Effect of polyethylene glycol on the kinetic behaviour of pyruvate kinase and other potentially regulatory liver enzymes

Abstract

Assay m the presence of 10% polyethylene glycol has been systematically used with potentially regulatory liver enzymes as an Indirect way to Induce aggregation of enzymes correspondmg to that which could occur at their phywologcal concentrations Pyruvate kmase L was markedly affected by polyethylene glycol, as was muscle phosphorylase a, while pyruvate kmase M as well as glucokmase, fructose-1,6-blsphosphatase and other liver enzymes examined were not affected Enzyme concentration Polyethylene glycol Liver enzyme Pyruvate kmase Glycogen phosphorylase a

Effect of polyethylene glycol on the activity, intrinsic fluorescence, and oligomeric structure of castor seed cytosolic fructose‐1, 6‐bisphosphatase

“…By contrast, most other enzymes that have been examined in the presence of PEG, or by various in situ approaches, do not display any significant alteration in their kinetic behaviour [5,6]. The exception appears to be certain regulatory oligomers such as mammalian phosphofructokinase and pyruvate kinase [5,6], as well as plant cytosolic pyruvate kinase, ribulose 1,5-P 2 carboxylase activase, and PPi-dependent phosphofructokinase [9][10][11]. This correlation places COS FBPase~ amongst the regulatory enzymes and thus further emphasizes its preeminent role in the regulation of the gluconeogenic pathway in the plant cytosol [1,3,181. …”

“…However, a PEG effect on any FBPase has not been documented [5,6]. In this report we demonstrate that the substrate saturation kinetics and intrinsic fluorescence of a plant FBPaseo are significantly altered by the addition of PEG to the assay medium and argue that these effects are elicited via stabilization of the enzyme's native tetrameric structure.…”

“…This suggests that an increase in homologous protein-protein interactions, probably promoting subunit aggregation, is responsible for the PEG-mediated activation and inhibition of COS FBPasec at low and high concentrations of F-1,6-P2, respectively. By contrast, most other enzymes that have been examined in the presence of PEG, or by various in situ approaches, do not display any significant alteration in their kinetic behaviour [5,6]. The exception appears to be certain regulatory oligomers such as mammalian phosphofructokinase and pyruvate kinase [5,6], as well as plant cytosolic pyruvate kinase, ribulose 1,5-P 2 carboxylase activase, and PPi-dependent phosphofructokinase [9][10][11].…”

“…Cytosolic enzymes such as FBPase, however, are present in vivo at far higher concentrations than are normally used during routine in vitro assays [4]. This difference is believed to be particularly important for regulatory oligomeric enzymes because their structure, and hence their kinetic properties, may be affected by both homologous and heterologous protein-protein interactions [4][5][6][7][8][9][10][11]. The interactions between proteins that probably exist at high protein concentrations prevailing in vivo can be specifically promoted in vitro by the addition of compatible solutes (i.e.…”

“…PEG) to the reaction mixture [6]. The mechanism involves exclusion of the protein from the binary solvent, thus increasing the local enzyme concentration and favouring protein aggregation [4][5][6][7]. The in vitro activity of homogeneous COS cytosolic pyruvate *Corresponding author.…”