Subcellular fractionation of bone marrow-derived macrophages: localization of phospholipase A1 and A2 and acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase.
Analysis of the subcellular distribution of lipid-metabolizing enzymes was carried out in bone marrow-derived macrophages with special respect to a comparison of the subcellular localisation of phospholipase A1 and A2 and to acyl-CoA:1-1-acylglycero-3-phosphorylcholine-0-acetyltransferase. After cell disruption differential centrifugation was followed by additional sucrose gradient purification of three main fractions. Satisfactory enrichment factors were obtained by this method for the following marker enzymes. The plasma-membrane enzyme alkaline phosphodiesterase I was enriched up to 25-fold and the acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase was enriched up to 30-fold. The marker enzyme for the endoplasmic reticulum, NADPH-cytochrome c reductase showed a similar enrichment and distribution as the acyltransferase. Therefore it was concluded that the acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase of bone marrow-derived macrophages is mainly located in the endoplasmic reticulum. Phospholipase A1 and A2 occurred in a high proportion together with the lysosomal marker enzyme N-acetyl-beta-glucosaminidase in the soluble supernatant and in the gradient fractions. In the endoplasmic reticulum phospholipase A2 occurred only in trace activities whereas phospholipase A1 was maximally enriched in this subcellular fraction. No subcellular fraction could be obtained where phospholipase A2 was enriched exclusively. However, it can be concluded that the two enzymes which are responsible for the balance of fatty acid liberation and re-acylation are located in two different cellular compartments. Furthermore it can be claimed that in the cell there has to exist an exchange of substrates and products between these compartments to achieve a complete metabolic cycle of the de- and re-acylation reaction of phospholipids in bone marrow-derived macrophages.
D000217:Acyltransferases / Q000032:analysis*; D000818:Animals; D001853:Bone Marrow / Q000201:enzymology; D002458:Cell Fractionation; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D005260:Female; D008264:Macrophages / Q000201:enzymology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010740:Phospholipases / Q000032:analysis*; D010741:Phospholipases A / Q000032:analysis*; D054466:Phospholipases A1; D054467:Phospholipases A2; D013347:Subcellular Fractions / Q000201:enzymology
D000217:Acyltransferases; C031076:2-acylglycerophosphocholine acyltransferase; D010740:Phospholipases; D010741:Phospholipases A; D054466:Phospholipases A1; D054467:Phospholipases A2