PMID- 29803814
OWN - NLM
STAT- MEDLINE
DCOM- 20181214
LR  - 20181214
IS  - 1873-488X (Electronic)
IS  - 1056-8719 (Linking)
VI  - 94
IP  - Pt 1
DP  - 2018 Nov-Dec
TI  - A non-radioactive in vitro CaMKII activity assay using HPLC-MS.
PG  - 64-70
LID - S1056-8719(18)30635-X [pii]
LID - 10.1016/j.vascn.2018.05.004 [doi]
AB  - INTRODUCTION: Calcium/Calmodulin-dependent protein kinase II (CaMKII) is a 
      multifunctional protein kinase that phosphorylates and regulates activity of many 
      substrates in various tissues. Traditional CaMKII activity assays rely on 
      incorporation of radioactivity onto a CaMKII substrate by utilizing gamma-(32)P ATP, 
      which has a short half-life and can pose health risks to the researchers. 
      METHODS: An 8-minute HPLC-MS method was developed to measure a CaMKII-specific 
      peptide substrate autocamtide-2 (AC-2) and its phosphorylated form, 
      phosphoautocamtide-2 (PAC-2). Degradation of AC-2 and PAC-2 in solutions and how 
      to stabilize them were studied. The method was validated according to FDA 
      guidelines for bioassays, and applied to determine CaMKII activity in a C2C12 
      cell lysate and IC50 of KN-93, a known CaMKII inhibitor. RESULTS: Simple 
      acidification with formic acid prevented AC-2 and PAC-2 from undergoing rapid 
      degradation in the CaMKII assay mixture and in diluted water solutions. LLOQ of 
      the HPLC-MS method was 0.26 muM and 0.12 muM for quantification of AC-2 and PAC-2, 
      respectively. Precision was within 15% and accuracy was within 100 +/- 15%. Using 
      the developed method, IC50 of KN-93 was measured to be 399 +/- 66 nM, which was 
      compatible to reported values. CONCLUSIONS: A validated HPLC-MS method provides 
      precise and accurate determination of AC-2 and PAC-2. This method enabled enzyme 
      activity assay and inhibitor IC50 determination for CaMKII without radioactive 
      labelled reagents.
CI  - Copyright (c) 2018 Elsevier Inc. All rights reserved.
FAU - Erwin, Tully
AU  - Erwin T
AD  - Department of Pharmaceutical Sciences, College of Pharmacy & Health Sciences, 
      Campbell University, Buies Creek, NC 27506, USA.
FAU - Rekulapally, Satish P
AU  - Rekulapally SP
AD  - Department of Pharmaceutical Sciences, College of Pharmacy & Health Sciences, 
      Campbell University, Buies Creek, NC 27506, USA.
FAU - Abraham, Thomas S
AU  - Abraham TS
AD  - Department of Pharmaceutical Sciences, College of Pharmacy & Health Sciences, 
      Campbell University, Buies Creek, NC 27506, USA.
FAU - Liu, Qinfeng
AU  - Liu Q
AD  - Department of Pharmaceutical Sciences, College of Pharmacy & Health Sciences, 
      Campbell University, Buies Creek, NC 27506, USA. Electronic address: 
      liuq@campbell.edu.
LA  - eng
PT  - Journal Article
DEP - 20180524
PL  - United States
TA  - J Pharmacol Toxicol Methods
JT  - Journal of pharmacological and toxicological methods
JID - 9206091
RN  - 0 (Benzylamines)
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Peptides)
RN  - 0 (Sulfonamides)
RN  - 0 (autocamtide-2)
RN  - 139298-40-1 (KN 93)
RN  - EC 2.7.11.17 (Calcium-Calmodulin-Dependent Protein Kinase Type 2)
SB  - IM
MH  - Animals
MH  - Benzylamines/chemistry/pharmacology
MH  - Biological Assay/*methods
MH  - Calcium-Calmodulin-Dependent Protein Kinase Type 2/*chemistry/*metabolism
MH  - Cells, Cultured
MH  - Chromatography, High Pressure Liquid
MH  - Enzyme Inhibitors/chemistry/pharmacology
MH  - Mass Spectrometry/methods
MH  - Mice
MH  - Peptides/chemistry/pharmacology
MH  - Radioactivity
MH  - Sulfonamides/chemistry/pharmacology
OTO - NOTNLM
OT  - Autocamtide-2
OT  - CaMKII
OT  - HPLC-MS method
OT  - IC50
OT  - Non-radioactive
OT  - Phosphoautocamtide-2
EDAT- 2018/05/29 06:00
MHDA- 2018/12/15 06:00
CRDT- 2018/05/28 06:00
PHST- 2018/04/18 00:00 [received]
PHST- 2018/05/16 00:00 [revised]
PHST- 2018/05/21 00:00 [accepted]
PHST- 2018/05/29 06:00 [pubmed]
PHST- 2018/12/15 06:00 [medline]
PHST- 2018/05/28 06:00 [entrez]
AID - S1056-8719(18)30635-X [pii]
AID - 10.1016/j.vascn.2018.05.004 [doi]
PST - ppublish
SO  - J Pharmacol Toxicol Methods. 2018 Nov-Dec;94(Pt 1):64-70. doi: 
      10.1016/j.vascn.2018.05.004. Epub 2018 May 24.