Protein Kinases

Molecular and Cellular Biochemistry

, Volume 127, Issue 1, pp 7-18

First online:

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

  • Chi-Ying F. HuangAffiliated withDepartment of Biochemistry and Biophysics, Iowa State University
  • , Chiun-Jye YuanAffiliated withDepartment of Biochemistry and Biophysics, Iowa State University
  • , Nataliya B. LivanovaAffiliated withA.N. Bach Institute of Biochemistry, Academy of ScienceDepartment of Biochemistry and Biophysics, Iowa State University
  • , Donald J. GravesAffiliated withDepartment of Biochemistry and Biophysics, Iowa State University

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Abstract

A catalytic fragment,γ 1-298, derived from limited chymotryptic digestion of phosphorylaseb 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 inE. 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 (γ 1-353 andγ 1-341) or less (γ 1-331 andγ 1-276) specific activity than does the full-length wild-type γ,γ 1-386. Three truncated forms,γ 1-316,γ 1-300, andγ 1-290 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 similarK m values for both substrates, i.e., about 18μM for phosphorylaseb and about 75 μM for MgATP. Three truncated γs,γ 1-316,γ 1-300, andγ 1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V max/K m) than that of the full-length wild-type γ and a 3.5- to 4.5-fold greater efficiency than that of the truncatedγ 1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of γ, which is located atγ 301-331·γ 1-290, but notγ 1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the γ subunit to exhibit phosphotransferase activity. Bothγ 1-290 andγ 1-300 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.

Key words

phosphorylase kinase γ subunit inhibitory domain