Platelet-activating factor (PAF)-dependent transacetylase and its relationship with PAF acetylhydrolases.
Platelet-activating factor (PAF)-dependent transacetylase (TA) is an enzyme that transfers an acetyl group from PAF to acceptor lipids such as lysophospholipids and sphingosine. This enzyme is distributed in membrane and cytosol of the cells. We previously revealed that TA purified from rat kidney membrane showed an amino acid sequence similarity to that of bovine PAF-acetylhydrolase (AH) (II). In the present study, we purified TA from the rat kidney cytosol and analyzed its amino acid sequence. The amino acid sequence of the cytosolic TA is similar to that of bovine PAF-AH (II) and membrane TA. To clarify the relationship between TA and PAF-AH (II), we isolated cDNA of rat PAF-AH (II). The predicted amino acid sequence of rat PAF-AH (II) from isolated cDNA included all the sequences found in TAs purified from the membrane and cytosolic TAs. In addition, monoclonal antibody to recombinant PAF-AH (II) cross-reacted with both cytosolic and membrane TAs. Consistent with sequence identity, recombinant PAF-AH (II) showed TA activity, whereas recombinant PAF-AH Ib, which is a different subtype of intracellular PAF-AHs, did not possess TA activity. Analysis of a series of site-directed mutant PAF-AH (II) proteins showed that TA activity was decreased, whereas PAF-AH activity was not affected in C120S and G2A mutant proteins. Thus, Cys(120) and Gly(2) are implicated in the catalysis of TA reaction in this enzyme. Furthermore, the transfer of acetate from PAF to endogenous acceptor lipids was significantly increased in a time-dependent manner in CHO-K1 cells transfected with PAF-AH (II) gene. These results demonstrate that PAF-AH (II) can function, as a TA in intact cells, and PAF-AH (II) and TA are the same enzyme.
Bae
K
K
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, the University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan.
Longobardi
L
L
Karasawa
K
K
Malone
B
B
Inoue
T
T
Aoki
J
J
Arai
H
H
Inoue
K
K
Lee
T
T
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000123:Acetyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000378:metabolism*; D051381:Rats
D018076:DNA, Complementary; D010972:Platelet Activating Factor; D000123:Acetyltransferases; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase