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Formation of bis(monoacylglycero)phosphate by a macrophage transacylase
1Department of Medicine (Neurology), University of Toronto, Ontario, Canada.
Abstract:
Formation of bis(monoacylglycero)phosphate (BMP) from lysophosphatidyl[U-14C]glycerol was studied in rabbit pulmonary alveolar macrophages. The majority of the activity was found in the particulate fraction (lysosome-enriched) sedimenting between 2000 and 12,000 rpm and it was maximal at pH 4.5. The activity in this fraction was stimulated by 2-mercaptoethanol and additional lipids from the fraction and inhibited by 5 mM CaCl2, 0.5 mM acyl-CoA, 1.0 mM chlorpromazine and by detergents, whereas chloroquine, cholesterol and butanol had no effect. The activity was retained by the particles after repeated freezing and thawing. After treatment with n-butanol, most of the activity was lost, but 84% could be recovered in the aqueous phase if the butanol-extracted lipids were added back giving an activity of 266 nmol/h per mg of protein. Lipids most effective in restoring activity were the total lipids extracted by butanol from the particulate fraction, fractions of the total lipids containing phospholipids and phosphatidylcholine from both native and commercial sources, with native BMP and commercial phosphatidylglycerol and sphingomyelin having a much smaller effect. The complexity of the lipid requirements was further indicated by the finding that addition of pure lipids to the total lipid extract reduced the efficacy of the latter. A direct transfer of [14C]oleic acid to BMP from labelled macrophage microsomal lipids was catalyzed by the soluble enzymes as was transfer from dioleoylphosphatidylcholine in the presence of lysophosphatidylglycerol. The particulate enzyme also catalyzed the transfer of [14C]oleic acid from 2-oleoylphosphatidylcholine to BMP in the presence of lysophosphatidylglycerol. These findings indicate that the transacylase involved in conversion of lysophosphatidylglycerol to BMP utilizes complex lipids other than phosphatidylinositol as acyl donors and has complex requirements for lipids as physicochemical activators. They further suggest that the transacylation might be catalyzed by lysosomal phospholipase A2.
Insights
Bis(monoacylglycero)phosphate (BMP) formation from lysophosphatidylglycerol in macrophages requires complex lipid activators and is catalyzed by a particulate enzyme, likely lysosomal phospholipase A2.
Area of Science:
- Biochemistry
- Cell Biology
- Lipid Metabolism
Background:
- Bis(monoacylglycero)phosphate (BMP) is a unique phospholipid found in lysosomes.
- Its synthesis pathway and enzymatic machinery remain incompletely understood.
Purpose of the Study:
- To investigate the formation of BMP from lysophosphatidylglycerol in rabbit pulmonary alveolar macrophages.
- To characterize the enzymatic activity and lipid requirements involved in BMP synthesis.
Main Methods:
- Subcellular fractionation of macrophages to isolate particulate and soluble enzyme fractions.
- Enzymatic assays using radiolabeled lysophosphatidylglycerol and oleic acid.
- Lipid extraction and supplementation experiments to determine cofactor requirements.
Main Results:
- BMP formation was localized to a lysosome-enriched particulate fraction, optimal at pH 4.5.
- The enzyme activity was stimulated by 2-mercaptoethanol and specific lipids, but inhibited by CaCl2 and acyl-CoA.
- Complex lipids, particularly phospholipids like phosphatidylcholine, were essential for enzyme activity, acting as activators rather than direct substrates.
- Evidence suggested a transacylase mechanism involving lysosomal phospholipase A2.
Conclusions:
- The synthesis of BMP from lysophosphatidylglycerol involves a transacylase with complex lipid requirements for activation.
- Lysosomal phospholipase A2 is a potential candidate enzyme catalyzing this transacylation reaction.
- Understanding BMP synthesis provides insights into lysosomal lipid metabolism and function.