title. enzymatic cycling assays for sensitivity t product nadh,h + o2o2 h2o2h2o2 dap g3p g3p ox g3p...
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title
Enzymatic Cycling Assays for sensitivity
t
Product
NADH,H+
O2 H2O2
DAPG3P
G3P OxG3P Ox
G3P DHG3P DH
O2 H2O2
DAPG3P
G3P OxG3P Ox
NAD+
GAPNTPTPITPIGKGK
Glycerol
NADH,H+
O2 H2O2
DAPG3P
G3P OxG3P Ox
G3P DHG3P DH
O2 H2O2
DAPG3P
G3P OxG3P Ox
NAD+
t
Product
[G3P]+[DAP]
Rate of reaction
ADP-Glc PPi
MPIMP-Golm:Enyzme Activity Profiling
‚Classic‘ enzymatic metabolite test
The rate depends on the amount of substrates in the cycle
Automation with a pipetting robot and a 96 plate reader
In the mid-term: complement/supplement with protemics (AG Weckwerth
Modular Organisation of the Enzyme Platform aids Sample Flow
DAP
NADH,H+
O2 H2O2
Gly3POX
Gly3PDHNAD+
G3P
NADPH,H+
MTTox
G6P 6PGG6PDH
PMSMTTred
NADP+
NADH,H+
MTTox
EtOH EthanalADH
PESMTTred
NAD+
PKATPADP + PEP
GKG3P
UMPKUDP
SPSF6P + UDPG
GKG3PATP
TPIGAP
TKE4P + X5P
GDHG3PDAP
AGPasePPi + ADPG ATP
GKG3P
GAP DHGAP
TPIDAPBPGA
PGKATP + 3PGA
ATP + GlyGK
G3P
Triose-PFBPPFP
PPi + F6PGDH
G3PTPIAld
G6PDHNADPHNADP+ + G6P
NADPHICDH
NADP+ + i-Cit
NADPHShikDH
NADP+ + Shik
PGIG6P
G6PDHNADPHF6P
FBPaseFBP
ATP + FruFruK
F6P
NADPHG6PGlcK
ATP + GlcG6PDH
G6PG6PDH
NADPHPGI
GLDHNAD+NADH + Glu
OxAPEPC
CO2 + PEP
LDHNAD+Pyr
AlaATAla +2OG
Asp + 2OGAspAT
OxA
NADHMalFum
Fum + H2OMDH
NAD+MDH
CS
NAD+MDH
Assay, Enzyme, Coupling Reactions Cycling System
Streamlining of Logistics
- All in ELISA format, with robot automation
- Programmed robotics tracks samples and automatises pipetting steps
- All pipetting volumes and incubation times are standardised
- Several ‚stopped‘assays flow into a shared product-determination
- Prepared Excel spread sheets allow automatic calculatin of results after pasting of raw data from the reader
- Automatised calculation of quality control parameters
Stoppedassay
Productdetermination
Automatisedcalculation
Manual extraction
and aliquoting
Dilution
No robot: 4 enzymes in 20 samples per person-day4-tip robot. 4 enzymes in 100 samples per person day 96 tip robot: expect a further 3-8 foled increase
Enzyme assay platform – performanceEnzyme Method Freeze/thawing
stability (% retained ± SD)
Dilution of FW Average activity (Arabidopsis rosette, nmol g-1 FW min-1)
Average coefficient of variation (%)
AGPase
Gly3POX / Gly3PDH
85 ± 5 1000 1255 12
NAD-GAPDH 90 ± 1 20000 36981 15
NADP-GAPDH 86 ± 3 10000 17041 14
PFP 26 ± 9 1000 191 16
PK 70 ± 9 1000 952 18
SPS n.d. 1000 540 8
TK 99 ± 3 1000 7462 17
GK 51 ± 9 500 18 33
cFBPase
Glc6PDH / (PMS+MTT)
55 ± 26 500 105 23
GlucoK 65 ± 13 1000 150 23
FructoK 65 ± 11 1000 179 18
Glc6PDH 37 ± 9 5000 849 13
NADP-IDH 97 ± 29 2000 1506 24
Shik DH 48 ± 5 2000 598 18
AlaAT
ADH / (PES+MTT)
79 ± 21 20000 5462 20
AspAT 81 ± 30 20000 5229 19
Fumarase 9 ± 3 20000 4875 10
GLDHam 97 ± 7 1000 876 11
PEPCase 126 ± 35 744 19
Acid Inv Glucose oxidase + Amplex Red
62 ± 14 1000 266 20
Fd-GOGAT Glutamate DH 37 ± 7 500 3370 13
NR SA + NNEDA175 ± 49 (Vsel)
93 ± 18 (Vmax)500
X575
128
GS PK + LDH 129 ± 16 1000 1867 14
Compare changes of enzyme activity and transcripts …
Time (h)0 4 8 12 16 20 24
Tra
ns
cri
pt
lev
el
0
2000
4000
6000
8000
10000
12000
14000
Activity (max)Activity (sel)At1g37130At1g77760
Time (h)0 4 8 12 16 20 24
Nitrate reductase (E.C. 1.7.1.1)
Time (h)72 96 120 144
0
100
200
300
400
500
600
700
800
Time (h)0 12 24 36 48
Time (h)0 4 8 12 16 20 24
0
1000
2000
3000
4000
5000
ActivityAt5g50950
Time (h)0 4 8 12 16 20 24
Time (h)72 96 120 144
0
2000
4000
6000
8000
Fumarase
Time (h)0 8 16 24 32 40 48
Time (h)0 4 8 12 16 20 24
0
2000
4000
6000
8000
10000
ActivityAt2g41220At5g04140
Time (h)0 4 8 12 16 20 24
Ferredoxin-dependent glutamate synthase
Time (h)72 96 120 144
0
1000
2000
3000
4000
Time (h)0 8 16 24 32 40 48
