Expression of the phosphorylase kinase gamma subunit catalytic domain in Escherichia coli.
The catalytic subunit of phosphorylase b kinase (gamma) and an engineered truncated form (gamma-trc, residues 1-297) have been expressed in Escherichia coli. The truncated protein included the entire catalytic domain as defined by sequence alignment with other protein kinases but lacked the putative calmodulin binding domain. Full-length protein was produced in insoluble aggregates. Some activity was regenerated by solubilization in urea and dilution into renaturating buffer but the activity was found to be associated with a smaller molecular weight component. Full-length protein could not be refolded successfully. The truncated gamma subunit was produced in the soluble fraction of the cell as well as in inclusion bodies. The insoluble protein was refolded by dilution from urea and purified to homogeneity, in a one step separation on DEAE-Sepharose to give a protein mol. wt 32,000 +/- 2000 with a high sp. act. of 5.3 mumol 32P incorporated into phosphorylase b(PPB)/min/nmol. Kinetic parameters gave Km for ATP 46 +/- 3 microM and Km for PPb 27 +/- 1 microM. The sp. act. and the Km values are comparable to those observed for the activated holoenzyme and indicate that the gamma-trc retains the substrate recognition and catalytic properties. The ratio of activities at pH 6.8/8.2 was 0.84. gamma-trc was inhibited by ADP with a Ki of 52 microM and was sensitive to activation by Mg2+ and inhibition by Mn2+, properties that are characteristic of the holoenzyme and the isolated gamma subunit. Calmodulin which confers calcium sensitivity on the isolated gamma subunit had no effect on the enzymic properties of gamma-trc.(ABSTRACT TRUNCATED AT 250 WORDS)
Cox
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Laboratory of Molecular Biophysics, University of Oxford, UK.
Johnson
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D000244:Adenosine Diphosphate / Q000494:pharmacology; D001665:Binding Sites; D002147:Calmodulin / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002451:Cell Compartmentation; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D010764:Phosphorylase Kinase / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects; D012710:Serum Albumin, Bovine / Q000494:pharmacology
D002147:Calmodulin; D002413:Cations, Divalent; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D000244:Adenosine Diphosphate; D010764:Phosphorylase Kinase