Biochemical basis for the specificity of alloxan inactivation of calmodulin-dependent protein kinase II

Abstract

The specificity and biochemical basis of inactivation of calmodulin-dependent protein kinase II by alloxan was studied in dispersed rat brain cells and a partially purified kinase preparation from an insulin-secreting tumor-cell line, RIN m5f. When mechanically dispersed rat brain cells were incubated with [ 32P]-phosphate to label endogenous ATP, depolarization with 44 mM KCl produced a significant ( P = 0.03) increase in phosphorylation of endogenous synapsin (132 ± 8 % of basal). Pre-treatment of the brain cells with 1.5 mM alloxan reduced depolarization-sensitive synapsin phosphorylation (109 ±5 %). Phosphopeptide mapping of depolarization-phosphorylated synapsin showed that alloxan pre-treatment reduced phosphorylation specifically at synapsin sites phosphorylated by calmodulin-dependent protein kinase II. The results demonstrate selective inactivation of calmodulin-dependent protein kinase II activity by alloxan in an intact cell system, which may be useful in the study of the Type II kinase in cells and tissues. Using a partially purified kinase preparation from RIN m5f cells, alloxan (100 μM) inactivated 76 ± 1 % calmodulin-dependent protein kinase II activity in 5 min at 37°C. Subsequent incubation with dithiothreitol restored most of the activity. 5,5′-Dithiobis (2-nitrobenzoic acid) (I 50 = 2.5 μM) also inactivated the kinase. These results suggested that a sulfhydryl group was involved at the inactivation site. Iodoacetamide (1.0 mM) had no inhibitory effect; however, preincubation with iodoacetamide protected the kinase activity from subsequent inactivation by alloxan. Covalent binding of [ 14C]-alloxan to calmodulin-dependent protein kinase was demonstrated. Alloxan and other bulky derivatizing agents may inactivate calmodulin-dependent protein kinase II by alkylating specific sulfhydryl groups. Iodoacetamide appears to protect the kinase by alkylating the same sulfhydryl groups, to yield a less bulky derivative which does not inactivate the kinase.