Protein mapping of two metallothionein-rich cell strains and their parent lines, using high-resolution two-dimensional electrophoresis.
A high-resolution two-dimensional gel electrophoresis (2-D) technique was used to characterize one human and one murine cadmium-resistant substrain and their parental wild-type lines. The substrains are cultured on 100 microM cadmium and contain high levels of the cysteine-rich protein metallothionein (MT). All four cell lines were labeled with [35S]methionine during growth. A remarkable consistency was found in the protein maps of the resistant strains compared to those obtained from their corresponding wild-type lines. Thus, in the maps from the human substrain only two spots were detected which were not found in the parent cells. In the murine substrain, two spots were more abundant and two diminished compared to the parent cells. No distinct spots corresponding to authentic MT were detected in any of the autoradiographs from the cadmium-resistant cells. The reason for this was found to be failure of the protein to focus in the first dimension. Purified [35S]cystine-labeled MT appeared as a diffuse labeling over the entire gel, and subsequently as wide horizontal bands in the second dimension. These bands were also clearly visible in the protein maps when MT-rich cells had been labeled with [35S]cysteine. This study shows that the standardized 2-D gel system used in many laboratories cannot be used to screen cell populations for MT.
D000818:Animals; D001345:Autoradiography; D002104:Cadmium / Q000494:pharmacology*; D002460:Cell Line; D002850:Chromatography, Gel; D003545:Cysteine / Q000378:metabolism; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004848:Epithelium / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008668:Metallothionein / Q000096:biosynthesis*; D051379:Mice; D009154:Mutation
D002104:Cadmium; D008668:Metallothionein; D003545:Cysteine