Cloning and expression of a cDNA encoding the beta-subunit (30-kDa subunit) of bovine brain platelet-activating factor acetylhydrolase
Cloning and expression of a cDNA encoding the beta-subunit (30-kDa subunit) of bovine brain platelet-activating factor acetylhydrolase
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DOI：
10.1074/jbc.270.52.31345
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发表时间：
1995-12-29
影响因子：
4.8
通讯作者：
Inoue, K
中科院分区：
文献类型：
--
作者：
Hattori, M;Adachi, H;Inoue, K
牛脑血小板活化因子（PAF）乙酰水解酶同工型Ib是一种异源三聚体酶，其γ亚基（以前我们称之为29-kDa亚基）作为催化亚基，而α-亚基（45 kDa）是人LIS-1产物的牛同源物，人LIS-1是Miller-Dieker无脑畸形的致病基因，在本研究中，我们克隆了牛脑PAF乙酰水解酶Ib β亚基（30 kDa）的cDNA。预测的229个氨基酸序列是同源的（63.2%的同一性），尤其是（86%的同源性），在大肠杆菌中产生的重组β蛋白显示出显著的PAF乙酰水解酶活性，是一种突变蛋白，其中Ser（48），对应于γ亚基的活性丝氨酸残基，虽然β-和γ-亚基在天然酶中形成异源复合物，但重组β-和γ-蛋白都以同源二聚体存在。纯化的重组β-蛋白易于用[1，3-H-3]二异丙基氟磷酸标记，而天然复合物中的β亚基仅被较高浓度的[1，3（3）H]二异丙基氟磷酸标记，标记程度低于γ亚基。结合我们以前的研究结果，本研究证明牛脑PAF乙酰水解酶Ib是一种独特的酶，具有两个催化亚基和另一个可能的调节亚基。
Bovine brain platelet-activating factor (PAF) acetyl hydrolase 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 Ser(48), which corresponds to the active serine residue of the gamma-subunit, was replaced with cysteine showed no enzymatic activity, suggesting Ser(48) 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-3]diisopropyl fluorophosphate, whereas the beta-subunit in the native complex was only labeled with higher concentrations of [1,3(3)H]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.