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Platelet-activating factor (PAF)-dependent transacetylase and its relationship with PAF acetylhydrolases
K Bae1, L Longobardi, K Karasawa
1Department of Health Chemistry, Faculty of Pharmaceutical Sciences, the University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan.
The Journal of Biological Chemistry
|June 27, 2000
Summary
Platelet-activating factor (PAF)-dependent transacetylase (TA) is the same enzyme as PAF-acetylhydrolase (AH) II. This enzyme transfers acetyl groups and is found in both cell membranes and cytosol, with specific amino acids crucial for its TA function.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Platelet-activating factor (PAF)-dependent transacetylase (TA) transfers acetyl groups from PAF to acceptor lipids.
- TA is found in both membrane and cytosol fractions of cells.
- Previous studies indicated sequence similarity between rat kidney membrane TA and bovine PAF-acetylhydrolase (AH) II.
Purpose of the Study:
- To purify and analyze the amino acid sequence of rat kidney cytosolic TA.
- To clarify the relationship between TA and PAF-AH (II).
- To investigate the enzymatic activity and cellular function of PAF-AH (II).
Main Methods:
- Purification and amino acid sequencing of rat kidney cytosolic TA.
- Isolation of rat PAF-AH (II) cDNA.
- Expression of recombinant PAF-AH (II) and PAF-AH Ib.
- Site-directed mutagenesis of PAF-AH (II).
- Enzyme activity assays (TA and PAF-AH).
- Transfection of CHO-K1 cells with PAF-AH (II) gene.
Main Results:
- Cytosolic TA shares amino acid sequence similarity with bovine PAF-AH (II) and membrane TA.
- Rat PAF-AH (II) cDNA encodes a protein whose sequence encompasses known TA sequences.
- Monoclonal antibodies against recombinant PAF-AH (II) cross-react with both cytosolic and membrane TAs.
- Recombinant PAF-AH (II) exhibits TA activity, while PAF-AH Ib does not.
- Specific mutations (C120S, G2A) in PAF-AH (II) reduce TA activity but not PAF-AH activity.
- Transfected cells show increased acetate transfer from PAF, indicating cellular TA activity.
Conclusions:
- PAF-AH (II) possesses transacetylase (TA) activity.
- Specific amino acid residues (Cys120, Gly2) are critical for the TA catalytic function of PAF-AH (II).
- PAF-AH (II) functions as a TA in intact cells.
- PAF-AH (II) and TA are the same enzyme.
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