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 Cl50 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.

Internal amino acid sequence analysis of the 80 kDa protein kinase C substrate from rat brain: Relationship to the 87 kDa substrate from bovine brain

“…Method II of purification yielded about 0.5 mg of homogeneous 80 kDa protein from 1 kg of frozen brains. This represents an increase of 3-fold over the yield obtained by method I [12]. The protein purified by method II had the same Retention time, min.…”

“…Method II was modified from method I as follows: heat-treated extracts were prepared as in method I [12] but the homogenization step was performed in the presence of 1 mM phenylmethylsulfonyl fluoride. The DEAE-cellulose step described in method I [12] was scaled up by mixing heat-treated extracts from 1 kg of frozen brains with 500 ml of DEAE-cellulose (Whatman). Proteins were eluted from the DEAE-cellulose column with a 2 1 linear gradient of 0-1 M NaCI in 20 mM Tris-HCl, pH 7.5, and 1 mM EDTA.…”

“…Proteins were eluted from the DEAE-cellulose column with a 2 1 linear gradient of 0-1 M NaCI in 20 mM Tris-HCl, pH 7.5, and 1 mM EDTA. The gel filtration step described in method I [12] was modified using a Sephadex G-150 superfine column (Pharmacia Biotechnology, Inc., 2.5 x 82 cm) pre-equilibrated and eluted with 1 M NaCI in 20 mM HCI. This column was loaded with half the amount of material obtained from the previous step.…”

“…This column was loaded with half the amount of material obtained from the previous step. The reverse-phase FPLC step described in method I [12] was replaced by preparative SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution as follows: the pooled material from the previous step was concentrated with Aquacide II (Calbiochem) and separated by SDS-PAGE according to Laemmli [17], using extensively polymerized 7.507o polyacrylamide separating gels (1 mm thick). The 80 kDa protein band was visualized by staining for 15 min with 0.207o Coomassie Blue R-250 and was excised from the gel.…”

“…umns [12]. It is likely that these protein kinase C substrates are part of a family of related proteins, a proposition that warrants considerable further work.…”