The 40 kDa 63Ni 2+-binding protein (pNiXc) on Western blots of Xenopus laevis oocytes and embryos is the monomer of fructose-1,6-bisphosphate aldolase A

Abstract

A Ni 2+-binding protein (pNiXc, 40 kDa), present in Xenopus laevis oocytes and embryos, was isolated from mature oocytes by chromatography on DEAE-cellulose and cellulose phosphate, followed by FPLC on Ni-iminodiacetate-Agarose, or reverse-phase HPLC on a C-4 column. Size-exclusion HPLC showed that intact pNiXc is ≈ 155 kDa, consistent with tetrametric structure, After cleavage with Lys-C proteinase or cyanogen bromide, six peptides were separated by HPLC and sequenced by Edman degradation, providing sequence data for 83 residues. Data-base search showed similarity of pNiXc to eukaryotic aldolases, with 96% identity to human aldolase A. pNiXc demonstrated aldolase activity with fructose 1,6-bisphosphate as substrate ( K m, 30 μM V max 26 μmol min −1mg −1); the aldolase activity was inhibited non-competitively by Cu 2+, Cd 2+, Co 2+, or Ni 2+. Equilibrium dialysis showed high affinity binding ( K D, 7μM) of 1 mole of Ni per mole of 40 kDa subunit. Based on metal-blot competition assays, the abilities of metals to compete with 63Ni 2+ for binding to pNiXc were ranked: Cu 2+ ⪢ Zn 2+ > Cd 2+ > Co 2+. This study identifies pNiXc as the monomer of fructose-1,6-bisphosphate aldolase A, and raises the possibility that aldolase A is a target enzyme for metal toxicity.