Purification of a 65-kiloDalton nuclear protein with structural homology to glutathione-S-transferase

Abstract

A 65-kDa subunit, nuclear protein from mung bean was previously identified using anti-idiotypic antibodies directed against IgG recognizing the auxin, indole-3-acetic acid. This protein was proposed to be a putative auxin-binding protein and was designated 65-kDa ABP (Prasad and Jones, Proc. Natl. Acad. Sci. USA 88 (1991) 5479–5483). Despite substantial effort, direct evidence for auxin-binding has not been demonstrated. Therefore, 65-kDa ABP is redesignated as p65 and herein is described its purification and the N-terminal sequence of this protein. Antibodies directed against a synthetic N-terminal peptide of p65 immunoprecipitate a 65-kDa peptide that is recognized by the anti-idiotypic antibodies indicating that the amino terminal sequence is of the originally described 65-kDa ABP. High sequence identity between p65 and the N-terminal sequence of several glutathione-S-transferases (GSTs) was found, although significant GST activity was not detected. We demonstrate that p65 is retained by both glutathione- and IAA-immobilized columns. These and previous observations indicate that p65 has structural homology, but not necessarily functional homology, to both GST and ABP.