Protein Kinases of Rat Liver Endoplasmic Reticulum. Solubilisation, Partial Characterisation and Comparison with Protein Kinases of Rat Liver Cytosol

Abstract

Protein kinase [EC 2.7.1.37] associated with rat liver microsomes was only partly extracted by treatment with 1.5 M KCl. The enzyme was solubilized by Triton X-100 or sodium deoxycholate at the same or slightly higher detergent concentrations than microsomal marker components. The enzyme activity increased 2-3-fold upon solubilization. Three peaks with protein kinase activity (fractions MI, MII and MIII) were resolved on DEAE-cellulose chromatography. Fraction MIII but not fractions MI or MII was activated by cyclic[c]AMO. All fractions catalyzed the phosphorylation of protamine and histones but not that of casein or phosvitin. Fractions MI and MIII had a similar substrate specificity and phosphorylated histones at a relatively much higher rate than did fraction MII. The isoelectric points (pI) were 8.1 for fraction MI, 5.5 for fraction MII and 4.9 for fraction MIII. On incubation of fraction MIII with cAMP it was split into 2 catalytically active components with pI 8.1 and 7.35. The component with pI 8.1 was predominant and corresponded to fraction MI. Five protein kinase peaks were resolved from rat liver cytosol by DEAE-cellulose chromatography. Three of them (fractions CIa, CIIb and CIII) had the same properties as each of the microsomal kinase fractions. A 4th fraction (CIIa) was cAMP-dependent and had the same substrate specificity as fractions MI and MIII. Its pI was 5.1, and it was split into 2 components by cAMP (pI 8.1 and 7.35). In binding studies fraction CIIb bound more efficiently to microsomes than fraction CIII, while fractions CIa, CIIa and the microsomal protein kinase fractions did not bind appreciably. When microsomes were treated with trypsin exposed protein kinase was inactivated and the latency of the remaining enzyme increased substantially. Most of fraction MII was inactivated by trypsin while fraction MIII was resistant. Possible orientation of protein kinase fractions MII and MIII in the microsomal membrane is discussed.