Alteration of nickel-binding proteins in nickel-resistant cells.
Proteins from wild-type and nickel-resistant cells (Balb/c-3T3 and B200, respectively) were studied by one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and detected either by standard protein staining or by fluorographic analysis of [63Ni] binding. Wild-type cells contained three major nickel-binding proteins with molecular masses of 68 kDa (p68), 55 kDa (p55), and 48 kDa (p48). The p55 was present in high concentrations in the microsomal fraction, whereas the p68 nickel-binding protein predominated in the cytosolic fraction. We were unable to demonstrate that p48 was localized in any subcellular fraction. Both the p55 and p48 proteins appeared to be present in similar amounts in wild-type and nickel-resistant cell lines, based upon silver staining of two-dimensional gels, yet in the nickel-resistant B200 cells, these proteins could not be visualized by [63Ni] binding. This suggested that if nickel binding to these proteins were important for nickel toxicity, then nickel resistance in the B200 cells would be associated with the observed loss of metal binding to these proteins. In addition to the changes in [63Ni]-binding proteins, the total concentration of a 44 kDa protein was increased in B200 cells, but its ability to bind nickel could not be established due to its rapid degradation. Among the nickel-binding proteins studied, the p55 contained nickel-binding sites that were the most resistant to exchange by excess nickel ions. The microsomal fraction that contained the highest concentration of p55 also had the highest nickel-binding activity when standardized for protein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Wang
X W
XW
Institute of Environmental Medicine, New York University Medical Center, Tuxedo 10987.
Costa
M
M
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000502:physiology*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D053487:DEAD-box RNA Helicases; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D008861:Microsomes / Q000378:metabolism; D009532:Nickel / Q000378:metabolism*; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000502:physiology*; D011494:Protein Kinases; D020365:RNA Helicases
D002352:Carrier Proteins; D009687:Nuclear Proteins; D009532:Nickel; D011494:Protein Kinases; C506751:Ddx5 protein, mouse; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases