum albumin (BSA) were all obtained from Sigma Lipids 32, 471–479 (1997). Chemical Company (St. Louis, MO). Standard phos- phatidylethanol and phosphatidylbutanol were prepared using A number of studies have shown that phosphatidic acid (PA) egg phosphatidylcholine and cabbage PLD in presence of is involved in cell signaling (1,2). PA can either be formed by ethanol or butanol (20). All other chemicals used were of an- the action of phospholipase D (PLD) on phospholipids or by alytical grade. phosphorylation of diacylglycerol. PLD can be activated in Preparation of mitochondria. Rats weighing 150–200 g, response to various stimuli and phorbol esters activate PLD fasted overnight, were decapitated, the small intestine re- through protein kinase C (3,4). Protein kinase C activation of moved and washed with ice-cold saline. Mitochondria were PLD takes place by both phosphorylation-dependent and in- isolated from enterocytes as described by Masola and Evered dependent mechanisms (5,6). Lipopolysaccharide stimulates (21). Isolated mitochondrial fraction was suspended in cellular PLD activity, and this requires an increase in intra- EGTA-free medium containing 250 mM sucrose/5 mM Hepes cellular Ca2+ (7). Exposure to oxygen-derived free radicals pH 7.4 and stored in ice at protein concentration of 8–10 also stimulates cellular PLD activity (8,9). Using PLD from mg/mL. Isolated mitochondria were relatively pure as judged various sources, it has been shown that divalent metal ions by the mitochondrial marker enzyme succinic dehydrogenase. can activate this enzyme (10,11). Microsomal and lysosomal contamination in the mitochon- Calcium plays an important role in many cellular func- drial preparation were checked by assaying marker enzymes, tions, and intracellular calcium is stored mainly in endoplas- namely, glucose-6-phosphatase, acid phosphatase and aryl- mic reticulum and to a small extent in the mitochondria (12). sulfatase, and these activities were not detectable. Protein was measured using BSA as standard (22). Enzyme assay. Mitochondrial PLD-like activity was mea- *To whom correspondence should be addressed. sured using endogenous mitochondrial phospholipids as sub- Abbreviations: PA, phosphatidic acid; pBPB, p-bromophenacylbromide; strate. Mitochondria (1 mg protein per mL in 0.25 M sucrose, PLD, phospholipase D; DAG, diacylglycerol; CHLP, chlorpromazine; TLC, thin-layer chromatography; HPLC, high-performance liquid chromatogra- 5 mM Hepes buffer pH 7.4) were incubated at 37°C for 30 phy; PE, phosphatidylethanolamine; PC, phosphatidylcholine. min. Following incubation, total lipids were extracted by Copyright © 1997 by AOCS Press 471 Lipids, Vol. 32, no. 5 (1997) 472 M. MADESH AND K.A. BALASUBRAMANIAN Bligh and Dyer’s method (23) and the PA was separated by by vol). This was identified after exposure to iodine vapor thin-layer chromatography (TLC) and quantitated by phos- (29). phate estimation. Extracted lipids were spotted on silica gel Lipid analysis. Neutral lipids were separated on silica gel G plates impregnated with 0.5 M oxalic acid and separated G plates using the solvent system hexane/diethyl ether/acetic using the solvent system chloroform/methanol/conc. HCl acid (80:20:1, by vol). Spots were identified by iodine expo- (85:13:0.5, by vol) (24). PA spots corresponding to standard sure, scraped, and eluted. Cholesterol (30), diglycerides, and were identified by iodine exposure, scraped, and eluted from triglycerides (31) were estimated as described. Free fatty the plates. PA was quantitated by phosphate estimation after acids were methylated and quantitated by gas chromatogra- acid digestion (25). PA formed was also separated and iden- phy after separation on a 5% EGSS-X column. Heptade- tified along with standard using two other solvent systems canoic acid was used as internal standard. Individual phos- (26,27). Formation of PA was further confirmed by separa- pholipids were separated on