Abstract
The time-dependent loss in enzyme activity associated with the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase) was altered by both pH and ATP concentration. These parameters also influenced the extent to which soluble CaM-kinase undergoes self-association to form large aggregates of sedimentable enzyme. Specifically, autophosphorylation of CaM-kinase in 0.01 mM ATP at pH 6.5 resulted in the formation of sedimentable enzyme and a 70% loss of enzyme activity. Under similar conditions at pH 7.5, the enzyme lost only 30% of its activity, and no sedimentable enzyme was detected. In contrast to 0.01 mM ATP, autophosphorylation of CaM-kinase at pH 6.5 in 1 mM ATP did not result in a loss of activity or the production of sedimentable enzyme, even though the stoichiometry of autophosphorylation was comparable. Electron microscopy studies of CaM-kinase autophosphorylated at pH 6.5 in 0.01 mM ATP revealed particles 100-300 nm in diameter that clustered into branched complexes. Inactivation and self-association of CaM-kinase were influenced by the conditions of autophosphorylation in vitro, suggesting that both the catalytic and physical properties of the enzyme may be sensitive to fluctuations in ATP concentration and pH in vivo.
Highlights
Ca2ϩ/calmodulin-dependent protein kinase II (CaM-kinase)1 is highly enriched in brain where it is thought to perform a multifunctional role in the transduction of Ca2ϩ signals
Activity measurements from enzyme autophosphorylated at pH 7.0 and 6.5 indicated that a greater percentage of enzyme activity was lost over time with a decrease in reaction pH (Fig. 1A)
Autophosphorylation of CaMkinase at pH 7.5 resulted in a 15% decrease in enzyme activity at 2 min, whereas pH 7.0 and pH 6.5 were both associated with a 40% loss of enzyme activity at 2 min
Summary
CaM-kinase, Ca2ϩ/calmodulin-dependent protein kinase II; CaM, calmodulin; AMP-PCP, adenylyl-(,␥-methylene)-diphosphonate; TEM, transmission electron microscopy; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid. Other parameters reported to influence the loss of enzyme activity and sites of autophosphorylation include reaction temperature [27] and the type and concentration of divalent ions [24, 27]. These findings indicate that the conditions of autophosphorylation are important determinants for alterations in the activity of CaM-kinase. We observed that pH and ATP concentration influenced the inactivation of CaM-kinase associated with autophosphorylation These parameters influenced the self-association of soluble CaM-kinase into sedimentable enzyme complexes, suggesting that cells undergoing fluctuations in pH and ATP concentration could produce autophosphorylation-associated changes in both the physical and catalytic properties of the enzyme in vivo
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