Purification and crystallization of bovine liver phosphorylase.
We have purified and crystallized bovine liver phosphorylase a. Starting from 2.5 kg of liver, we obtain 250 mg of phosphorylase a, with a specific activity of 90 units/mg, representing 15% recovery. SDS polyacrylamide gels show three bands, a 95 kDa band with the same mobility as muscle phosphorylase, and two smaller bands of 55 kDa and 40 kDa, which are probably proteolytic fragments. These fragments remain associated and have native conformation and catalytic activity. Crystals which diffract to 2.8 A resolution, were prepared by the hanging drop method using polyethylene glycol PEG 4000 as precipitant. The crystals were prepared in the presence of activators maltotriose and phosphite and crack when placed in solutions containing the inhibitors glucose and caffeine. This suggests phosphorylase is present in an active conformation.
D000818:Animals; D002417:Cattle; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010710:Phosphates; D010762:Phosphorylase a / Q000302:isolation & purification* / Q000378:metabolism; D006005:Phosphorylases / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D011188:Potassium; D017680:Potassium Compounds; D011487:Protein Conformation; D014312:Trisaccharides
D010446:Peptide Fragments; D010710:Phosphates; D017680:Potassium Compounds; D014312:Trisaccharides; D011092:Polyethylene Glycols; C008317:maltotriose; C013216:potassium phosphate; D010762:Phosphorylase a; D006005:Phosphorylases; D011188:Potassium