Purification of Protein Kinase C and Phorbol Ester Receptor Using Polyacrylamide-Immobilized Phosphatidylserine

  • Charles R. Filburn
  • Tsutomu Uchida
Chapter
Part of the Experimental Biology and Medicine book series (EBAM, volume 6)

Summary

A \(C{{a}^{{{{2}^{ + }}}}}\) + phospholipid-dependent protein kinase and a phorbol ester receptor were purified from a cytosolic extract of rabbit renal cortex by ion exchange chromatography on DEAE-cellulose and affinity chromatography on polyacrylamide-immobilized phosphatidylserine.Protein kinase activity was monitored by assaying \(C{{a}^{{{{2}^{ + }}}}}\) + phospholipid-dependent phosphorylation of histone H1, phorbol ester binding by measuring specific binding of [3H]- phorbol dibutyrate in the presence of \(C{{a}^{{{{2}^{ + }}}}}\) and phosphatidylserine. Both activities eluted together from the DEAE-cellulose column, bound to the affinity column in the presence of \(C{{a}^{{{{2}^{ + }}}}}\), and eluted symmetrically from the affinity column upon application of EGTA. Recovery from the affinity column was the same for both activities (usually 20–30%) and resulted in a high degree of purification.

Keywords

Cholesterol Cellulose Sucrose Cytosol Settling 

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Copyright information

© The Humana Press Inc. 1985

Authors and Affiliations

  • Charles R. Filburn
    • 1
  • Tsutomu Uchida
    • 1
  1. 1.Laboratory of Molecular Aging, National Institute on AgingNIH, Gerontology Research Center, Baltimore City HospitalsBaltimoreUSA

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