Journal of Chromatography B: Biomedical Sciences and Applications
Interaction between phosphofructokinase and aldolase from Saccharomyces cerevisiae studied by aqueous two-phase partitioning
Introduction
In the living cell enzymes are part of multienzyme systems responsible for running biochemical pathways. These enzyme complexes are characterized by a high degree of organization and regulation to make their function optimized [1]. One very important and highly regulated process in the cytosol is the glycolysis, and in yeast cells, glycolytic enzymes constitute almost half of the total soluble protein [2]. High metabolic activity may require that the active sites of two sequential enzymes are close to each other and these proteins interact to channel intermediates. An enzyme–enzyme complex formation would have several biological advantages for the living cell such as regulation of metabolism, direct transfer of metabolites between enzymes and shielding metabolites from the aqueous cytosol. It is also possible that enzyme activities are controlled by association–dissociation and regulated through induced conformational changes. Evidences for the sequential enzyme–enzyme interactions with clearly advantageous overall effects on metabolism have been previously documented [3], [4], [5], [6], [7] using phase partitioning, fluorescent anisotropy and isoelectric-focusing experiments.
Phase partitioning in aqueous two-phase systems can be used to detect minute changes in surface properties, such as hydrophobicity and charge, as the partition is determined by the surface properties of the partitioned substance. Preliminary attempts to purify enzymes from commercially available Bakers’ yeast (Saccharomyces cerevisiae) provided us with an indication of mutual influences of several sequential glycolytic enzymes on each other’s behaviour in aqueous two-phase systems. We found that the partitioning of aldolase and phosphofructokinase changed with increasing degree of enzyme purification, and hence decreasing degree of contaminating enzymes. In the present study we have found an influence of highly purified phosphofructokinase (EC 2.7.1.11) and highly purified aldolase (EC 4.1.2.13) isolated from Saccharomyces cerevisiae (strain CBS 8066), on each other’s activity and partitioning in aqueous polymer two-phase systems. Multiple sequence alignment of Class II aldolases along with studies of enzyme 3D structure provided informations about two conserved residues, Glu 182 and Asp 183 (in the sequence E. coli; the Swissprotein Database number P11604) on the surface exposed loop. Their involvement in the interaction with phosphofructokinase is suggested.
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Microorganism
Saccharomyces cerevisiae, strain CBS 8066 (kindful gift from Professor Bärbel Hahn-Hägerdahl, Department of Applied Microbiology, Lund University, Lund, Sweden) was cultivated on Petri dishes on a medium with the following composition (per liter): 20 g glucose, 10 g yeast extract, 20 g proteous peptone and 20 g bacto agar at 30°C for 4 days. Cells were then transferred to 200 ml medium (similar composition as above, but without agar) in 1-l buffled Erlenmeyer flasks and grown on a rotary shaker
Results
The modified protocol for yeast phosphofructokinase purification gave purification factor and yield of 190 and 11%, respectively (Table 1), which is comparable with results reported earlier [10]. Much time was saved by the precipitation with polyethylene glycol 8000 (Table 1, steps 2 and 6) instead of two precipitation steps with ammonium sulfate, since, the dialysis prior to the ion-exchange chromatography (step 5) could be omitted.
The modified protocol for yeast aldolase purification resulted
Discussion
We here show that phosphofructokinase and aldolase interact to form a complex with different partitioning in Dextran–polyethylene glycol two-phase systems, compared to that of either enzyme. Complex formation took place both in the presence and absence of AMP, but presence of AMP was obligatory to get enhanced activities of the individual enzymes. The concentration of aldolase needed to get maximum change in K(phosphofructokinase) was around 0.025 μM, whereas that needed to get stimulation of
Acknowledgements
We thank Professor Bärbel Hahn–Hägerdahl, Department Applied Microbiology, Lund University for providing us with the yeast strain.
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Deceased, November 1999.