Abstract
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.
Highlights
From the Gerontology Research Center, Laboratory of Molecular Aging,National Institute of Aging, Baltimore, Maryland 21224 esters cause a redistribution of the enzyme from cytosol to membranes inresponsive cells [13] has heightened interest in this enzyme
Protein kinase activityand phorbol ester binding were monitored by assaying Ca2+ plus phosphatidylserine-dependent phosphorylationof 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
Renal cortices dissected from kidneys of New Zealand to the affinity column in the presence of Ca2+,and White rabbits were minced and homogenized with a glass homogeeluted symmetrically upon application of EGTA
Summary
+fractions of the DEAE-cellulose column eluant containing the Ca2+ phospholipid-dependent protein kinase and phorbol ester binding activities. This mixture was pumped a t 15 ml/h into a closed 0.5-ml mixing chamber, into which a solution containing 10 mM MES, pH. The resulting mixture was pumped at 30 ml/h directly onto the affinity column and, after the total sample was applied, wasfollowed by mlof column buffer, which was followed by 45 ml of the same buffer but with CaC12 reduced to 0.1 mM At this point, eluting buffer, identical to thecolumn buffer but with 2 mM EGTA in place of CaC12, was applied and additional 3.0-ml fractions were collected. The reaction mixture contained 25 mM Tris, pH 7.5, 10 mM MgAc, 1.4 mM CaC12, 0.4 mM EGTA and EDTA (DEAEcolumn eluate), or 0.4 mM EGTA
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