Purification and properties of protein kinase C from rabbit reticulocyte lysates.
We have previously reported that addition of Ca2+ and phospholipid (PL) inhibits translation in hemin-containing reticulocyte lysates through activation of a eukaryotic protein synthesis initiation factor (eIF-2) kinase. The possibility that this activation was mediated by a Ca2+-PL-dependent protein kinase (protein kinase C, PKC) appeared unlikely by the observation that it was prevented or reversed by NADPH-generating systems. Nevertheless, reticulocyte lysates contain a potent PKC activity and we deemed it desirable to isolate this enzyme to answer unequivocally the question whether it does or does not activate eIF-2 alpha kinase. We have purified reticulocyte PKC to near homogeneity with Mr 95,500 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme absolutely depended upon both Ca2+ and phosphatidylserine for activity on histone H1 or the beta-subunit of initiation factor eIF-2 and underwent autophosphorylation in a Ca2+- and PL-dependent manner. Mild treatment with trypsin yielded an Mr 82,000 polypeptide that still required Ca2+ and PL for activity. This Mr agrees with that reported for other PKCs, suggesting that these enzymes may undergo limited degradation during isolation. Further proteolytic treatment converted the reticulocyte enzyme into a Ca2+- and PL-dependent form, as is known for PKCs from other sources. The highly purified PKC had no effect on translation in hemin-supplemented reticulocyte lysates.
Pelaez
F
F
Centro de Biología Molecular, CSIC-Universidad Autónoma, Madrid, Spain.
de Herreros
A G
AG
de Haro
C
C
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002499:Centrifugation, Density Gradient / Q000379:methods; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D011493:Protein Kinase C / Q000097:blood* / Q000302:isolation & purification; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*
D006882:Hydroxyapatites; D017886:Durapatite; D011493:Protein Kinase C; D002118:Calcium