Cloning and expression of a cDNA encoding the β-subunit (30-kDa subunit) of bovine brain platelet-activating factor acetylhydrolase

Mitsuharu Hattori, Hideki Adachi, Junken Aoki, Masafumi Tsujimoto, Hiroyuki Arai, Keizo Inoue

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83 Citations (Scopus)

Abstract

Bovine brain platelet-activating factor (PAF) acetylhydrolase isoform Ib is a heterotrimeric enzyme. Its γ-subunit (which, formerly, we called the 29-kDa subunit) acts as a catalytic subunit, whereas the α-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 β-subunit (30 kDa) of bovine brain PAF acetylhydrolase Ib. The predicted 229-amino acid sequence was homologous (63.2% identity) to that of the γ-subunit, especially (86% identity) in the catalytic and PAF receptor homologous domains. The recombinant β-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 γ-subunit, was replaced with cysteine showed no enzymatic activity, suggesting Ser48 is the active serine residue. Although the β- and γ-subunits form a heterocomplex in the native enzyme, both recombinant β- and γ-proteins exist as a homodimer. The purified recombinant β- protein was labeled readily with [1,3-3H]diisopropyl fluorophosphate, whereas the β-subunit in the native complex was only labeled with higher concentrations of [1,33H]diisopropyl fluorophosphate to a lesser extent than the γ-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.

Original languageEnglish
Pages (from-to)31345-31352
Number of pages8
JournalJournal of Biological Chemistry
Volume270
Issue number52
DOIs
Publication statusPublished - 1995 Dec 29

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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