Macromolecular interactions, Consequences on carbohydrate metabolism

Abstract: Most cells are crowded with solutes, enzymes, nucleic acids, structural proteins and membranes. Crowding promotes macromolecular interactions and determines major properties of the cellular environment such as viscosity, diffusion and inhomogeneity. Macromolecular interactions that involve enzymes can affect the kinetic and regulatory properties of these, hence the interactions are part of the metabolic regulation. The interaction between two sequential glycolytic enzymes from Saccharomyces cerevisiae, phosphofructokinase and aldolase, was characterized using aqueous polymer two-phase partitioning. Complex formation took place both in the presence and absence of AMP but activities were only enhanced in its presence. The metabolic advantage arising from this interaction is the channeling of fructose 1,6-bisphosphate. Sucrose metabolism was studied in BY-2 tobacco cell suspensions, with focus on sucrose synthase (SuSy). Two isoforms, SuSy1 and SuSy2 with distinct catalytic properties could be isolated. They were differently regulated by the actin cytoskeleton. Furthermore, fructose 2,6-bisphosphate (F26BP), a powerful metabolic regulator, exerted its effect in an isoform-specific manner and more strongly so in the presence of actin. An indication for isoform specific SuSy association with plasma membrane (PM) was obtained. To gain insight into existing regulatory mechanisms that are inevitably affected upon rupture of cellular structures and dilution of the cytosol, an in situ method was developed, based on the selective cell permeabilization with the channel forming peptide alamethicin. The method was found to be very useful in studies of the cytochrome and alternative pathway capacities and measurements of enzyme activities inside permeabilized cells, especially since the tonoplast remained intact.