Inactivation and self-association of Ca2+/calmodulin-dependent protein kinase II during autophosphorylation.
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.
Hudmon
A
A
Department of Neurobiology, University of Texas Health Science Center, Houston, 77225, USA.
Aronowski
J
J
Kolb
S J
SJ
Waxham
M N
MN
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats
D046911:Macromolecular Substances; D010455:Peptides; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases