Mechanism of aggregation of UV-irradiated glycogen phosphorylase b at a low temperature in the presence of crowders and trimethylamine N-oxide.

To characterize the initial stages of protein aggregation, the kinetics of aggregation of UV-irradiated glycogen phosphorylase b (UV-Phb) was studied under conditions when the aggregation proceeded at a low rate (10°C, 0.03M Hepes buffer, pH6.8, containing 0.1M NaCl). Aggregation of UV-Phb was induced by polyethylene glycol and Ficoll-70, acting as crowders, or a natural osmolyte trimethylamine N-oxide (TMAO). It has been shown that the initial rate of the stage of aggregate growth is proportional to the protein concentration squared, suggesting that the order of aggregation with respect to the protein is equal to two. It has been concluded that the aggregation mechanism of UV-Phb at 10°C in the presence of crowders includes the nucleation stage and stages of protein aggregate growth (the basic aggregation pathway). The aggregation mechanism is complicated in the presence of TMAO, and the stage of aggregate-aggregate assembly induced by TMAO should be added to the basic aggregation pathway. It has been shown that the ability of TMAO at a low concentration (0.05M) to induce aggregation of UV-Phb is due to the decrease in the absolute value of zeta potential of the protein in the presence of TMAO.

Kinetic regime of thermal aggregation of holo- and apoglycogen phosphorylases b.

To characterize the role of pyridoxal 5'-phosphate in stabilization of the conformation of muscle glycogen phosphorylase b (Phb), the mechanism of thermal aggregation for holo- and apoforms of Phb has been studied using dynamic light scattering. The order of aggregation with respect to the protein (n) for aggregation of holoPhb at 48°C is equal to 0.5 suggesting that the dissociative mechanism of denaturation is operative and denaturation is followed by rapid aggregation stage. In the case of aggregation of apoPhb at 37°C n=2 and the rate-limiting stage is aggregation of unfolded protein molecules.

C-Glucosylated malonitrile as a key intermediate towards carbohydrate-based glycogen phosphorylase inhibitors.

Glycogen utilization involves glycogen phosphorylase, an enzyme which appears to be a potential target for the regulation of glycaemia, as the liver isoform is a major player for hepatic glucose output. A single C-glucosylated malonitrile allowed for the synthesis of three glucose-based derivatives namely bis-oxadiazoles, bis-amides and a C-glucosylated tetrahydropyrimidin-2-one. When evaluated as glycogen phosphorylase inhibitors, two of the synthesized compounds displayed inhibition in the sub-millimolar range. In silico studies revealed that only one out of the bis-amides obtained and the C-glucosylated tetrahydropyrimidin-2-one may bind at the catalytic site.

Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: does the nitration of Tyr613 in the allosteric inhibition site control enzymatic function?

There is increasing evidence that sequence-specific formation of 3-nitrotyrosine (3-NT) may cause functional changes in target proteins. Recently, the nitration of Tyr residues in glycogen phosphorylase b (Ph-b) was implicated in the age-associated decline of protein function [Sharov et al., Exp. Gerontol. 41 (2006) 407-416]; in another report, the nitration of one specific residue, Tyr613, located in the allosteric inhibition site was hypothesized as a rationale for peroxynitrite inactivation [Dairou et al., J. Mol. Biol. 372 (2007) 1009-1021]. In this study, we have optimized the analysis of in-gel Ph-b digests by high performance liquid chromatography-electro spray ionization-tandem mass spectrometry, in order to achieve a quantitative analysis of nitration of individual Tyr residues at a high coverage of Tyr-containing sequences (92%). Our data do not confirm the role of Tyr613 nitration in the control of enzymatic function. Furthermore, we show here that the enzymatic activity of Ph-b does not directly correlate with the protein nitration levels, and that the modification of Cys and, potentially, other amino acid residues can better rationalize Ph-b inactivation by peroxynitrite.

Natural flavonoids are potent inhibitors of glycogen phosphorylase.

There is little known about effects to be expected from high intake of flavonoids with respect to regulation of glucose/glycogen homeostasis. Glucose/glycogen homeostasis is mainly regulated by glycogen synthase (GS) and glycogen phosphorylase (GP). We investigated effects of naturally occurring flavonoids with varying substitution pattern on the activity of isolated muscle GP. Almost all flavonoids tested inhibited phosphorylated, active GPa, as well as unphosphorylated, adenosine monophosphate-activated GPb. However, inhibition of GPa was two-to-fourfold stronger than that of GPb. The flavonol quercetin and the anthocyanidins cyanidin and delphinidin turned out to be the most potent inhibitors of GPa, with concentration values where enzymatic activity is 50% of the respective control in the low micromolar range (<5 microM). Furthermore, by comparing GPa inhibitory activity of typical representatives from all known flavonoid classes, structural elements of flavonoids required for effective GP inhibition could be identified.