Platelet-activating factor (PAF) acetylhydrolase, which inactivates PAF by removing the acetyl group at the sn-2 position, is distributed widely in plasma and tissues. In a previous study, we demonstrated that the PAF acetylhydrolase activity present in the soluble fraction of bovine brain cortex could be separated chromatographically into three peaks (tentatively designated isoforms Ia, Ib, and II) (Hattori, M., Arai, H., and Inoue, K. (1993) J. Biol. Chem. 268, 18748 -18753). In this study, these three isoforms were also detected in kidney and liver cytosols, although their relative activity ratios in these tissues differed. In particular, isoform II was responsible for the majority of the bovine liver PAF acetylhydrolase activity. We purified isoform II from bovine liver cytosol to near homogeneity and demonstrated that it is a single 40-kDa polypeptide. This enzyme was inactivated by diisopropyl fluorophosphate and 5,5-dithiobis(2-nitrobenzoic acid), suggesting that both serine and cysteine residues are required for the enzyme activity, and [ 3 H]diisopropyl fluorophosphate labeled only the 40-kDa polypeptide, confirming the enzyme's identity. Isoform II showed a comparatively broader substrate specificity than isoform Ib. Isoform II hydrolyzed propionyl and butyroyl moieties at the sn-2 position approximately half as effectively as it did PAF, whereas isoform Ib hardly hydrolyzed these substrates.Taken together with previous data, the current findings indicate that tissue cytosol contains at least two types of PAF acetylhydrolase with respect to polypeptide composition, substrate specificity, and tissue distribution and suggest that these two enzymes may share distinct physiological functions in tissues.

Platelet-activating factor-acetylhydrolase (PAF-AH), which removes the acetyl group at the sn-2 position of PAF, is distributed widely in tissues and plasma. Tissue cytosol contains at least two types of PAF-AH, isoforms Ib and II. Isoform Ib is a tertiary G-protein complex-like heterotrimeric enzyme that is involved in brain development such as formation of the brain cortex. Isoform II (PAF-AH(II)), however, is a 40-kDa monomer and has an amino acid sequence that exhibits a 41% identity with that of plasma PAF-AH. Although PAF-AH(II) preferentialy hydrolyzes oxidized phospholipids as well as PAF in vitro, the function of this enzyme has not, as yet, been elucidated. Here, we report that PAF-AH(II) functions as an anti-oxidant phospholipase. PAF-AH(II) was found to be an N-myristoylated enzyme that has never been reported among lipases and phospholipases. In MDBK cells treated with oxidants, PAF-AH(II) translocated from cytosol to membranes within 20 min, whereas in cells treated with anti-oxidants, it translocated, conversely, from membranes to cytosol. Overexpression of PAF-AH(II) in Chinese hamster ovary-K1 cells suppressed oxidative stress-induced cell death, which occurs by apoptosis. These findings suggest that intracellular PAF-AH(II) translocates between cytosol and membranes in response to a redox state of the cell and protects the cell against oxidative stress most probably by hydrolyzing oxidized phospholipids.Platelet-activating factor (PAF) 1 (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid involved in diverse physiological events such as inflammation, anaphylaxis, and the reproductive system (1, 2). PAF is inactivated by a specific enzyme, PAF-acetylhydrolase (PAF-AH), that removes the acetyl moiety at the sn-2 position of the glycerol backbone (3, 4).Mammalian PAF-AH is classified into two types (5, 6), plasma (extracellular) and tissue (intracellular). Plasma PAF-AH is a 43-kDa monomeric enzyme that effectively abolishes the inflammatory effects of PAF on leukocytes and the vasculature, indicating involvement of the enzyme in the maintenance of plasma PAF at certain levels (7). Recently, we succeeded in purifying and cloning of intracellular PAF-AHs. Tissue cytosol contains at least two types of intracellular PAF-AH, isoforms Ib and II (8). Isoform Ib is a tertiary G-protein complex-like heterotrimeric enzyme (9) that is involved in brain development since the 45-kDa-subunit was identical to the product of the causative gene for Miller-Dieker lissencephaly, a malformation of the brain cortex (10). On the other hand, isoform II (PAF-AH(II)) is a 40-kDa monomer and has an amino acid sequence that exhibits a 41% identity with that of plasma PAF-AH (7,11,12). It is expressed most abundantly in liver and kidney in bovine. Furthermore, PAF-AH(II), as well as plasma PAF-AH, has the ability to hydrolyze short chain phospholipids and oxidized fragments of polyunsaturated fatty acids at the sn-2 position as well as PAF while isoform Ib behaves as a specific acetylhydro...

No abstract

No abstract

Platelet-activating factor (PAF) acetylhydrolase, which inactivates PAF by removing the acetyl group at the sn-2 position, is widely distributed in plasma and tissues. We previously demonstrated that tissue cytosol contains at least two types of PAF acetylhydrolase, isoforms Ib and II, and that isoform Ib is a heterotrimer comprising 45-, 30-, and 29-kDa subunits, whereas isoform II is a 40-kDa monomer.In this study, we isolated cDNA clones of bovine and human PAF acetylhydrolase isoform II. From the longest open reading frame of the cloned cDNAs, both bovine and human PAF acetylhydrolases II are predicted to contain 392 amino acid residues and to exhibit 88% identity with each other at the amino acid level. Both enzymes contain a Gly-X-Ser-X-Gly motif that is characteristic of lipases and serine esterases. Expression of isoform II cDNA in COS7 cells resulted in a marked increase in PAF acetylhydrolase activity. An immunoblot study using an established monoclonal antibody against the bovine enzyme revealed that the recombinant protein exists in the membranous fraction as well as the soluble fraction. Isoform II is expressed most abundantly in the liver and kidney in cattle, but low levels were also observed in other tissues. The amino acid sequence deduced from the cDNA of isoform II had no homology with any subunit of isoform Ib. Interestingly, however, the amino acid sequence of isoform II showed 41% identity with that of plasma PAF acetylhydrolase. Combined with previous data demonstrating that isoform II shows similar substrate specificity to plasma PAF acetylhydrolase, these results indicate that tissue type isoform II and the plasma enzyme may share a common physiologic function.