title. enzymatic cycling assays for sensitivity t product nadh,h + o2o2 h2o2h2o2 dap g3p g3p ox g3p...

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