Monitoring The Level of The Universal Nucleotide AMP in Diverse Enzymatic Reactions Using
Bioluminescent Homogenous Assay Platform
Said A. Goueli1,2, Kevin Hsiao1, Subhanjan Mondal1
1Cell
Signaling group, Research and Development, Promega Corp. and 2Dept. of Pathology and laboratory Medicine, University of Wisconsin Medical School, Madison, WI 53711
Abstract 3735
2. Assay Principle & Protocol for AMP-Glo™
ADP conversion &
ATP detection
Net RLU
Light
ADP
0
12.5µl
5µl
< 2.5µl
2X Substrate
12.5µl
5µl
< 2.5µl
AMP-Glo™ Reagent I
25µl
10µl
< 5µl
AMP Detection Solution
50µl
20µl
< 10µl
Km
(A T P )
= 2 1 .7 8 µ M
0
200
400
600
800
1000
A T P (µ M )
2
3
4
Peptides, H3,
H4, dsDNA,
Nucleosome
200
400
Ub+ ATP
All
p53+ ATP
E1+ Ub+ ATP
600
800
CARP2 Titration with 100µM ATP Using AMP-Glo™
500
1000
Asp-AA tRNA Synthetase (ng)
U s in g A M P -G lo ™
1 .5  1 0
7
1 .0  1 0
7
5 .0  1 0
6
RLU
2.0E+06
0
1500
200
400
600
800
1,000
1,200
CARP2 (nM)
250,000
SUMOylation of RanGAP (1µM) Study with 10µM ATP Using AMP-Glo™
RLU
RLU
0
- 1 .5
SUMO + ATP
0
- 1 .0
- 0 .5
0 .0
0 .5
1 .0
0
20
L o g 1 0 [ P D E 4 B 2 ] , n g /R x n
40
60
80
610
6
410
6
E1 + SUMO + RanGAP + ATP
E1 + E2 + SUMO + RanGAP + ATP
E1 + E2 + SUMO + RanGAP
NEDDylation of p53
7,000,000
5,000,000
15,000
10,000
0
0
1,000
1,500
DNMT1 (ng/Rxm)
2,000
150,000
50,000
0
0
6
SAH (µM)
EZH1 Complex
140,000
9
3,000,000
R² = 0.9955
100,000
80,000
60,000
40,000
R² = 0.9902
20,000
0
0
10
20
30
40
50
0
PRMT5/MEP50 (ng/Rxn)
200
400
600
800
Enzyme Complex (ng/Rxn)
9. Features of Promega Luminescent AMP-Glo™
•Universal: AMP Detection in the Presence or Absence of ATP as Substrate
•HTS Formatted: 96-, 384-, and 1536-well Plates
•Stable Signal
1,000,000
210
6
0
E1 + ATP
0
-2
-1
0
1
L o g 1 0 [ R o lip r a m ] , µ M
2
3
E1 + NEDD8 + ATP
E1 + E2 + p53 +
NEDD8 + ATP
E1 + E2 + E3 + p53 + E1 + E2 + E3 + p53 + E1 + E2 + E3 + p53 +
NEDD8 + ATP
ATP
NEDD8
www.promega.com
12
EZH2 Complex
120,000
R² = 0.9983
3
EZH1 & EZH2 Complex Titration Using H3
peptide and Methyltransferase-Glo™
160,000
20µM H4 (His-tag Full Length)
180,000
90,000
500
200,000
100,000
270,000
5,000
0
Light
20µM H4 (1-20) Peptide
360,000
R² = 0.9939
R² = 0.9961
250,000
•Ultrasensitive: pmoles of nucleotides
4,000,000
2,000,000
IC 5 0 ( µ M ) = 2 . 7 7
Detection Solution
•Robust: Z’>0.8
6,000,000
U s in g A M P -G lo ™
6
E1 + SUMO + ATP
100
cA M P, µM
R o lip r a m T itr a tio n in P D E 4 B 2 (1 .5 n g /R x n )
810
(384LV Format)
300,000
•Luminescent: Free of Fluorescent Compounds Interference
50,000
K d = 1 3 .1 5 µ M
E C 5 0 (n g /R x n ) = 0 .5
0
SAH Titration Using MTase-Glo™
•Homogenous, Nonradioactive and Antibody free
100,000
6
12
•Broad Applications in Diverse Biochemical Reactions (See Fogures)
200,000
150,000
6
10
350,000
PRMT5/MEP50 Titration using two different substrates
(20µM SAM) Using Methyltransferase-Glo™
450,000
20,000
3.0E+06
8
MTase-Glo™
Reagent
25,000
(SUMOylation & NEDDylation)
c A M P T itr a tio n in P D E 4 B 2 F u ll L e n g th
6
(Luciferin/Luciferase)
R² = 0.9848
8. Quantification of AMP in Biochemical Reactions
P D E 4 B 2 F u ll L e n g t h T it r a t i o n
4
MTase-Glo™
AMP/ADP
DNMT1 Titration Using Methyltransferase-Glo™
2Hrs Rxn with 0.15µM E1 (Ube1), 0.6µM E2 (UbcH5c), & 40µM Ubiquitin-WT
RLU
0
2
CH3
Peptides,
H3, H4, DNA,
Nucleosome
E1+E2+ Ub E1+ E2+ E3+ E1+ E2+ E3+ E1+ E2+ E3+
+p53+ ATP Ub+ p53+ ATP p53+ ATP
Ub+ p53
5.0E+06
RLU
2 .0  1 0
E1+E2+Ub
0.0E+00
( 1 0 µ M c A M P ) U s in g A M P - G lo ™
RLU
RLU
4 .0  1 0
E1+E2
1.0E+06
7. Quantification of AMP in Biochemical Reactions
(cAMP-Phosphodiesterase PDE4B2)
6
E2+Ub
0
Thr-AA tRNA Synthetase (ng)
6 .0  1 0
E2 Alone
90,000
0
6
E1+Ub
180,000
0
8 .0  1 0
E1 Alone
270,000
90,000
0
Nicotinamide Nucleotide (µM)
SAH
MTase-Glo™ Reagent can be added during MT Rxn
4.0E+06
180,000
Methyltransferases
SAM: S-Adenosyl-Methionine
SAH: S-Adenosyl-Homocysteine
360,000
270,000
3
(Methyltransferases)
SAM
