Biochemical basis for the specificity of alloxan inactivation of calmodulin-dependent protein kinase II.
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, RINm5f. 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 RINm5f cells, alloxan (100 microM) inactivated 76 +/- 1% calmodulin-dependent protein kinase II activity in 5 min at 37 degrees C. Subsequent incubation with dithiothreitol restored most of the activity. 5,5'-Dithiobis (2-nitrobenzoic acid) (I50 = 2.5 microM) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Kloepper
R F
RF
Department of Pediatrics, Washington University School of Medicine, St Louis, MO.
Norling
L L
LL
McDaniel
M L
ML
Landt
M
M
D000496:Alloxan / Q000494:pharmacology*; D000704:Analysis of Variance; D000818:Animals; D001921:Brain / Q000201:enzymology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D005260:Female; D047428:Protein Kinase Inhibitors; D051381:Rats
D047428:Protein Kinase Inhibitors; D000496:Alloxan; D017871:Calcium-Calmodulin-Dependent Protein Kinases