Xenopus lipovitellin 1 is a Zn(2+)- and Cd(2+)-binding protein.

This report discusses the identification of a Zn(2+)- and Cd(2+)-binding protein of Xenopus laevis that is abundant in vitellogenic oocytes and in embryos from fertilization to stage 46. Oocyte or embryo homogenates were fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with 65Zn2+ or 109Cd2+. The resulting autoradiograms showed binding of both radionuclides to a protein, designated pCdZn. Freon extraction of oocyte and embryo homogenates showed pCdZn to be a yolk protein. When pCdZn was isolated from oocyte homogenates by ammonium sulfate precipitation, delipidation, and chromatography, it co-purified with lipovitellin 1. The amino acid composition of pCdZn closely resembled the reported composition of lipovitellin 1 and the molecular weight of purified pCdZn (approximately 115 kD) corresponded to reported values for lipovitellin 1 (111-121 kD). Amino acid sequence analyses of five peptides derived from pCdZn yielded 94% identity to the reported sequence of lipovitellin 1, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Based on these findings, pCdZn was identified as lipovitellin 1. This study suggests that lipovitellin 1 is the major storage protein for zinc in mature oocytes and developing embryos of Xenopus laevis.

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.

Lipovitellin 2 beta is the 31 kD Ni(2+)-binding protein (pNiXb) in Xenopus oocytes and embryos.

An Ni(2+)-binding protein (pNiXb, 31 kD) present in mature Xenopus laevis oocytes and in embryos from fertilization in N/F stage 42, was isolated and characterized. After oocytes or embryos were fractionated by PAGE, electroblotted onto nitrocellulose, and probed with 63Ni2+, pNiXb was detected by autoradiography. pNiXb, a yolk protein located in the embryonic gut, was purified from yolk platelets by ammonium sulfate precipitation, delipidation, gel filtration chromatography, and HPLC analysis. During these steps, pNiXb copurified with lipovitellin 2. The N-terminal sequence of purified pNiXb exactly matched that of Xenopus lipovitellin 2 beta, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Since pNiXb and lipovitellin 2 beta agree in N-terminal sequence, amino acid composition, and apparent molecular weight, they appear to be identical. Based on a metal-blot competition assay, the abilities of metal ions to compete with 63Ni2+ for binding to pNiXb were ranked: Zn2+ approximately Cu2+ approximately Co2+ > Cd2+ approximately Mn2+ > Sn2+. This study shows that Xenopus lipovitellin 2 beta is a metal-binding protein in vitro, and raises the possibility that it may function similarly in vivo.