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Detection of two Zn-finger proteins ofXenopus laevis, TFIIIA, and p43, by probing western blots of ovary cytosol with65Zn2+,63Ni2+, or109Cd2+

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Abstract

Tow Zn-finger proteins, TFIIIA (a constituent of 7S RNP particles) and p43 (a constituent of 42S RNP particles), were detected in ovary extracts of juvenileXenopus laevis females by in vitro binding of radiolabeled divalent metals. Proteins fractionated by SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) were transferred by Western blotting onto nitrocellulose membranes, probed with65Zn2+,63Ni2+, or109Cd2+, and visualized by autoradiography. Detection limits for TFIIIA were approx 0.07 μg/well by109Cd2+-probing, 0.13 μg/well by65Zn2+-probing, and 0.26 μ/well by63Ni2+-probing. Protein p43 was more clearly visualized by probing with63Ni2+ than with65Zn2+ or109Cd2+. After purified TFIIIA was cleaved with cyanogen bromide,65Zn2+,109Cd2+, and63Ni2+ distinctly labeled the 22 kDa middle fragment;65Zn2+ and109Cd2+ also labeled the 11 kDa N-terminal fragment, but didnot label the 13 kDa C-terminal fragment. These results are consistent with the notion that the radioligands were bound to finger-loop domains of TFIIIA, which occur in the middle and N-terminal fragments. Based on the abilities of nonradioactive metal ions to compete with65Zn2+ for binding to TFIIIA on Western blots, the relative affinities of the metals for TFIIIA were ranked as follows: Zn2+=Cu2+≥Hg2+>Cd2+>Co2+≥Ni2+. Even at a 1000-fold molar excess, Mn2+ did not compete with65Zn2+ for binding to TFIIIA. Probing Western blots with the radiolabeled metal ions greatly facilitates the detection, isolation, and quantitation of TFIIIA and p43.

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Author notes

  1. Shan -Meei Lin

    Present address: Kaohsiung Medical College, Kaohsiung, Taiwan, Republic of China

Authors and Affiliations

  1. Department of Laboratory Medicine, University of Connecticut School of Medicine, 06032, Farmington, Connecticut

    Gregory S. Makowski, Shan -Meei Lin, Sidney M. Hopfer & F. William Sunderman

  2. Department of Anatomy, University of Connecticut School of Medicine, 06032, Farmington, Connecticut

    Sean M. Brennan

  3. Department of Pharmacology, University of Connecticut School of Medicine, 06032, Farmington, Connecticut

    Henry M. Smilowitz, Sidney M. Hopfer & F. William Sunderman

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  2. Shan -Meei Lin
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Makowski, G.S., Lin, S.M., Brennan, S.M. et al. Detection of two Zn-finger proteins ofXenopus laevis, TFIIIA, and p43, by probing western blots of ovary cytosol with65Zn2+,63Ni2+, or109Cd2+ . Biol Trace Elem Res 29, 93–109 (1991). https://doi.org/10.1007/BF03032687

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  • DOI: https://doi.org/10.1007/BF03032687

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