title. enzymatic cycling assays for sensitivity t product nadh,h + o2o2 h2o2h2o2 dap g3p g3p ox g3p...
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![Page 1: Title. Enzymatic Cycling Assays for sensitivity t Product NADH,H + O2O2 H2O2H2O2 DAP G3P G3P Ox G3P DH O2O2 H2O2H2O2 DAP G3P G3P Ox NAD + GAPNTPTPIGK](https://reader030.vdocuments.site/reader030/viewer/2022032805/56649edc5503460f94bed7e2/html5/thumbnails/1.jpg)
title
![Page 2: Title. Enzymatic Cycling Assays for sensitivity t Product NADH,H + O2O2 H2O2H2O2 DAP G3P G3P Ox G3P DH O2O2 H2O2H2O2 DAP G3P G3P Ox NAD + GAPNTPTPIGK](https://reader030.vdocuments.site/reader030/viewer/2022032805/56649edc5503460f94bed7e2/html5/thumbnails/2.jpg)
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
![Page 3: Title. Enzymatic Cycling Assays for sensitivity t Product NADH,H + O2O2 H2O2H2O2 DAP G3P G3P Ox G3P DH O2O2 H2O2H2O2 DAP G3P G3P Ox NAD + GAPNTPTPIGK](https://reader030.vdocuments.site/reader030/viewer/2022032805/56649edc5503460f94bed7e2/html5/thumbnails/3.jpg)
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
![Page 4: Title. Enzymatic Cycling Assays for sensitivity t Product NADH,H + O2O2 H2O2H2O2 DAP G3P G3P Ox G3P DH O2O2 H2O2H2O2 DAP G3P G3P Ox NAD + GAPNTPTPIGK](https://reader030.vdocuments.site/reader030/viewer/2022032805/56649edc5503460f94bed7e2/html5/thumbnails/4.jpg)
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
![Page 5: Title. Enzymatic Cycling Assays for sensitivity t Product NADH,H + O2O2 H2O2H2O2 DAP G3P G3P Ox G3P DH O2O2 H2O2H2O2 DAP G3P G3P Ox NAD + GAPNTPTPIGK](https://reader030.vdocuments.site/reader030/viewer/2022032805/56649edc5503460f94bed7e2/html5/thumbnails/5.jpg)
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
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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
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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
![Page 8: Title. Enzymatic Cycling Assays for sensitivity t Product NADH,H + O2O2 H2O2H2O2 DAP G3P G3P Ox G3P DH O2O2 H2O2H2O2 DAP G3P G3P Ox NAD + GAPNTPTPIGK](https://reader030.vdocuments.site/reader030/viewer/2022032805/56649edc5503460f94bed7e2/html5/thumbnails/8.jpg)
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
![Page 9: Title. Enzymatic Cycling Assays for sensitivity t Product NADH,H + O2O2 H2O2H2O2 DAP G3P G3P Ox G3P DH O2O2 H2O2H2O2 DAP G3P G3P Ox NAD + GAPNTPTPIGK](https://reader030.vdocuments.site/reader030/viewer/2022032805/56649edc5503460f94bed7e2/html5/thumbnails/9.jpg)
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.
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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