Detection of two Zn-finger proteins of Xenopus laevis, TFIIIA, and p43, by probing western blots of ovary cytosol with 65Zn2+, 63Ni2+, or 109Cd2+.
Two Zn-finger proteins, TFIIIA (a constituent of 7S RNP particles) and p43 (a constituent of 42S RNP particles), were detected in ovary extracts of juvenile Xenopus 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 with 65Zn2+, 63Ni2+, or 109Cd2+, and visualized by autoradiography. Detection limits for TFIIIA were approx 0.07 micrograms/well by 109Cd(2+)-probing, 0.13 micrograms/well by 65Zn(2+)-probing, and 0.26 mu/well by 63Ni(2+)-probing. Protein p43 was more clearly visualized by probing with 63Ni2+ than with 65Zn2+ or 109Cd2+. After purified TFIIIA was cleaved with cyanogen bromide, 65Zn2+, 109Cd2+, and 63Ni2+ distinctly labeled the 22 kDa middle fragment; 65Zn2+ and 109Cd2+ also labeled the 11 kDa N-terminal fragment, but did not 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 with 65Zn2+ for binding to TFIIIA on Western blots, the relative affinities of the metals for TFIIIA were ranked as follows: Zn2+ = Cu2+ greater than or equal to Hg2+ greater than Cd2+ greater than Co2+ greater than or equal to Ni2+. Even at a 1000-fold molar excess, Mn2+ did not compete with 65Zn2+ for binding to TFIIIA. Probing Western blots with the radiolabeled metal ions greatly facilitates the detection, isolation, and quantitation of TFIIIA and p43.
Makowski
G S
GS
Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington 06032.
Lin
S M
SM
Brennan
S M
SM
Smilowitz
H M
HM
Hopfer
S M
SM
Sunderman
F W
FW
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002106:Cadmium Radioisotopes; D003488:Cyanogen Bromide; D003600:Cytosol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D066298:In Vitro Techniques; D009532:Nickel; D010053:Ovary / Q000737:chemistry; D011485:Protein Binding; D011868:Radioisotopes; D012261:Ribonucleoproteins / Q000032:analysis* / Q000378:metabolism; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000032:analysis* / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics*; D016335:Zinc Fingers; D015037:Zinc Radioisotopes
D002106:Cadmium Radioisotopes; D011868:Radioisotopes; D012261:Ribonucleoproteins; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D015037:Zinc Radioisotopes; D009532:Nickel; D003488:Cyanogen Bromide