Inhibition mechanisms of .DELTA.-aminolevulinic acid dehydratase by heavy metals. I. Application of immobilized enzyme.

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  • 重金属によるδ‐Ａｍｉｎｏｌｅｖｕｌｉｎｉｃ　Ａｃｉｄ　Ｄｅｈｙｄｒａｔａｓｅの阻害機構の解明　Ｉ　　固定化酵素の利用
  • ジュウキンゾク ニ ヨル デルタ Aminolevulinic Acid
  • I. Applicatian of immobilized enzyme
  • I.固定化酵素の利用

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By using an immobilized enzyme, restoration mechanisms were investigated for δ-aminolevulinic acid dehydratase (ALAD; EC 4. 2. 1. 24) activity when it was inhibited by various heavy metals. The immobilized ALAD was prepared by binding glass beads (O. D. 7mm) with the enzyme highly purified from human erythrocytes, using a glutaraldehyde reagent. This immobilized ALAD was inhibited by incubation with various heavy metals until the enzyme was almost inactive, and then restored by the addition of dithiothreitol (DTT), DTT+Zn2+ and EDTA or heating (60°C, 5min) in this order. ALAD inhibited by Zn2+, Mn2+ or Pb2+ was reactivated easily in over 90% of the cases if these metals were removed from the reaction mixture following the addition of DTT or DTT+Zn2+. Hg2+ and SeO32+ showed irreversible inhibition, reactivating only up to 50%, even if DTT+Zn was added. The levels of restoration activity inhibited by Cd2+, Sn2+ and Sn4+ were different due to the kinds of buffer solutions used. Other reactivations were attempted with heating or by adding EDTA for the enzyme restored by DTT+Zn2+, but further restorations were not observed. For the immobilized enzyme, it was feared that its original properties might be lost, but the Km, the Vmax and the optimum pH of the immobilized ALAD were 0.27mM, 22.5units/mg and pH 6.4-6.8, respectively. These values almost agree with the values of previous reports. Immobilization of the enzyme makes it possible to readily separate the enzyme from the reaction mixture without the use of gel filtration or other complicated procedures. For this reason, the use of immobilized enzyme seems to be a good method for the analysis of enzyme properties.

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