Rapid inactivation of rat liver phospho enolpyruvate carboxykinase by microsomes and reversal by reductants

Abstract

The hepatic gluconeogenic enzyme phospho enolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lase (transphosphorylating), EC 4.1.1.32) is activated 3–4-fold by micromolar quantities of Fe 2+ and ferroactivator, a cytosol protein that is necessary for this activation. After the carboxykinase in liver cytosol, or the purified carboxykinase, was activated by adding Fe 2+, rat liver microsomes were added to duplicate their ratio to the carboxykinase in liver. Microsomes caused a biphasic inactivation of the carboxykinase in cytosol. All of the activity corresponding to Fe 2+ activation was lost within 10 min ( t 1 2 equaled 3–4 min) and the remaining (basal) activity was lost over hours ( t 1 2 equaled 3–4 h). Adding the reducing agent dithiothreitol (1 mM) after 10 or 60 min of exposure of cytosol to microsomes caused a complete and three-fourths complete restoration of Fe 2+ activation. In preventing the microsome-induced loss of Fe 2+ activation, ascorbate and cysteine were also effective. When the purified carboxykinase was activated by Fe 2+ and the synthetic ferroactivator 3-aminopicolinate, microsomes interfered with the enzyme activity in a manner identical to their effect on the enzyme in cytosol, suggesting that they did not act on ferroactivator protein. When Fe 2+ was added to the crude or the purified carboxykinase 5–60 min after microsomes, the carboxykinase was not activated. If the microsomes were acting by oxidizing or sequestering Fe 2+, the carboxykinase would have been at least transiently activated because it takes only seconds for Fe 2+ to fully activate the enzyme and minutes for microsomes to cause a complete loss of Fe 2+ activation. By the process of elimination, microsomes probably act on the carboxykinase itself. Microsomes did not inactivate five other glycolytic/gluconeogenic enzymes in liver cytosol, including some that have sulfhydryls which are very sensitive to oxidation. The microsomal factor was inactivated by the proteases, chymotrypsin, trypsin and pronase. The activity of the microsomal factor was highest in liver and kidney; almost as high in heart as in liver; and low or negligible in pancreas, spleen, lung and skeletal muscle. The microsomal activity was decreased in livers of alloxan diabetic rats, and the decrease was proportional to their blood glucose concentrations — such that severely diabetic rats had almost none of the activity. Liver microsomes from rats treated with the antioxidant, vitamin E, were completely without effect on Fe 2+ activation. The results indicate that it is possible to alter rapidly the Fe 2+ activation of phospho enolpyruvate carboxykinase by mechahisms that probably involve oxidation/reduction. The decreases in the microsomal factor induced by alloxan diabetes; its high content in the gluconeogenic organs, liver and kidney; and its apparent specificity for the carboxykinase are preliminary evidence of the microsomal factor having a physiological role in the regulation of the carboxykinase.