Why in vivo may not equal in vitro – new effectors revealed by measurement of enzymatic activities under the same in vivo-like assay conditions

Authors

  • Rodolfo García-Contreras,

    1. Section of Molecular Cell Physiology, Netherlands Institute for Systems Biology, VU University Amsterdam, The Netherlands
    2. Departamento de Bioquímica, Instituto Nacional de Cardiología, Mexico City, México
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  • Paul Vos,

    1. Department of Methodology and Applied Biostatistics, VU University Amsterdam, The Netherlands
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  • Hans V. Westerhoff,

    1. Section of Molecular Cell Physiology, Netherlands Institute for Systems Biology, VU University Amsterdam, The Netherlands
    2. Manchester Centre for Integrative Systems Biology, The University of Manchester, UK
    3. Synthetic Systems Biology, University of Amsterdam, The Netherlands
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  • Fred C. Boogerd

    Corresponding author
    • Section of Molecular Cell Physiology, Netherlands Institute for Systems Biology, VU University Amsterdam, The Netherlands
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Correspondence

F. C. Boogerd, Section of Molecular Cell Physiology, Netherlands Institute for Systems Biology, VU University Amsterdam, The Netherlands

Fax: +31 20 5987229

Tel.: +31 20 5987194

E-mail: fred.boogerd@falw.vu.nl

Website: http://www.bio.vu.nl/vakgroepen/mcp

Abstract

Does the understanding of the dynamics of biochemical networks in vivo, in terms of the properties of their components determined in vitro, require the latter to be determined all under the same conditions? An in vivo-like assay medium for enzyme activity determination was designed based on the concentrations of the major ionic constituents of the Escherichia coli cytosol: K+, Na+, Mg2+, phosphate, glutamate, sulfate and Cl. The maximum capacities (Vmax) of the extracted enzymes of two pathways were determined using both this in vivo-like assay medium and the assay medium specific for each enzyme. The enzyme activities differed between the two assay conditions. Most of the differences could be attributed to unsuspected, pleiotropic effects of K+ and phosphate. K+ activated some enzymes (aldolase, enolase and glutamate dehydrogenase) and inhibited others (phosphoglucose isomerase, phosphofructokinase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase), whereas phosphate inhibited all glycolytic enzymes and glutamine synthetase but only activated glutamine 2-oxoglutarate amidotransferase. Neither a high glutamate concentration, nor macromolecular crowding affected the glycolytic or nitrogen assimilation enzymes, other than through the product inhibition of glutamate dehydrogenase by glutamate. This strategy of assessing all pathway enzymes kinetically under the same conditions may be necessary to avoid inadvertent differences between in vivo and in vitro biochemistry. It may also serve to reveal otherwise unnoticed pleiotropic regulation, such as that demonstrated in the present study by K+ and phosphate.

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