Identification of Protein Disulfide Isomerase as a Phorbol Ester-Binding Protein

Abstract

A specific binding protein for 12-O-tetradecanoylphorbol 13-acetate (TPA), different from protein kinase C (PKC) and histone H1, was purified from HeLa cell extract by the use of affinity gel pendanted with phorbol ester (TPA-GEL). The purified binding protein was identified as protein disulfide isomerase (PDI, EC 5.4.3.1) by peptide sequence analysis. The dissociation constants (K_d's) of TPA to PDI, histone H1 and PKCα were determined to be 1.03×10^-6M, 5.70×10^-7M, and 4.00×10^-7M, respectively, by the surface plasmon resonance (SPR) method. TPA moderately inhibited PDI activity assessed in terms of reactivation of denatured RNase A.