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

  title={Expression, purification, characterization, and deletion mutations of phosphorylase kinase $\gamma$ subunit: identification of an inhibitory domain in the $\gamma$ subunit},
  author={Chi-Ying F. Huang and Chiun-Jye Yuan and Nataliya B. Livanova and Donald J. Graves},
  journal={Molecular and Cellular Biochemistry},
A catalytic fragment,γ1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, 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… 
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The regulatory beta subunit of phosphorylase kinase interacts with glyceraldehyde-3-phosphate dehydrogenase.

The interactions described herein between the beta subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.

The Regulatory β Subunit of Phosphorylase Kinase Interacts with Glyceraldehyde‐3‐phosphate Dehydrogenase

Interactions between PhK and GAPDH provide a possible mechanism for linking glycogenolysis and glycolysis in muscle.

Physicochemical changes in phosphorylase kinase induced by its cationic activator Mg2+

In this study, changes in the physicochemical properties of PhK induced by Mg2+ under nonactivating and activating conditions were investigated by circular dichroism spectroscopy, zeta potential analyses, dynamic light scattering, second derivative UV absorption, negative stain electron microscopy, and differential chemical crosslinking.

The crystal structure of human muscle glycogen phosphorylase a with bound glucose and AMP: An intermediate conformation with T‐state and R‐state features

The Crystal Structure of Human Muscle Glycogen Phosphorylase a with Bound Glucose and AMP: An Intermediate Conformation with T-State and R-State Features Christine M. Lukacs,,* Nikos G. Oikonomakos,



Expression of the phosphorylase kinase γ subunit catalytic domain in Escherichia coli

The catalytic subunit of phosphorylase b kinase and an engineered truncated form (gamma-trc) have been expressed in Escherichia coli and properties that are characteristic of the holoenzyme and the isolated gamma subunit are observed.

Isolation and properties of the active gamma subunit of phosphorylase kinase.

Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells.

It is concluded that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma- Subunit associated with cellular calmodulin, which replaces the delta-sub unit normally associated with the gamma- subunit in the holoenzyme.

The subunit structure of rabbit-skeletal-muscle phosphorylase kinase, and the molecular basis of its activation reactions.

  • P. Cohen
  • Biology, Chemistry
    European journal of biochemistry
  • 1973
Phosphorylase kinase was isolated from rabbit skeletal muscle in a state approaching homogeneity as judged by the criteria of ultracentrifugal analysis, ion-exchange chromatography, and antigen-antibody precipitation in agar, and the evidence suggests that the α and β subunits may be structurally related.

Inhibition of the catalytic subunit of phosphorylase kinase by its alpha/beta subunits.

Analysis by mutagenesis of the ATP binding site of the gamma subunit of skeletal muscle phosphorylase kinase expressed using a baculovirus system.

Two unexpected findings are that for one residue that is very conserved (Gly26) there is some flexibility of substitution not apparent from the evolutionary conservation and that a second quite conserved residue in protein kinases does not produce a protein optimized for nucleotide binding.

Homology of the gamma subunit of phosphorylase b kinase with cAMP-dependent protein kinase.

The complete amino acid sequence of the catalytic subunit (gamma subunit) of rabbit skeletal muscle phosphorylase b kinase was determined and compared with bovine cAMP-dependent protein kinase and with tyrosine-specific kinases of viral origin revealed a significant degree of sequence identity.