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.
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 approximately 155 kDa, consistent with tetrameric 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 (Km, 30 microM Vmax 26 mumol min-1 mg-1); the aldolase activity was inhibited non-competitively by Cu2+, Cd2+, Co2+, or Ni2+. Equilibrium dialysis showed high affinity binding (Kd, 7 microM) of 1 mole of Ni per mole of 40 kDa subunit. Based on metal-blot competition assays, the abilities of metals to compete with 63Ni2+ for binding to pNiXc were ranked: Cu2+ >> Zn2+ > Cd2+ > Co2+. 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.
Antonijczuk
K
K
Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington 06030.
Kroftova
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OS
Varghese
A H
AH
Antonijczuk
A
A
Henjum
D C
DC
Korza
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G
Ozols
J
J
Sunderman
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D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry*; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry*; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry*; D009865:Oocytes / Q000201:enzymology; D011868:Radioisotopes; D015843:Serpins; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000196:embryology / Q000378:metabolism*
D002352:Carrier Proteins; D011868:Radioisotopes; D015843:Serpins; D029867:Xenopus Proteins; C077804:SERPINA6 protein, Xenopus; D009532:Nickel; D005634:Fructose-Bisphosphate Aldolase