Abstract
We have previously identified two enzyme activities that transfer the acetyl group from platelet-activating factor (PAF) in a CoA-independent manner to lysoplasmalogen or sphingosine in HL-60 cells, endothelial cells, and a variety of rat tissues. These were termed as PAF:lysoplasmalogen (lysophospholipid) transacetylase and PAF:sphingosine transacetylase, respectively. In the present study, we have solubilized and purified this PAF-dependent transacetylase 13,700-fold from rat kidney membranes (mitochondrial plus microsomal membranes) based on the PAF:lysoplasmalogen transacetylase activity. The mitochondria and microsomes were prepared and washed three times, then solubilized with 0.04% Tween 20 at a detergent/protein (w/w) ratio of 0.1. The solubilized fractions from mitochondria and microsomes were combined and subjected to sequential column chromatographies on DEAE-Sepharose, hydroxyapatite, phenyl-Sepharose, and chromatofocusing. The enzyme was further purified by native-polyacrylamide gel electrophoresis (PAGE) and affinity gel matrix in which the competitive inhibitor of the enzyme, 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphoethanolamine was covalently attached to the CH-Sepharose. On SDS-PAGE, the purified enzyme showed a single homogeneous band with an apparent molecular mass of 40 kDa. The purified enzyme catalyzed transacetylation of the acetyl group not only from PAF to lysoplasmalogen forming plasmalogen analogs of PAF, but also to sphingosine producing N-acetylsphingosine (C2-ceramide). In addition, this enzyme acted as a PAF-acetylhydrolase in the absence of lipid acceptor molecules. These results suggest that PAF-dependent transacetylase is an enzyme that modifies the cellular functions of PAF through generation of other diverse lipid mediators.
Highlights
Ero-3-phosphocholine, is a potent lipid mediator with a wide variety of biological activities related to physiological and pathological phenomena [1, 2]
Materials—l-O-Hexadecyl-2-acetyl-GPC was obtained from Sigma. 1-O-Hexadecyl-2-N-methylcarbamyl-GPC and C2-ceramide were purchased from Biomol. 1-O-Alkyl-2-acetyl-GPC was from Avanti
We found that the platelet-activating factor (PAF)-dependent transacetylase contains PAF hydrolyzing activities in crude membrane fractions and purified preparations; the ratios of lysoplasmalogen transacetylase/acetylhydrolase were 0.74, 0.84, and 1.04 in the mitochondria, microsomes, and purified enzyme, respectively
Summary
Materials—l-O-Hexadecyl-2-acetyl-GPC was obtained from Sigma. 1-O-Hexadecyl-2-N-methylcarbamyl-GPC and C2-ceramide were purchased from Biomol. 1-O-Alkyl-2-acetyl-GPC was from Avanti. Active fractions of DEAE-Sepharose were applied onto a column of hydroxyapatite (1.5 ϫ 8.49 cm, 15 ml), which was equilibrated with 20 mM Tris-HCl (pH 7.4), 1 mM DTT, 1 g/ml leupeptin, and 0.02% Tween 20. The column was washed with the same solution, and the enzyme activity was eluted with 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM DTT, 1 g/ml leupeptin and 0.02% Tween 20. Purification of the Enzyme by Affinity Gel Matrix—The active fractions of native-PAGE were combined with 50 l of affinity gel matrix in a microcentrifuge tube, which was equilibrated with 20 mM Tris-HCl (pH 7.4) containing 0.1 M NaCl, 1 mM DTT, and 0.02% Tween 20 and mixed gently at 4 °C for 30 min. The amount of protein in the final enzyme preparation (Table III, step 8) was estimated by comparing the intensity of the protein band with those of standard proteins after SDS-PAGE and silver staining
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