Partial purification of protein kinase C isoenzymes from rat liver

Abstract

Due to the growing number of recently cloned isoenzymes, purification and assay of protein kinase C (PKC) have become increasingly cumbersome. This paper reports the development of a shortened protocol for partial purification and assay of alpha, beta, delta and zeta PKC from rat liver, allowing the determination of a PKC subspecies activity pattern on a single tissue preparation. Calcium-dependent alpha and beta PKC subspecies were resolved by application of a DEAE eluate to a hydroxylapatite column, delta PKC was separated with SP-Sepharose and phenyl-Sepharose chromatography, whereas three column passages were necessary to isolate zeta PKC: DEAE-Sepharose, phenyl-Sepharose and heparin-Sepharose. This procedure allows reproducible separation and assay as well as constant recovery of the four liver PKC isoenzymes.