Effect of alkylation on the physical properties of simian virus 40 T-antigen species

Abstract

Large and small species of T[tumor]-antigen were analyzed by immunoprecipitation and 2-dimensional gel electrophoresis. The T-antigen species were subjected to electrophoresis directly or after reduction and alkylation with N-ethylmaleimide. Treatment with N-ethylmaleimide improved the resolution of large-T by 2-dimensional gel electrophoresis and was a requirement for the resolution of small-t antigen on 2-dimensional gels. Large-T did not form a discrete protein spot, but formed a streak from approximately pH 6.5-6.9 on isoelectric focusing gels. Small-t formed a sharp protein spot at approximately pH 7.2 when subjected to electrophoresis under non-equilibrium conditions which extended the pH gradient to include proteins with basic isoelectric points. Treatment with N-ethylmaleimide decreased the mobility of the T-antigen species during sodium dodecyl sulfate gel electrophoresis. The apparent increase in MW was probably due to the association of N-ethylmaleimide with cysteine-rich regions of these proteins. Viable deletion mutants of SV40 which do not induce the synthesis of small-t but produce small-t-related polypeptides were used to localize the cysteine-rich region of small-t to between 0.54 and 0.59 on the genetic map of SV40.