*4.7. Kinetic Characterization of Pyruvate Kinases*

The activity of purified cPK isoenzymes was assayed in a coupled reaction system involving lactate dehydrogenase (LDH) according to a previously described method by Plaxton [19], based on photometric detection of NAD+ formation following LDH-mediated NADH oxidation. PK activity measurement was performed in a total volume of 200 μL reaction solution (50 mM TRIS-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2, 5% *w*/*v* PEG 200, 1 mM DTT, 0.15 mM NADH) containing LDH (20 U/mL LDH, Roche) applying 5-10 μL of freshly purified PK protein in diverse concentrations. The shift in absorption was detected at ݠ =340 nm in a 96-well format in a microplate reader (Infinite M200, Tecan, Männedorf, Schweiz) at 25 ◦C. To start reactions, substrates and potential effectors of PK enzymes were added to all reaction batches simultaneously applying a house made applicator with 96 spatulas.

**Author Contributions:** For research articles with sever Conceptualization, S.K.; methodology and investigation, S.W., S.S. and S.K.; validation and interpretation, S.W., and S.K.; formal analysis, S.S. and S.K.; resources, S.K.; writing (original draft), S.W. and S.K.; writing (review and editing), all co-authors; supervision, S.K., All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by German Science Foundation, grant number Kr4245/1-1 and Kr4245/2-1.

**Acknowledgments:** We acknowledge Diana Vogelmann for technical assistance and Rainer Schwacke (Research Centre, Jülich) for critical comments and discussion.

**Conflicts of Interest:** The authors declare no conflict of interest.
