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title. ADP-Glc PPi. G3P Ox. G3P Ox. G3P Ox. G3P Ox. Product. Product. Rate of reaction. O 2. O 2. O 2. O 2. H 2 O 2. H 2 O 2. H 2 O 2. H 2 O 2. GK. TPI. NTP. GAP. DAP. DAP. DAP. DAP. G3P. G3P. G3P. G3P. Glycerol. NADH,H +. NADH,H +. NAD +. NAD +. t. t.

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  2. ADP-Glc PPi G3P Ox G3P Ox G3P Ox G3P Ox Product Product Rate of reaction O2 O2 O2 O2 H2O2 H2O2 H2O2 H2O2 GK TPI NTP GAP DAP DAP DAP DAP G3P G3P G3P G3P Glycerol NADH,H+ NADH,H+ NAD+ NAD+ t t [G3P]+[DAP] G3P DH G3P DH Automation with a pipetting robot and a 96 plate reader MPIMP-Golm:Enyzme Activity Profiling Enzymatic Cycling Assays for sensitivity ‚Classic‘ enzymatic metabolite test The rate depends on the amount of substrates in the cycle In the mid-term: complement/supplement with protemics (AG Weckwerth

  3. AGPase GK PPi + ADPG ATP G3P PGK GAP DH TPI ATP + 3PGA BPGA GAP DAP GK ATP + Gly G3P Ald PFP TPI Gly3POX PPi + F6P FBP Triose-P G3P O2 H2O2 GDH PK GK ADP + PEP ATP G3P SPS UMPK GK G3P DAP F6P + UDPG UDP ATP G3P TK TPI GDH E4P + X5P GAP DAP G3P NADH,H+ NAD+ Gly3PDH FBPase PGI G6PDH FBP F6P G6P NADPH G6PDH G6PDH PGI FruK G6P NADPH G6P 6PG ATP + Fru F6P G6PDH GlcK ATP + Glc G6P NADPH NADP+ NADPH,H+ G6PDH NADP+ + G6P NADPH MTTox MTTred ICDH NADP+ + i-Cit NADPH PMS ShikDH NADP+ + Shik NADPH AlaAT LDH Ala +2OG Pyr NAD+ ADH MDH AspAT EtOH Ethanal NAD+ Asp + 2OG OxA MDH Fum NAD+ Fum + H2O Mal NADH NADH,H+ CS GLDH NADH + Glu NAD+ MTTox MTTred PEPC MDH PES CO2 + PEP OxA NAD+ Assay, Enzyme, Coupling Reactions Cycling System Modular Organisation of the Enzyme Platform aids Sample Flow

  4. Manual extraction and aliquoting Stopped assay Product determination Automatised calculation Dilution 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 No robot: 4 enzymes in 20 samples per person-day 4-tip robot. 4 enzymes in 100 samples per person day 96 tip robot: expect a further 3-8 foled increase

  5. Enzyme assay platform – performance

  6. Glucose-1-phosphate adenylyltransferase (E.C. 2.7.7.27) Nitrate reductase (E.C. 1.7.1.1) 3000 2000 14000 800 2500 12000 1500 Activity 700 2000 At1g27680 Activity (max) 10000 600 Transcript level At2g21590 Activity (sel) At4g39210 1500 1000 At1g37130 500 8000 At5g19220 At1g77760 400 At5g48300 1000 6000 300 500 4000 500 200 2000 100 0 0 20 16 120 144 0 5 10 15 0 4 8 12 20 24 10 72 96 0 20 30 40 50 0 0 Time (h) Time (h) Time (h) Time (h) 0 16 0 12 96 4 8 12 24 4 8 16 20 24 72 120 144 20 0 12 24 36 48 Time (h) Time (h) Time (h) Time (h) Fumarase 5000 8000 4000 Activity 6000 At5g50950 3000 4000 2000 2000 1000 0 0 16 144 0 4 8 12 20 24 0 4 8 12 16 20 24 72 96 120 0 8 16 24 32 40 48 Time (h) Time (h) Time (h) Time (h) Ferredoxin-dependent glutamate synthase 10000 4000 8000 Activity At2g41220 3000 6000 At5g04140 2000 4000 1000 2000 0 0 0 4 8 12 16 20 24 0 4 8 12 16 20 24 0 8 16 24 32 40 48 72 96 120 144 Time (h) Time (h) Time (h) Time (h) Compare changes of enzyme activity and transcripts … Diurnal changes in pgm Diurnal changes in wild-types Extended night in wild-types

  7. Extension of the night leads to - large and rapid changes of transcript levels, - smaller and delayed changes of enzyme activites A 2h E 48h B 4h F 72h Transcripts Enzyme activities C 8h G 148h ‚Global‘ analysis of data for 23 enzymes and the corresponding transcripts D 24h H

  8. Amplitude of diurnal activity change Amplitude of diurnal transcript change There no relation between the amplitudes of the diurnal change of an enzymes and the diurnal change of the encoding transcripts is Wild-type pgm Enyzmes with a very smooth curve 23 Enzymes

  9. AlaAT Fd-GOGAT GDH GS Fructokinase NIA Fumarase AGPase PFP 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 activtyafter a given delay (see x-axis) for each of the six 4 h time intervals.

  10. 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 Almost all pgm samples are classified as like a wild-type plant that has been in the dark for 3 days Enzyme activities are a quasi-stable parameter that integrates changes over time and regulation at several levels, and can be measured precisely

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