P-enolpyruvate carboxykinase ferroactivator. Purification and some properties.
P-enolpyruvate carboxykianse ferroactivator was purified from rat liver cytosol to apparent homogeneity. This protein has an S20 of 4.69. High speed sedimentation equilibrium indicated a molecular weight of 82,000; that obtained by gel filtration was 126,000. this discrepancy in molecular weight by the two methods may indicate that this protein has a high axial ratio. A subunit molecular weight of 23,600 was obtained by sodium dodecyl sulfate electrophoresis. Synthesis of P-enolpyruvate is stimulated 3-fold when P-enolpyruvate carboxykinase is incubated in the presence of ferroactivator plus Fe2+; activity with Fe3+, Mn2+, Co2+, Cd2+, Mg2+, or Ca2+ was not affected by the ferroactivator. P-enolpyruvate synthesis by carboxykinase was activated by Mn2+ in the absence of ferroactivator. Quinolinic acid strongly inhibits carboxykinase activated by ferroactivator and Fe2+, but does not inhibit the enzyme activated by Mn2+. In the reverse reaction, ferroactivator and Fe2+ stimulated oxalacetate synthesis only briefly and carboxykinase activity rapidly returned to control rates. Increased oxalacetate synthesis by carboxykinase that had been incubated with ferroactivator and Co2+ was sustained. Stimulation of P-enolpyruvate synthesis by enzyme incubated in the presence of sulfate and Fe2+ rapidly diminished as a function of incubation time. Under similar conditions, P-enolpyruvate carboxykinase ferroactivator maintained the enzyme in the Fe2+-activated state for at least 4 h. The amounts of purified ferroactivator required to stimulate P-enolpyruvate carboxykinase maximally and the amount present in liver cytosol are sufficient to activate the enzyme maximally in vivo if adequate Fe2+ were present. It is possible that the rate of P-enolpyruvate synthesis in gluconeogenic tissues may be regulated by the availability of intracellular Fe2+ to the ferroactivator and P-enolpyruvate carboxykinase.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003600:Cytosol / Q000502:physiology; D004789:Enzyme Activation; D007501:Iron / Q000032:analysis / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008667:Metalloproteins / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism*; D051381:Rats
D000596:Amino Acids; D008667:Metalloproteins; D007501:Iron; D010729:Phosphoenolpyruvate Carboxykinase (GTP)