Purification of a phosphoprotein from rat brain closely related to the 80 kDa substrate of protein kinase C identified in Swiss 3T3 fibroblasts

Abstract

A phosphoprotein expressed in rat brain is closely related to the 80 kDa substrate of protein kinase C present in 3T3 cells. The protein kinase C substrates from both sources migrate identically on two-dimensional gel electrophoresis and give similar phosphopeptide fragments when digested with protease. Using a series of chromatographic steps, including DEAE-cellulose chromatography, Sephadex G150 gel filtration and reverse phase fast protein liquid chromatography, this phosphoprotein was purified 3800-fold from rat brain. The preparation appears homogenous by one- and two-dimensional gel electrophoresis, is an effective substrate of protein kinase C and contains a high proportion of the acidic amino acids glutamate and aspartate, and of alanine.