Association of poly(A)polymerase with tryptophan receptor in rat hepatic nuclei

Abstract

This study conducted experiments with two rat liver nuclear envelope proteins, a tryptophan receptor glycoprotein and poly(A)polymerase, and compared the structural similarities by using affinity chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and antibody specificity. Earlier, a rat hepatic nuclear envelope glycoprotein that is present in limited quantity and binds tryptophan with high affinity was identified and characterized. Also, analysis of eluted nuclear membrane proteins bound to either tryptophan-agarose or concanavalin A-agarose columns by SDS-PAGE previously revealed a major protein of similar molecular weight after staining with Coomassie blue. Polyclonal antibodies were raised in rabbits by injecting the excised gel protein band. The second protein, an enzyme, poly(A)polymerase, was previously purified from rat liver nuclei by sequential chromatography on DEAE-Sephadex, QAE-Sephadex, phosphocellulose, hydroxyapatite, and DNA-cellulose columns. Analysis of eluate from the final column by SDS-PAGE and Coomassie blue staining revealed a single band. Antibodies to the purified enzyme were raised in chickens. In the present study, using anti-tryptophan receptor antibodies and anti-poly(A)polymerase antibodies, we observed that each recognized the same protein by immunoblot analysis. The anti-tryptophan receptor antibodies did not recognize the catalytic site of poly(A)polymerase but did reduce the enzymatic activity when the antigen-antibody complex was sedimented using Pansorbin ( Staphyloccus aureus cells wearing a coat of protein A). However, antipoly(A)polymerase antibodies inhibited binding of 3H-tryptophan to the receptor site. Furthermore, tryptophan could elute poly(A)polymerase from both tryptophan agarose and poly(A)sepharose columns. Analysis of eluates from these columns by SDS-PAGE followed by silver staining of the gel revealed the presence of a major band with an apparent molecular weight of about 65,000–67,000. Our present findings suggest that the tryptophan receptor and poly(A)polymerase share structural homology.