6.0E+06
450,000
RLU
RLU
1
2.5
6. Quantification of AMP in Biochemical Reactions
8.0E+06
0.0E+00
0.0E+00
(1mg/ml Calf Liver t-RNA, 1mM Asp, & 100µM ATP)
360,000
2
2.0E+06
Human Asp-tRNA Synthetase Using AMP-Glo™
450,000
1.5
1.0E+07
4.0E+06
L o g 1 0 [ A m in o a c y l t R N A s y n th e t a s e - E .c o li] , n g
Human Thr-tRNA Synthetase Using AMP-Glo™
1
E. coli DNA Ligase, U/Rxn/5µl
5.0E+05
0
(1mg/ml Calf Liver tRNA,1mM Thr, & 100µM ATP)
0.5
1.2E+07
0
0
0
0
1Hr Rxn with 0.1µM E1 (Ube1), 1.0µM E2 (UbcH5c), 0.2µM Mdm2, & 10µM Ubiquitin-WT
1.0E+06
R² = 0.9985
10 µM Oligos
50,000
0
1.6E+07
E C 5 0 (n g ) = 1 6 4
100000
NADP + Oligos + Ligase (20min Rxn)
100,000
20,000
Ubiquitination of p53 (0.5µM) Study with 50µM ATP Using AMP-Glo™
1.5E+06
200000
200000
60,000
5. Quantification of AMP in Biochemical Reactions
(CARP2 & Mdm2 – Ubiquitin Ligase)
1Hrs Rxn with 0.15µM E1 (Ube1), 0.6µM E2 (UbcH5c), & 40µM Ubiquitin-WT
12
NADPH + Oligos + Ligase (20min Rxn)
80,000
40,000
CARP2 (2µM) Study with 100µM ATP Using AMP-Glo™
10
NAD + Oligos + Ligase (20min Rxn)
150,000
100,000
384LV- or
1536-well
well
8
NADH + Oligos + Ligase (20min Rxn)
R² = 0.9919
RLU
384-well
Compds/Enzyme
2.0E+06
300000
6
200,000
120,000
RLU
400000
4
T4 Ligase, U/Rxn
Nicotinamide Nucleotide specificity as Substrate
for E. coli DNA Ligase Using AMP-Glo™
E. coli DNA Ligase Titration Using AMP-Glo™
AMP-Glo™ Reaction Format
96-well
RLU
600000
2
(10µM Oligos & 10µM NAD)
2.5E+06
400000
1.0E+05
140,000
RLU
500000
RLU
RLU
600000
800000
1.5E+05
0.0E+00
1.4E+07
1000000
R² = 0.9984
2.0E+05
5.0E+04
AMP
Detection
Solution
AMP-Glo™
Reagent I
Ubiquitin Ligase
Aminoacyl tRNA Synthetase,
DNA Ligase(Mammalian)
Succ CoA Synthetase, etc.
2.5E+05
RLU
U s in g A M P -G lo ™ a s s a y
ATP depletion &
AMP conversion
AMP
(+/- ATP)
RLU
E . c o li ( K m A T P )
(0.5µg DNA (pBR322)/Rxn with 100µM ATP)
3.0E+05
cAMP
RNA
NAD
(Total E. Coli tRNA Synthetases and Human Asp and Thr AA t-RNA Synthetases)
A m in o a c y l t-R N A s y n th e ta s e ( E . c o li) A c tiv ity S tu d y
T4 DNA Ligase (ATP substrate) & E. coli DNA Ligase (NAD substrate)
T4 DNA Ligase Titration Using AMP-Glo™
cAMP-PDE
Poly A-Deadenylases
Ribonucleases
DNA ligase (bacterial)
4. Quantification of AMP in Biochemical Reactions
K in e tic P a r a m e te r F o r A m in o a c y l tR N A s y n th e ta s e
3. Quantification of AMP in Biochemical Reactions
RLU
Adenine nucleotides are major determinants of the energy status of the cell and thus any
modulation of their cellular concentration has significant consequences to cellular
metabolism, cellular growth and cell death. Many biochemical reactions that generate AMP
as a reaction product are pivotal players in a variety of signaling pathways and thus are
considered validated drug targets. These include enzymes involved in protein ubiquitination
(Ubiquitin ligases), protein synthesis (aminoacyl tRNA synthetases), second messenger
signaling (cAMP-phosphodiesterases), and DNA synthesis and repair (prokaryotic and
eukaryotic DNA ligases). Therefore, an assay that monitors the activity of these enzymes is
desirable in the search for selective modulators and the development of novel therapeutics.
As AMP is a common product of these enzymatic reactions, the development of an assay
that monitors AMP in a homogenous and sensitive fashion will have significant impact in
these diverse areas of research. In addition, enzyme reactions that generate products which
can be converted to AMP can be also monitored using this assay. We demonstrate the utility
of this assay platform in monitoring the activity of cAMP-PDE, aminoacyl tRNA synthetases,
ubiquitin ligases, DNA ligases. Furthermore, enzyme reactions such as methyltransferases
that produce product (SAH) which can be converted to AMP can be monitored using this
assay platform.
AMP Generating Reactions
1. Abstract
For technical information: [email protected]
March 2014
1000