Expression, purification, characterization, and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

Chi Ying F. Huang, Chiun-Jye Yuan, Nataliya B. Livanova, Donald J. Graves*

*Corresponding author for this work

Research output: Contribution to journalArticle

25 Scopus citations

Abstract

A catalytic fragment, γ1-298, derived from limited chymotryptic digestion of phosphorylase b kinase (Harris, W.R. et al., J. Biol. Chem., 265: 11740-11745, 1990), is reported to have about six-fold greater specific activity than does the γ subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the γ subunit, full-length wild-type and seven truncated forms of γ were expressed in E. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar (γ1-353 and γ1-341) or less (γ1-331 and γ1-276) specific activity than does the full-length wild-type γ, γ1-386. Three truncated forms, γ1-316, γ1-300, and γ1-290 have molar specific activities approximately twice as great as those of the full-length wild-type γ and the nonactivated holoenzyme. All recombinant γs exhibit similar Km values for both substrates, i.e., about 18μM for phosphorylase b and about 75 μM for MgATP. Three truncated γs, γ1-316, γ1-300, and γ1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (Vmax/Km) than that of the full-length wild-type γ and a 3.5- to 4.5-fold greater efficiency than that of the truncated γ1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of γ, which is located at γ301-331·γ1-290, but not γ1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the γ subunit to exhibit phosphotransferase activity. Both γ1-290 and γ1-300 have several properties similar to full-length wild-type γ, including metal ion responses (activation by free Mg2+ and inhibition by free Mn2+) pH dependency, and substrate specificities.

Original languageEnglish
Pages (from-to)7-18
Number of pages12
JournalMolecular and Cellular Biochemistry
Volume127-128
Issue number1
DOIs
StatePublished - 1 Nov 1993

Keywords

  • inhibitory domain
  • phosphorylase kinase γ subunit

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