Affinity chromatography of protein kinase C-phorbol ester receptor on polyacrylamide-immobilized phosphatidylserine.
An affinity column, prepared by immobilizing phosphatidylserine and cholesterol in polyacrylamide, was utilized in the purification of protein kinase C. Protein kinase activity and phorbol ester binding were monitored by assaying Ca2+ plus phosphatidylserine-dependent phosphorylation of histone H1 and [3H]phorbol dibutyrate binding, respectively. Both activities were present in a cytosolic extract of rabbit renal cortex, eluted together from a DEAE-cellulose column, bound to the affinity column in the presence of Ca2+, and eluted symmetrically upon application of EGTA. Recovery from the affinity column was high (30-50%) and resulted in as much as a 6000-7700-fold purification, depending on the region of the DEAE-cellulose peak that was applied. Following affinity column purification, protein kinase and phorbol ester binding activity eluted symmetrically upon gel filtration, with a molecular weight of approximately 80 kDa. A protein of the same size was present in silver-stained gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity column purified samples from the DEAE-cellulose peak. From 2-4 other, smaller proteins were also present, their number and relative amounts depending on the region of the DEAE-cellulose peak used. These data indicate that Ca2+-dependent/binding to a polyacrylamide-immobilized phospholipid provides a useful technique for purification of protein kinase C as well as other, unidentified proteins exhibiting a Ca2+ plus phospholipid-dependent interaction.
D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D002846:Chromatography, Affinity / Q000379:methods; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D007672:Kidney Cortex / Q000378:metabolism*; D010703:Phorbol Esters / Q000378:metabolism; D010718:Phosphatidylserines; D011493:Protein Kinase C; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D011955:Receptors, Drug; D011971:Receptors, Immunologic / Q000302:isolation & purification* / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D010703:Phorbol Esters; D010718:Phosphatidylserines; D011955:Receptors, Drug; D011971:Receptors, Immunologic; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D011494:Protein Kinases; D011493:Protein Kinase C