Molecular and Cellular Biochemistry

, Volume 127, Issue 1, pp 7–18

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

Authors

  • Chi-Ying F. Huang
    • Department of Biochemistry and BiophysicsIowa State University
  • Chiun-Jye Yuan
    • Department of Biochemistry and BiophysicsIowa State University
  • Nataliya B. Livanova
    • A.N. Bach Institute of BiochemistryAcademy of Science
    • Department of Biochemistry and BiophysicsIowa State University
  • Donald J. Graves
    • Department of Biochemistry and BiophysicsIowa State University
Protein Kinases

DOI: 10.1007/BF01076753

Cite this article as:
Huang, C.F., Yuan, C., Livanova, N.B. et al. Mol Cell Biochem (1993) 127: 7. doi:10.1007/BF01076753

Abstract

A catalytic fragment,γ1-298, 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 (γ1-353 andγ1-341) or less (γ1-331 andγ1-276) specific activity than does the full-length wild-type γ,γ1-386. Three truncated forms,γ1-316,γ1-300, andγ1-290 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 similarKm values for both substrates, i.e., about 18μM for phosphorylaseb and about 75 μM for MgATP. Three truncated γs,γ1-316,γ1-300, andγ1-290, 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 truncatedγ1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of γ, which is located atγ301-331·γ1-290, but notγ1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the γ subunit to exhibit phosphotransferase activity. Bothγ1-290 andγ1-300 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 subunitinhibitory domain

Copyright information

© Kluwer Academic Publishers 1993