Effect of membrane lipids on phosphofructokinase from Bacillus stearothermophilus using a novel 33P radiochemical assay

Tsaloglou, Maria-Nefeli, Dymond, Marcus K. and Attard, George S. (2007) Effect of membrane lipids on phosphofructokinase from Bacillus stearothermophilus using a novel 33P radiochemical assay. Chemistry and Physics of Lipids, 149 (Supplement 1), S46-S47. (doi:10.1016/j.chemphyslip.2007.06.105).

Abstract

Phosphofructokinase (PFK-1), a major regulatory enzyme of glycolysis, catalyses the phosphorylation of d-fructose 6-phosphate by ATP to yield d-fructose 1,6-bisphosphate. Mammalian PFK-1 is known to bind to biomembranes with a regulatory effect on enzyme activity (Karadsheh and Uyeda, 1977). We have conducted a study into whether PFK-1 activity depends on membrane lipid composition and whether this enzyme is regulated via membrane stored curvature elastic stress, in an analogous manner to CTP:phosphocholine cytidylytransferase (CCT) (Attard et al., 2000 G.S. Attard, R.H. Templer, W.S. Smith, A.N. Hunt and S. Jackowski, PNAS 97 (16) (2000), p. 9032. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (82)[Attard et al., 2000] and [Attard et al., 2006]). As part of this investigation, we developed a novel 33P radiochemical assay for PFK-1. The assay combines the benefits of a previously available 32P assay (Sola-Penna et al., 2002), with the advantage of a markedly smaller decay energy. Kinetic studies yielded comparable results to both the spectrophotometric and 32P radiochemical assays, confirming the reliability of the method. The effect of vesicles composed of dioleoyl phosphatidylcholine (DOPC) and oleic acid (OA) on PFK-1 from Bacillus stearothermophilus was studied. OA was found to increase catalytic activity twofold and threefold compared to DOPC and no lipid present, respectively. While these results suggest that stored curvature elastic energy may play a role in modulating enzyme activity, they are significantly different from the results obtained with CCT. Therefore, interesting questions arise about the fundamental mechanisms that underlie the effect of membrane lipid composition on PFK-1 activity.

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Contributors

Author: George S. Attard

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