, Volume 7, Issue 1, pp 1-12

The Ca2+/calmodulin-dependent protein kinase II-associated protein complex isolated from chicken retina

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Abstract

Retinal cytosolic Ca2+/calmodulin-dependent protein kinase II (CaM KII) was isolated from hatched 6-wk chicken retinae by ultracentrifugation and affinity chromatography using calmodulin (CaM) and anti-CaM KII-α columns. Samples from different fractions were examined with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining or immunoblotting. Comparisons were made between the final antibody affinity eluates from retina and forebrain. Silver-stained gels showed that multiple proteins were present in the antibody affinity eluates from retina, including major proteins of 178, 56, and 45 kDa and several minor proteins. Immunoblots revealed that CaM KII-α was present in eluates from the retina and forebrain. CaM KII-β was present in the antibody eluate from forebrain but not retina. The latter subunit was present in the crude homogenates of the retina. Regarding the antibody eluate from retina, the possibility that the major 56 kDa protein was tubulin was ruled out, but protein tau (τ) and synapsin I were present. The presence of multiple proteins in the antibody affinity eluate indicates that these proteins were coisolated in a CaM KII-α-associated protein complex. The finding that protein τ and synapsin I are associated with retinal CaM KII provides further insight into the mechanisms underlying the function of the kinase in this tissue. The lack of cytosolic CaM KII-β subunit in the antibody affinity eluate from retina is indicative of a brain region-specificity in subunit composition of the kinase.