Purification and characterization of the Ca2+/calmodulin-dependent protein kinase II from chicken forebrain
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CaM kinase II is known to be enriched in mammalian and avian brains. To determine the holoenzymic composition and functional characteristics of this kinase, a new approach for isolation was applied to isolate it from the chicken forebrain. Forebrains of hatched 45-d chicken were dissected, homogenized, and centrifuged. The supernatant was loaded onto a CaM-agarose affinity column and the calmodulin-binding proteins were eluted with EGTA. Selected eluates were loaded onto the antibody-agarose affinity column, which was prepared with monoclonal antibody (MAb) (6G9) to the CaM kinase II α subunit. Samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and either silver-stained or blotted onto a nitrocellulose membrane. The protein composition and the immunoreactivity of the antibody-agarose affinity eluate fractions were analyzed with a densitometric scanner. Silver staining of gels showed that the β subunit doublet, the β′ subunit, and a putative substrate were coeluted with the α subunit from the antibody affinity column although only the α subunit bound the 6G9 antibody. Scintillation counting showed that the autophosphorylation of the kinase was significantly reduced in the eluate from the antibody affinity column. Whereas silver staining indicated an increase in the relative amount of α subunit had occurred during purification, phosphorylation assays indicated an increase in the relative amount of the α subunit after the last purification step. A possible reason for this is discussed. The presence of β/β′ subunits in the antibody-agarose affinity eluate indicated the existence of an αβ/β′ heteropolymer. The phosphorylation assay was not a good indication of the amount of purification because of the loss of enzyme activity following purification. In contrast, the immunoassay indicated a 97-fold purification from the cytosolic fraction was achieved using the method. In conclusion, the data indicate the existence of the CaM kinase II αβ/β′ heteropolymer in the chicken forebrain.
Index EntriesMultifunctional CaM kinase heteropolymer autophosphorylation antibody affinity chromatography 6G9 monoclonal antibody
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