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The H-Ras-like suppressor (HRASLS) is a protein family consisting of five members in humans. Despite their discovery as tumor suppressors, we demonstrated that all these proteins are phospholipid-metabolizing enzymes, such as phospholipase (PL) A1 /A2 and acyltransferase. We thus proposed to rename HRASLS1-5 as PLA/acyltransferase (PLAAT)-1-5. Notably, PLAATs exhibit N-acyltransferase activity to biosynthesize N-acylated ethanolamine phospholipids, including N-acyl-plasmalogen, which serve as precursors of bioactive N-acylethanolamines. Furthermore, the overexpression of PLAAT-3 in animal cells causes disappearance of peroxisomes and a remarkable reduction in plasmalogen levels. This finding might be related to the inhibitory effect of PLAAT-3 on the chaperone activity of the peroxin PEX19. In this article, we will review our recent findings about PLAAT proteins, with special reference to their roles in peroxisome biogenesis and plasmalogen metabolism.

Azithromycin accumulates in lysosomes where it causes phospholipidosis. In homogenates prepared by sonication of fibroblasts incubated for 3 days with azithromycin (66 μM), the activities of sulfatase A, phospholipase A1, N-acetyl-β-hexosaminidase and cathepsin B increased from 180 to 330%, but not those of 3 non-lysosomal enzymes. The level of cathepsin B mRNA was unaffected. The hyperactivity induced by azithromycin is non-reversible upon drug withdrawal, prevented by coincubation with cycloheximide, affects the Vmax but not the Km, and is not reproduced with gentamicin, another drug also causing lysosomal phospholipidosis. The data therefore suggest that azithromycin increases the level of lysosomal enzymes by a mechanism distinct from the stimulation of gene expression but requiring protein synthesis, and is not in direct relation to the lysosomal phospholipidosis.

Low density lipoproteins (LDL) as well as isolated apolipoprotein B (ApoB) have been shown to exhibit phospholipase A2 (PLA2) activity toward phospholipids containing an oxidized or short fatty acyl chain at position 2. Some of these studies employed the fluorescent analogue of phosphatidyl choline (PC), C6-NBD-PC, containing NBD-caproic acid (C6-NBD-FA) at position 2 as a substrate, representative of short fatty acyl chains. The release of NBD-caproic acid from position 2 is attributed to PLA2-catalysed hydrolysis. However, this fatty acid can be released also by other enzymatic pathways. In the present study we examined, and ruled out, other enzymatic pathways which may be responsible for the hydrolysis of fatty acids from position 2 of phospholipids. On the other hand, we found that LDL as well as isolated ApoB hydrolyse C6-NBD-FA from both carbon 1 and carbon 2 of these phospholipids, thus exhibiting independent and simultaneous activities of phospholipase A1 and phospholipase A2.

Mammals have three members of the intracellular phospholipase A 1 protein family (phosphatidic acid preferring-phospholipase A 1 , p125, and KIAA0725p). In this study, we showed that KIAA0725p is localized in the Golgi, and is rapidly cycled between the Golgi and cytosol. Catalytic activity is important for targeting of KIAA0725p to Golgi membranes. RNA interference experiments suggested that KIAA0725p contributes to efficient membrane trafficking from the Golgi apparatus to the plasma membrane, but is not involved in brefeldin A-induced Golgi-to-endoplasmic reticulum retrograde transport. Structured summary MINT- 8019765 : KIAA0725 (uniprotkb: O94830 ) and Beta-COP (uniprotkb: P53618 ) colocalize (MI: 0403 ) by fluorescence microscopy (MI: 0416 ) MINT- 8019775 : KIAA0725 (uniprotkb: O94830 ) and GM130 (uniprotkb: Q5PXD5 ) colocalize (MI: 0403 ) by fluorescence microscopy (MI: 0416 )

In Arabidopsis, there are at least seven class I acylhydrolase members, which have a putative N-terminal chloroplast-targeting signal. Here, we show that all seven class I proteins are localized to the chloroplasts and hydrolyze phosphatidylcholine at the sn-1 position. However, based on their activities toward various lipids, Arabidopsis class I enzymes could be further divided into three sub-groups by substrate specificity, one with phospholipase-specific activity, another with phospholipase and galactolipase activities, and the other with broad lipolytic activity toward phosphatidylcholine, galactolipids, and triacylglycerol. These results suggest that the three sub-groups of class I acylhydrolases have specific roles in chloroplasts.

