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Addition of glucagon to isolated hepatocytes reduced the activity of 6-phosphofructokinase (ATP:D-fructose--phosphate 1-phosphotransferase, EC 2.7.1.11) and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) Phosphorylation contributed to the inhibition of pyruvate kinase, but several lines of evidence indicated that this reaction was not responsible for the inhibition of phosphofructokinase. First, the increase in phosphorylation in intact cells induced by increasing the concentration of glucagon did not correlate well with the decrease in enzyme activity. Second, phosphorylation of phosphofructokinase induced by addition of cyclic AMP and Mg2 -ATP or by addition of Mge+-ATP and the catalytic subunit of the cyclic AMP-dependent protein kinase to hepatocyte extracts had no effect on enzyme activity. Third, ammonium sulfate precipitation of the enzyme from extracts of cells incubated with glucagon abolished the hormone effect. The effect could be restored, however, by the addition of a phosphofructokinase-free extract from glucagon-treated cells to the ammonium sulfate-treated enzyme from either untreated or glucagontreated cells. These results suggest that the inhibition of phosphofructokinase by glucagon is due to changes in the level of an allosteric effector(s). The addition of glucagon to isolated rat hepatocytes inhibits the activity of both phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.11.1) (1-3) and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) (4-8). These effects account in part for the stimulation of gluconeogenesis by the hormone. The inhibition of both enzymes is characterized by increases in the substrate concentration needed for half-maximal activity (So.5) (1,2,(4)(5)(6)(7)(8) and in sensitivity to inhibition by ATP (3,5, 7). Glucagon also stimulates the in vivo phosphorylation of both enzymes (3, 9), and this has led to the hypothesis that enzyme inhibition by phosphorylation is the mechanism by which the hormone stimulates gluconeogenesis (10). The results we report here support this hypothesis in the case of pyruvate kinase, but not in the case of phosphofructokinase.MATERIALS AND METHODS Preparation and Incubation of Hepatocytes. Isolated hepatocytes were prepared from fed rats (male SpragueDawley, 200-300 g) as described (8). The cells were suspended to a final concentration of 50 mg of liver/ml in Krebs-Henseleit buffer that contained 0.5% bacitracin. In experiments in which only phosphofructokinase activity was measured, heated extracts from 5-ml aliquots of cell suspension were prepared as described (1), except that the cells were homogenized in 1.5 ml of buffer (50 mM potassium phosphate, pH 7.5/100 mM NaF/1 mM EDTA/20 mM 3-mercaptoethanol/0. 1 mM phenylmethylsulfonyl fluoride). In experiments in which 32p incorThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this...

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