A recombinant form of the catalytic subunit of phosphorylase kinase that is soluble, monomeric, and includes key C-terminal residues.
Residues 302-326 of the catalytic (gamma) subunit of phosphorylase kinase (PhK) may comprise an autoinhibitory, pseudosubstrate domain that binds calmodulin. To study this, the cDNA corresponding to rabbit muscle PhKgamma was expressed using Escherichia coli. This yielded two stable, high-activity PhKgamma forms (35 and 42 kDa by SDS-PAGE) that were smaller than an authentic sample of rabbit muscle PhKgamma (45 kDa by SDS-PAGE). Each recombinant form was purified to homogeneity. The N-terminal sequence of the larger, 42-kDa form (pk42) matched that of the rabbit muscle enzyme. This suggested that pk42 consisted of PhKgamma residues 1-362, including the putative calmodulin-binding, autoinhibitory domain. Kinetic parameters obtained for pk42 were like those previously reported for the intact gamma subunit. This implied that the lack of 25 PhKgamma C-terminal residues did not affect phosphorylase kinase activity, but greatly improved enzyme stability. An additional 60 residues were removed from the C-terminus of pk42 using the protease m-calpain. This increased the kinase activity 1.5-fold. Consistent with this, the activity of a mutant PhKgamma that consisted of residues 1-300, denoted gamma1-300, was like that of the m-calpain-treated enzyme. Therefore, although the effect was small, some influence by the C-terminus of pk42 was noted. Moreover, when pk42 was incubated with ATP alone, a C-terminal threonine residue became phosphorylated. Although the influence of this autophosphorylation cannot be inferred from this data, it was evidence that the C-terminus accessed the enzyme's active site. Taken together, these data imply that pk42 will be useful to study phosphorylase kinase structure/activity relationships.
Pete
M J
MJ
Signal Transduction Training Group, Iowa State University, Ames, Iowa, 50011, USA.
Liao
C X
CX
Bartleson
C
C
Graves
D J
DJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002154:Calpain / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics* / Q000502:physiology; D002853:Chromatography, Liquid; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010764:Phosphorylase Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010763:Phosphorylase b / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013696:Temperature
D002147:Calmodulin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D010763:Phosphorylase b; D010764:Phosphorylase Kinase; D002154:Calpain