Time (h)0 5 10 15 20
0
500
1000
1500
2000
2500
3000
ActivityAt1g27680At2g21590At4g39210At5g19220At5g48300
Time (h)0 4 8 12 16 20 24
Time (h)72 96 120 144
0
500
1000
1500
2000
Glucose-1-phosphate adenylyltransferase (E.C. 2.7.7.27)
Time (h)0 10 20 30 40 50
Extended night in wild-types
Diurnal changes in wild-types
Diurnal changes in pgm
Extension of the night leads to
- large and rapid changes of transcript levels,
- smaller and delayed changes
of enzyme activites
0.00
0.10
0.20
0.30
0.40
0.50
0.60
2 4 8 24 48 72 148
Extended night (h)
T-T
est
valu
e (a
gai
nst
en
d o
f th
e n
igh
t d
ata)
-5 -4 -3 -2 -1 0 1 2 3 4 5
Transcripts
Activities
-5 -4 -3 -2 -1 0 1 2 3 4 5
Transcripts
Activities
-5 -4 -3 -2 -1 0 1 2 3 4 5
Transcripts
Activities
-5 -4 -3 -2 -1 0 1 2 3 4 5
Transcripts
Activities
-5 -4 -3 -2 -1 0 1 2 3 4 5
Transcripts
Activities
-5 -4 -3 -2 -1 0 1 2 3 4 5
Activities
-5 -4 -3 -2 -1 0 1 2 3 4 5
Activities
A
F
G
E
D
C
B
H
2h
4h
8h
24h
48h
72h
148h
‚Global‘ analysis of data for 23 enzymes and the corresponding transcripts
Transcripts
Enzyme activities
There no relation between the amplitudes of the diurnal change of an enzymes and the diurnal change of the encoding transcripts is
23 Enzymes
Wild-typepgm
Enyzmes with avery smoothcurve
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100
Amplitude summed transcripts
Am
plitu
de a
cti
vit
ies
WT pgm
Amplitude of diurnal transcript change
Am
plit
ude
of d
iurn
al a
ctiv
ity
chan
ge
GDH
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
0 4 8 12 16 20 24
Lag between transcript levels and activity
R2
WT
pgm
GDHAla AT
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
0 4 8 12 16 20 24
Lag between transcript levels and activity
R2
WT
pgm
AlaATGS
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
0 4 8 12 16 20 24
Lag between transcript levels and activity
R2
WT
pgm
GS
PFP
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
0 4 8 12 16 20 24
Lag between transcript levels and activity
R2
WT
pgm
PFP
Fd-GOGAT
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
0 4 8 12 16 20 24
Lag between transcript levels and activity
R2
WT
pgm
Fd-GOGAT
Fructokinase
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
0 4 8 12 16 20 24
Lag between transcript levels and activity
R2
WT
pgm
Fructokinase
NR
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
0 4 8 12 16 20 24
Lag between transcript levels and activity
R2
WT
pgm
NIA
AGPase
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
0 4 8 12 16 20 24
Lag between transcript levels and activity
R2
WT
pgm
AGPaseFumarase
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
0 4 8 12 16 20 24
Lag between transcript levels and activity
R2
WT
pgm
FumaraseNADP-GAPDH
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
0 4 8 12 16 20 24
Lag between transcript levels and activityR
2
WT
pgm
NADP-GAPDH
Transcript changes are followed only after a delay by changes of enzyme activity, and the delay varies from enzyme to enzyme
Fig. 6J. Time lag of the response of individual enzyme activities in wildtpye polants ( ) and pgm ( ). Regression coefficnets were estimated as in panels I-J, and are here plotted for 10 selected enzymes. Each point rtepresents the mean of 6 regression
coefficents, calculated from a plot of the change of transcript against the change of enzyme activty after a given delay (see x-axis) for each of the six 4 h time intervals.
Enzyme activities are a quasi-stable parameter that integrates changes over time and regulation at several levels, and can be measured precisely
Almost all pgm samples are classified as like a wild-type plant that has been in the dark for 3 days
Decision tree constructed using samples from wild-type plants during the diurnal cycle, and after 6 h, and 1, 2, 3 and 7 days in the dark