Outer membrane phospholipase A (OMPLA) activity is regulated by reversible dimerisation with the dimer being the active species. Observed lag phases in activity indicated that dimerisation may be slow relative to turnover. A covalent OMPLA dimer indeed did not display lag phase behaviour. A model for OMPLA kinetics was proposed accounting for a slow dimerisation step. Preincubation conditions determined the initial amount of monomer and influenced both lag times and final activities. Under the conditions used, substrate concentrations higher than 50 mol% inhibited OMPLA activity and increased lag times. Our results may shed more light on mechanisms controlling OMPLA activity in vivo.

Phosphatidylcholine with docosahexaenoic acid at both sn-1 and sn-2 positions occurs in relatively high abundance in bonito muscle. To explore a possible route for the dipolyunsaturated molecular species, phosphatidylcholine formation from 2-[1-14C]linoleoyl lysophosphatidylcholine was examined using a cytosolic fraction from bonito muscle. The formation of radiolabeled phosphatidylcholine was greatest at 15 degrees C and did not require the presence of cofactors such as CoA and calcium. By DEAE-cellulofine column chromatography, the activity to form phosphatidylcholine was separated from that of phospholipase A1, and the specific activity increased by about 100-fold. The possible involvement of cytosolic lysophosphatidylcholine/transacylase in synthesis of dipolyunsaturated phosphatidylcholine is discussed.

We have previously shown that unsaturated phosphatidylserines inhibit mitogen-induced T cell activation. We now report that the inhibitory action requires a protein present in bovine and human serum. Partial purification and phospholipase assay show that this protein has phospholipase A activity on phosphatidylserine but not phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol. In short incubations (1–3 h) 2-acyl lysophosphatidylserine is produced but in longer incubations the cis-unsaturated fatty acid is also released. Experiments on peripheral blood mononuclear cells indicate that the unsaturated fatty acid becomes the main responsible for the PS-induced inhibition and that 2-acyl lysophosphatidylserine enhances the inhibitory effect of fatty acid.

A member of the annexin family (the heterotetrameric annexin II2pl 12 complex purified from porcine intestinal epithelium) was tested for its ability to affect different calcium-dependent intrinsic lipolytic activities of rat liver hepatic lipase (HL). Whereas annexin II in the presence of calcium failed to interfere with HL triacyl glycerol lipase (EC 3.1.1.3) activity, it inhibited HL phospholipase A1 (EC 3.1.1.32) and lysophospholipase (EC 3.1.1.5) activities. Inhibition could be overcome by increasing the substrate concentration. Under phospholipase A1 assay conditions, annexin II did not bind to the purified HL enzyme. These results therefore suggest that only inhibitor/substrate interactions lead to inhibition of HL phospholipase A1 and lysophospholipase activities, an obviously general mechanism of phospholipase inhibition by annexins. Possible implications of HL inhibition in vivo by annexins are discussed.

Cultured rat hepatocytes exhibit acid phospholipase A activity. On the basis of product formation from stereospecifically radiolabeled phosphatidylethanolamine substrates, phospholipases A1 and A2 have been identified with optimal activities at pH 4.5. According to subcellular fractionation studies, the acid phospholipases in hepatocytes appear to be located in the lysosomal compartment. Application of specific inhibitors of the biosynthesis, glycosylation, and translocation of lysosomal enzymes in hepatocyte cultures suggests a half-life of approx. 1 day for the acid lysosomal phospholipase A1. About the same value for the half-life was obtained for the lysosomal marker enzymes, acid phosphatase and β-N-acetyl-D-hexosaminidase.