Abstract
The catalytic activity of phosphoenolpyruvate carboxykinase in rat liver cytosol is stimulated by incubating with Fe2+, Mn2+, Co2+, and Cd2+. When purified, the enzyme no longer responds to Fe2+, Co2+, or Cd2+ but retains a response to Mn2+. Low concentrations of SO4(2-) in the incubation medium with enzyme and divalent transition metal allow stimulation by Fe2+ and Co2+ and enhance the response to Mn2+. Under identical conditions, orthophosphate with Fe2+ is a potent inhibitor of the enzyme (half-maximal inhibition at 50 muM). A thiol is required in the incubation medium for the effects of Fe2+ plus sulfate or orthophosphate to be expressed. The magnitude of these effects depends on the thiol concentration. Dithiothreitol is more effective than GSH and activation by sulfate plus Fe2+ appears to require the reduced form of dithiothreitol. Sulfate ion is not considered to be the physiological Fe2+-activator of P-enolpyruvate carboxykinase in rat liver cytosol, as this function is fulfilled by a newly discovered liver protein. Knowledge concerning the interaction of Fe2+ and sulfate with the enzyme may be useful in examining their interaction between the enzyme, ferrous ion, and this activator protein.
Highlights
From the Department of Biochemistry Madison, Wisconsin 53706 and the Institute for Enzyme Research, University of Wisconsin, The catalytic activity of phosphoenolpyruvate carboxykinase in rat liver cytosol is stimulated by incubating with Fez+, Mn2+, Co*+, and Cd*+
Ammonium sulfate, when incubated with the pure enzyme plus 30 /LM Fe*+, restored the response to Fez+. This necessitated the removal of ammonium sulfate prior to the assay of enzymatic activity
The purified enzyme was prepared as needed from the 70% ammonium sulfate suspension by extensive dialysis uer.sus the glycerol-containing buffer described above
Summary
From the Department of Biochemistry Madison, Wisconsin 53706 and the Institute for Enzyme Research, University of Wisconsin, The catalytic activity of phosphoenolpyruvate carboxykinase in rat liver cytosol is stimulated by incubating with Fez+, Mn2+, Co*+, and Cd*+. Low concentrations of SOa2m in the incubation medium with enzyme and divalent transition metal allow stimulation by Fe’+ and Co2+ and enhance the response to Mn’+. A thiol is required in the incubation medium for the effects of Fe’+ plus sulfate or orthophosphate to be expressed. The magnitude of these effects depends on the thiol concentration. Fe*+-activator of P-enolpyruvate carboxykinase in rat liver cytosol, as this function is fulfilled by a newly discovered liver protein. Knowledge concerning the interaction of Fez+ and sulfate with the enzyme may be useful in examining the interaction between the enzyme, ferrous ion, and this activator protein
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