Cloning and expression of a cDNA encoding the beta-subunit (30-kDa subunit) of bovine brain platelet-activating factor acetylhydrolase.
Bovine brain platelet-activating factor (PAF) acetylhydrolase isoform Ib is a heterotrimeric enzyme. Its gamma-subunit (which, formerly, we called the 29-kDa subunit) acts as a catalytic subunit, whereas the alpha-subunit (45 kDa) is the bovine homolog of the product of human LIS-1, the causative gene of Miller-Dieker lissencephaly, indicating that this intracellular PAF acetylhydrolase plays a key role in brain development. In the current study, we cloned the cDNA for the beta-subunit (30 kDa) of bovine brain PAF acetylhydrolase Ib. The predicted 229-amino acid sequence was homologous (63.2% identity) to that of the gamma-subunit, especially (86% identity) in the catalytic and PAF receptor homologous domains. The recombinant beta-protein produced in Escherichia coli showed significant PAF acetylhydrolase activity. A mutant protein, in which Ser48, which corresponds to the active serine residue of the gamma-subunit, was replaced with cysteine showed no enzymatic activity, suggesting Ser48 is the active serine residue. Although the beta- and gamma-subunits form a heterocomplex in the native enzyme, both recombinant beta- and gamma-proteins exist as a homodimer. The purified recombinant beta-protein was labeled readily with [1,3-H]diisopropyl fluorophosphate, whereas the beta-subunit in the native complex was only labeled with higher concentrations of [1,3-3H]diisopropyl fluorophosphate to a lesser extent than the gamma-subunit. Combined with our previous data, the present study demonstrated that bovine brain PAF acetylhydrolase Ib is a unique enzyme possessing two catalytic subunits and another, possibly regulatory, subunit.
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
M
M
Hattori
H
H
Adachi
J
J
Aoki
M
M
Tsujimoto
H
H
Arai
K
K
Inoue
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007531:Isoflurophate / Q000737:chemistry; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011994:Recombinant Proteins; D007531:Isoflurophate; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase