Phosphoglucomutase from rabbit muscle

Phosphoglucose isomerase from rabbit muscle (commercially available from Sigma Aldrich code P9544-1KU), phosphomannose isomerase from E. coli, glucose-6-phosphate dehydrogenase from bakers’ yeast (S. cerevisiae), phosphoglucomutase from rabbit muscle (commercially available from Sigma Aldrich code P3397-1KU), α-D-glucose 1,6-bisphosphate potassium salt hydrate, α-D-glucose 1-phosphate disodium salt hydrate, and β-nicotinamide adenine dinucleotide phosphate sodium salt were purchased from Sigma-Aldrich. α-D-mannose 1,6-bisphosphate was prepared and purified as described [18 (link)]. SYPRO Orange was from Invitrogen Molecular Probes. All other reagents were of analytical grade.

The activity of HAPS_0849 was confirmed with a Glucose-6-Phosphate Assay Kit (Sigma, MAK014) according to the manufacturer’s manual, with some modifications. Briefly, 10 µL of 1 mM G1P was added to a 96-well plate, and then G6P assay buffer was added to each well to bring the volume to 50 µL. Another 50-µL reaction mix containing 45 µL of G6P Assay Buffer, 2 µL of G6P Enzyme Mix, 2 µL of G6P Substrate Mix and 1 µL of protein sample or phosphate buffered saline (PBS) as a negative control was prepared. Next, 50 µL of the reaction mix was added to 50 µL of the 1 mM G1P solution described above and mixed well by pipetting. The reaction was incubated for 30 min at room temperature, and then the absorbance was measured at 450 nm (A450). To eliminate the effect of background NADH or NADPH, a blank sample was prepared for each sample by omitting the glucose-6-phosphate enzyme mix. The blank readings were subtracted from the sample readings. Activity was calculated as U (units) = (A_s − A_b)/(T₁ − T₀) × df (where A_s, A450 of sample after 30 min; A_b, A450 of sample without G6P enzyme mix as a blank; T₁, time reaction was stopped; T₀, time reaction began; df, dilution factor of protein). Phosphoglucomutase from rabbit muscle (Sigma Aldrich, USA) was used as positive control.

DEAE-Sepharose ff and Superdex-75 were purchased from GE Healthcare Life Sciences, Milan, Italy. Phosphoglucose isomerase from rabbit muscle, phosphomannose isomerase from E. coli, glucose 6-phosphate dehydrogenase from baker’s yeast (S. cerevisiae), phosphoglucomutase from rabbit muscle, α-d(+)Mannose 1-phosphate sodium salt hydrate, and β-Nicotinamide adenine dinucleotide phosphate sodium salt, were purchased from Sigma-Aldrich, Milan, Italy. Sypro Orange was from Invitrogen Molecular Probes, Monza, Italy. Mannose-1,6-bisphosphate was synthesized and purified, as described [3 (link)]. All of the other reagents were of analytical grade.