THE 40-KDA NI-63(2-BINDING PROTEIN (PNIXC) ON WESTERN BLOTS OF XENOPUS-LAEVIS OOCYTES AND EMBRYOS IS THE MONOMER OF FRUCTOSE-1,6-BISPHOSPHATE ALDOLASE-A())

A Ni2+-binding protein (pNiXc, 40 kDa) present in Xenopus laevis oocyt es and embryos, was isolated from mature oocytes by chromatography on DEAE-cellulose and cellulose phosphate, followed by FPLC on Ni-iminodi acetate-Agarose, or reverse-phase HPLC on a C-4 column. size-exclusion HPLC showed that intact pNiXc is approximate to 155 kDa, consistent w ith tetrameric structure. After cleavage with Lys-C proteinase or cyan ogen bromide, sir peptides were separated by HPLC and sequenced by Edm an degradation, providing sequence data for 83 residues. Data-base sea rch showed similarity of pNiXc to eukaryotic aldolases, with 96% ident ity to human aldolase A. pNiXc demonstrated aldolase activity with fru ctose 1,6-bisphosphate as substrate (K-m, 30 mu M V-max 26 mu mol min( -1)mg(-1)); the aldolase activity was inhibited non-competitively by C u2+, Cd2+, Co2+, or Ni2+. Equilibrium dialysis showed high affinity bi nding (K-D, 7 mu M) of 1 mole of Ni per mole of 40 kDa subunit. Based on metal-blot competition assays, the abilities of metals to compete w ith Ni-63(2+) for binding to pNiXc were ranked: Cu2+ >> Zn2+ > Cd2+ > Co2+. This Study identifies pNiXc as the monomer of fructose-1,6-bisph osphate aldolase A, and raises the possibility that aldolase A is a ta rget enzyme for metal toxicity.