Purification and characterization of calmodulin-stimulated protein kinase II from two-day and adult chicken forebrain.
Soluble calmodulin-stimulated protein kinase II has been purified from 2-day and adult chicken forebrain. At both ages the holoenzyme eluted from a Superose-6B column with an apparent molecular weight of approximately 700,000 daltons and contained three subunits. The subunits were found to be the counterparts of the alpha, beta, and beta' subunits of the enzyme purified from adult rat brain in that they had one-dimensional phosphopeptide maps that were indistinguishable from those of the corresponding subunit in the rat enzyme and they migrated in SDS-polyacrylamide gels with the same apparent molecular weights. However, the doublet formed by the beta subunit was much more clearly resolved in the chicken enzyme and the beta' subunit, which was much more abundant in the adult chicken than in the adult rat, was also found to be a doublet. The ratio of the concentrations of the alpha and beta subunits changed during development. By autoradiography following autophosphorylation, the alpha:beta ratios of the 2-day and adult enzymes were 0.89 +/- 0.07 and 1.92 +/- 0.26, respectively; by silver staining the alpha:beta ratios were 0.95 +/- 0.11 and 1.85 +/- 0.17, respectively. The concentration of the beta' subunit was equal to that of the beta subunit at both ages. Autophosphorylation produced a decrease in the electrophoretic mobility of the alpha and beta subunits in SDS-polyacrylamide gels and a marked decrease in the calcium dependence of the substrate phosphorylation activity of the enzyme at both ages. The purified enzyme from chicken brain appeared to be more stable under standard in vitro assay conditions than the rat enzyme, and this was particularly so for the enzyme from 2-day forebrain.
Rostas
J A
JA
Neuroscience Group, Faculty of Medicine, University of Newcastle, N.S.W., Australia.
Brent
V A
VA
Seccombe
M
M
Weinberger
R P
RP
Dunkley
P R
PR
D000375:Aging; D000818:Animals; D001921:Brain / Q000201:enzymology* / Q000254:growth & development; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002645:Chickens; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism
D046911:Macromolecular Substances; D010748:Phosphopeptides; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases