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

Abstract

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.

• Authors: M Hattori, H Adachi, J Aoki, M Tsujimoto, H Arai, K Inoue
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 270 Issue 52 Pg. 31345-52 (Dec 29 1995) ISSN: 0021-9258 [Print] United States
• PMID: 8537406 (Publication Type: Journal Article)

Chemical References

• DNA, Complementary
• Recombinant Proteins
• Isoflurophate
• Phospholipases A
• 1-Alkyl-2-acetylglycerophosphocholine Esterase

Topics

• 1-Alkyl-2-acetylglycerophosphocholine Esterase
• Amino Acid Sequence
• Animals
• Base Sequence
• Blotting, Western
• Brain (enzymology)
• Cattle
• Cloning, Molecular
• DNA, Complementary
• Escherichia coli (genetics)
• Humans
• Isoflurophate (chemistry)
• Molecular Sequence Data
• Phospholipases A (chemistry, genetics)
• Recombinant Proteins (chemistry, genetics)
• Sequence Homology, Amino Acid