title: Purification of glycogen phosphorylase isozymes by metal-affinity chromatography.
abstract: Mammalian phosphorylase isozymes from muscle, brain and liver were expressed in Escherichia coli and purified from the crude bacterial cell extracts in one step using a copper-loaded, metal-affinity matrix. Good chromatographic behavior, enzyme activity and protein stability were maintained by judicious choice of pH and buffer which contained 250 mM sodium chloride and 25 mM beta-glycerophosphate at pH 7.0. Small amounts of beta-mercaptoethanol and EDTA in the buffers further stabilized the enzymes, but stripped some of the metal from the column which, nonetheless, retained good chromatographic characteristics. Owing to the presence of multiple surface histidine residues in the phosphorylase dimers, good enzyme purities (90-98%) and recoveries (>90%) were routinely obtained from crude bacterial lysates after two passes through the copper column. Of the various metal ions which were investigated, Cu2+ gave the best chromatographic results. Imidazole gradients at constant pH were used to selectively desorb the phosphorylase from the metal column whose capacity for phosphorylase binding in the presence of bacterial proteins exceeded 30 mg enzyme per milliliter of matrix.
authors: Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
authors: C B
authors: CB
authors: Luong
authors: M F
authors: MF
authors: Browner
authors: R J
authors: RJ
authors: Fletterick
authors: B L
authors: BL
authors: Haymore
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007098:Imino Acids; D007527:Isoenzymes / Q000302:isolation & purification*; D008670:Metals; D006005:Phosphorylases / Q000302:isolation & purification*; D011817:Rabbits
chemical_list: D007098:Imino Acids; D007527:Isoenzymes; D008670:Metals; C030699:copper(II)-iminodiacetate; D006005:Phosphorylases; D003545:Cysteine