Abstract
A synergistic activation of phosphorylase kinase by Ca2+ plus Mg2+ was found to be the primary cause of the hysteresis, or lag, in the phosphorylase kinase reaction. Preincubation of the enzyme for short times with Ca2+ plus Mg2+ resulted in an approximately 7-fold increase in the kinase activity in subsequent assays with phosphorylase b or phosphorylase kinase as substrates, whereas preincubation with each metal ion by itself had no effect. Maximal activation through preincubation with Ca2+ plus Mg2+ occurred in 1 min 45 s and was readily reversed by chelation of both metal ions. As a result of the activation, the progress curve of phosphorylase b conversion at pH 6.8 was found to be nearly linear. Activation by Ca2+ plus Mg2+ was not apparent when subsequent assays were carried out at pH 8.2, or when previously autophosphorylated enzyme was used. Furthermore, the synergistic activation was found to occur significantly slower and/or to decrease in the presence of ATP, phosphorylase b, beta-glycerophosphate, and inorganic phosphate. How the synergistic activation by Ca2+ plus Mg2+ relates to autophosphorylation and the lag in the phosphorylase kinase reaction is discussed.
Highlights
Ca” plus M$+ was found to be the primary cause of assays of phosphorylase b conversion couldperhaps be caused the hysteresis, or lag, in the phosphorylase kinase re- by autophosphorylation
We have looked in further detail at the activation of phoswith Ca” plus M$’ resulted in an approximately 7-fold phorylase kinase by reaction components and have found a increase in the kinase activity in subsequent assays with phosphorylaseb or phosphorylase kinaseas substrates, whereas preincubation with each metal bioyn itself had no effect
The individual subunits were present in thermore, the synergistic activation was fotounocdcur approximately equimolar concentrations as determined with a Gelsignificantly slower and/or to decreaisnethe presence man Automatic Computing Densitometer after Coomassie brilliant of ATP, phosphorylase b, &glycerophosphate, and in- blue staining of SDS gels
Summary
Ca” plus M$+ was found to be the primary cause of assays of phosphorylase b conversion couldperhaps be caused the hysteresis, or lag, in the phosphorylase kinase re- by autophosphorylation. Removal of the lag is so slow under normal assay conditions, When the time dependence of the activation by Ca2+plus those in which the enzyme is not preincubated with the pair M F was carried out in the presence of phosphorylase b
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