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

AUTOR(ES)

Crawford, L V

RESUMO

We analyzed large and small species of T-antigen by immunoprecipitation and two-dimensional gel electrophoresis. The T-antigen species were subjected to electrophoresis either directly or after reduction and alkylation with N-ethylmaleimide. Treatment with N-ethylmaleimide improved the resolution of large-T by two-dimensional gel electrophoresis and was a requirement for the resolution of small-t antigen on two dimensional gels. Large-T did not form a discrete protein spot, but rather formed a streak from approximately pH 6.5 to 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. We suggest that the apparent increase in molecular weight was due to the association of N-ethylmaleimide with cysteine-rich regions of these proteins. Viable deletion mutants of simian virus 40 which do not induce the synthesis of small-t but product 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 simian virus 40.

Documentos Relacionados

• Dimerization of simian virus 40 T-antigen hexamers activates T-antigen DNA helicase activity.
• Localization of T-antigen on simian virus 40 minichromosomes by immunoelectron microscopy.
• Inhibition of simian virus 40 T-antigen expression by cellular differentiation.
• DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication.
• Cooperative assembly of simian virus 40 T-antigen hexamers on functional halves of the replication origin.