p53 undergoes epitopic changes in vitro by sodium-vanadate.
p53 has at least two conformations that differ in their immunoreactivity. They consist of the functional tumor suppressor form, characteristic of the wild type p53 and the mutant form, generated by changes in the primary amino acid sequence of the protein. It has been previously shown that the wild type p53 protein also acquires the mutant conformation upon certain changes in growth conditions. Here we report that similar epitopic changes can be induced in crude cell lysate by addition of vanadate anions at 1 mM final concentration. A panel of anti p53 antibodies was used to discriminate between the different immunoreactive forms of wild type p53 in SV80 fibroblasts. It was found that addition of sodium vanadate to the lysis buffer converted part of p53 molecules into a mutant conformation that is recognized by the PAb 240 monoclonal antibodies. The effect of vanadate on p53 conformation was prominent even if it was added to the cell lysates after 15 min of pre-incubation at 37 degrees C. This further excluded its possible role as phosphatase inhibitor in the system and suggested a direct interaction with the p53 protein itself. Based on these data we recommend to avoid using sodium vanadate as a phosphatase inhibitor in experiments where in vivo conformational changes of wild type p53 are studied.
Landesman
Y
Y
Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, Israel.
Bringold
F
F
Kimchi
A
A
D000911:Antibodies, Monoclonal; D002461:Cell Line, Transformed; D000939:Epitopes; D016158:Genes, p53; D006801:Humans; D009154:Mutation; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D014638:Vanadates / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D014638:Vanadates