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

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.