JBC, Vol. 251, Issue 23, 7480-7486, Dec, 1976
Stimulation of phosphorylase kinase autophosphorylation by peptide analogs of phosphorylase
G. M. Carlson and D. J. Graves
Autoactivation of phosphorylase kinase in the presence of substrates has
been studied to determine the cause of the hysteresis, or lag, in the
phosphorylase kinase reaction. Peptide analogs corresponding to the
convertible serine region of phosphorylase have been used as low molecular
weight alternative substrates. Autophosphorylation of the kinase molecule
was measured under conditions that favored autoactivation. Phosphorylase b
and a tetradecapeptide, which was found to be a good model of
phosphorylase, stimulated autoactivation by 86- and 37-fold, respectively.
The tetradecapeptide also stimulated autophosphorylation of subunits A and
B of the kinase molecule. This increased autophosphorylation coincided with
an increased ability to convert phosphorylase. This finding supports the
hypothesis that autophosphorylation is responsible for the lag in the
phosphorylase kinase reaction. No evidence was obtained to suggest that the
lag could be due to dissociation of the kinase. The stoichiometry of
phosphate incorporation into phosphorylase kinase subunits by
autophosphorylation was much greater than that reported to occur by protein
kinase phosphorylation. Multiple phosphorylation sites in subunit A
accounted for most of the phosphate incorporation during
autophosphorylation. Saturating levels of hexa- and octapeptide analogs
also caused stimulation of autophosphorylation. Possible mechanisms and
experimental implications of substrate-stimulated autophosphorylation are
discussed. Consideration also is given to the possible role of effectors in
autophosphorylation in vivo.
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