silica gel H plates using the sol- tion of extracted lipids on TLC followed by isolated PA spot vent system chloro-form/methanol/acetic acid/water separated by high-performance liquid chromatography (25:15:4:2, by vol) (32) and quantitated by phosphate esti- (HPLC) (28). A normal-phase silica 5-µm column was used mation after acid hydrolysis. Individual aminophospholipids with the solvent gradient of 1–9% water in hexane/2- were also quantitated after derivatization with fluorescamine propanol (3:4, vol/vol) at a flow rate of 1 mL/min and the and separation on silica gel H plates impregnated with eluents monitored at 220 nm. Transphosphatidylation was 3% magnesium acetate using the solvent system chloro- checked by inclusion of 300 mM ethanol or butanol in the form/methanol/NH4OH/water (60:40:5:2, by vol) (33). incubation mixture, and extracted lipids were separated on Eluted individual spots were quantitated using Shimadzu SF silica gel G plates using the upper phase of ethyl ace- 5000 spectrofluorometer (Tokyo, Japan) with excitation at tate/isooctane/acetic acid/water (13:2:3:10, by vol) (27). PA 395 nm and emission at 468 nm. formation in the presence of ethanol was further checked by Separation and analysis of ethanolamine. Following incu- HPLC. Metal ion stimulation of phosphatidylcholine degra- bation, enzyme activity was terminated with 10% trichloro- dation generated choline which was separated by TLC, using acetic acid. After cooling on ice, the mixture was transferred the solvent system methanol/0.6% NaCl/ammonia (50:50:5, FIG. 2. Thin-layer chromatographic separation of phosphatidic acid (PA) and phosphatidylethanol. Silica gel G plate was developed using the upper phase of the solvent system ethylacetate/isooctane/acetic FIG. 1. Effect of various metal ions (at 0.1 and 1 mM final concentra- acid/water (13:2:3:10, by vol). Lane 1 contains PA and phos- tion) on phospholipase D activity of intestinal mitochondria. Each value phatidylethanol standard. Lane 2 represents Ca2+-activated mitochon- represents mean ± SEM of three separate estimations. *P < 0.05 versus drial PA formation in the absence of ethanol. Lane 3 represents PA for- control (CON) incubated mitochondria. mation by Ca2+ activation in presence of 200 mM ethanol. Lipids, Vol. 32, no. 5 (1997) METAL ION STIMULATION OF PHOSPHOLIPASE D-LIKE ACTIVITY 473 FIG. 3. High-performance liquid chromatography separation of phosphatidic acid formed in mitochondria; (A) standard phosphatidic acid and phosphatidylethanol; (B) unincubated mito- chondria; (C) mitochondria incubated with 1 mM Ca2+; (D) mitochondria incubated with 1 mM Ca2+ + 200 mM ethanol. Total lipids were extracted. Phosphatidic acid separated by thin- layer chromatography was eluted and run on high-performance liquid chromatography as de- scribed in the text. Lipids, Vol. 32, no. 5 (1997) 474 M. MADESH AND K.A. BALASUBRAMANIAN TABLE 1 Composition of Isolated Phosphatidic Acida 2+ Co C30 +Ca Phosphate (nmole) 10.50 20.98 60.70 Fatty acids (nmole) 16:0 10.00 18.75 57.45 18:0 and 18:1 5.70 10.10 28.45 18:2 6.26 8.45 15.20 20:4 3.10 5.40 19.00 Total fatty acid 25.56 42.70 120.05 Ratio of phosphate to fatty acid 1:2.24 1:2.03 1:1.97 a Co: Control unincubated mitochondria; C30: mitochondria incubated for 30 min without any acti- vator. For calcium activation, mitochondria were incubated with 1 mM Ca2+ for 30 min. to a test tube, shaken on a vortex mixer for a minute, left for presence of Mg2+ + ethanol and 53.9 ± 1.9 in presence of Mg2+ 15 min and then centrifuged. The sediment was washed twice alone). Figure 2 shows the TLC separation of PA and phos- with 2 mL of 3% trichloroacetic acid and the supernatants phatidylethanol after Ca2+ activation of mitochondrial PLD- pooled together. Trichloroacetic acid from the supernatant like activity which showed the absence of phosphatidylethanol was removed by repeated extraction with diethyl ether, and formation in the presence of ethanol.