Measurement of activities of autophosphorylated/non-autophosphorylate CaMKII

CaMKII activity was measured utilizing syntideII as a substrate. Purified CaMKII was pre-incubated in the assay mixture (35 mM Hepes-Na ( pH 8.0), 10 mM MgC12, 0.5 μM CaM, 5 μM ATP, 1 mM CaCl2 or 1 mM EGTA, total 25 μL) at 30°C for 2 mins. After this pre-incubation, the protein substrate/radioactive ATP mixture was added to the same test tube and the preparation was further incubated at 30°C, for 5 mins (final assay condition: 35 mM Hepes-Na (pH 8.0), 10 mM MgCl2, 0.125 μM CaCl2, 20 μM syntideII, 11.25 μM [γ-32P] ATP, 10 % DMSO and indicated concentrations of KN-93, supplemented with 0.25 mM CaCl2 and 2 mM EGTA (for autophosphorylated samples) or 0.25 mM EGTA and 2 mM CaCl2 (for nonautophosphorylated samples ), total 100 μL). The reaction was terminated by adding of 25 μL of 100 % (w/v) ice-cold TCA. After centrifugation, 80 μL of the supernatant was applied to phosphocellulose paper. The filters were then washed with 75 mM H3PO4 for 15 mins with continuous agitation. After 4-cycles of washing, the radioactivity retains on the filter paper was quantified in a liquid scintillation counter.

Cell lines

NIH 3T3 fibroblasts

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

~ 24 μM; 70 hrs

Applications

KN-93 inhibited serum-induced fibroblast cell growth with an IC50 value of 8 μM, and induced apoptosis after prolonged G1 arrest at the concentration of 24 μM.

Animal models

Parkinson’s disease (PD) rats

Dosage form

1, 2 or 5 μg; intrastriatal administration; b.i.d., for 21 days

Applications

In PD rats, KN-93 (5 μg) ameliorated levodopa-induced dyskinesia through lowering the expression of pGluR1S845.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Sumi M, Kiuchi K, Ishikawa T, Ishii A, Hagiwara M, Nagatsu T, Hidaka H. The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells. Biochem Biophys Res Commun. 1991 Dec 31;181(3):968-75.

[2]. Tombes RM, Grant S, Westin EH, Krystal G: G1 cell cycle arrest and apoptosis are induced in NIH 3T3 cells by KN-93, an inhibitor of CaMK-II (the multifunctional Ca2+/CaM kinase). Cell Growth Differ 1995, 6(9):1063-1070.

[3]. Yang X, Wu N, Song L, Liu Z. Intrastriatal injections of KN-93 ameliorates levodopa-induced dyskinesia in a rat model of Parkinson"s disease. Neuropsychiatr Dis Treat. 2013;9:1213-20.