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Catecholamines and Ca2+ mediate an increase in reactive-thiol status of phosphofructokinase, associated with an increase in activation of the enzyme. A correlation was observed between the number of reactive thiols and activation (r = 0.878; P less than 0.001) for 14 different treatments, with approx. 1 group per 85,000-Mr subunit becoming available over the range from the lowest to the highest state of activation.

Perfusion of the isolated rat heart with Ca2+ concentrations exceeding 3 mM activated phosphofructokinase and phosphorylase, and decreased the concentration of cyclic AMP. Half-maximal activation of phosphofructokinase occurred at 5 mM-CaCl2; significant activation of phosphorylase did not occur until the concentration of CaCl2 exceeded 12 mM. The time course for the activation of phosphofructokinase at 12 mM-CaCl2 indicated that maximal activation occurred within 2 min; when the perfusion-medium Ca2+ concentration was re-adjusted to 3 mM, the phosphofructokinase activity returned to pre-activation values within 30 s. The addition of Ca2+ to extracts of heart did not activate phosphofructokinase. The activation of phosphofructokinase by sub-maximal doses of adrenaline and Ca2+ were not additive. The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol and by 1 microM-isoprenaline was inhibited by high concentrations of K+ (22-56 mM). The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol, 12 mM-CaCl2 and by 1 microM-isoprenaline was blocked by the slow Ca2+-channel blocker nifedipine. These findings suggest that both the beta- and alpha-adrenergic mechanisms for the activation of rat heart phosphofructokinase involve an increase in the myoplasmic Ca2+ concentration. This increase may result from an inhibition of Ca2+ efflux or a stimulation of Ca2+ influx.

Phosphofructokinase was determined at low substrate concentration using a new isotopic assay in extracts of perfused rat heart. Epinephrine treatment of the perfused heart resulted in an activation of the enzyme. Half-maximal activation of phosphofructokinase occurred at 5 X 10(-7) M epinephrine, which was approximately that required to produce half-maximal activation of phosphorylase. Time course studies indicated that epinephrine-mediated changes in beating rate, cyclic AMP concentration, and phosphorylase a activity were maximal at 1 to 2 min and preceded maximal activation of phosphofructokinase by approximately 3 min. the activated form of the enzyme as expressed in heart extracts was sensitized to the activators, cyclic AMP, AMP, glucose 1,6-bisphosphate, and fructose 1,6-bisphosphate. Passage of control extract that was untreated, activated by AMP, or inhibited by citrate through Sephadex G-25 columns gave eluate activities approaching control extract values. The epinephrine-activated form of the enzyme remained activated following similar treatment. The data suggest that epinephrine mediates a modification of phosphofructokinase that is independent of changes in intracellular effector concentration.

A high molecular-weight protein was found in heart extracts which, in the assay for phosphofructokinase, artificially activated the enzyme. The protein could be removed by gel-exclusion chromatography or high-speed centrifugation. The mechanism of activation appeared to be due to the hydrolysis of ATP to ADP, AMP and inorganic phosphate which was inhibited by Mn2+. Phosphofructokinase is thus activated by the production of activators and by the lowered inhibitory concentration of ATP. Since the adrenergic/Ca2+-activated form of the enzyme is the more sensitive to activators, the difference between the two forms of phosphofructokinase is amplified in the presence of the ATPase and diminished upon its removal or its inhibition by Mn2+. The Mn2+-sensitive ATPase appears to play no part in the adrenergic/Ca2+-mediated control of cardiac phosphofructokinase or the interconverting reactions.

The activity ratio of phosphofructokinase in perfused rat heart and its activation by epinephrine was examined in non-obese, fat-fed obese, and genetically obese rats. For non-obese colony rats there was an age-dependent increase in the activity ratio of phosphofructokinase from 0.2 at 40 days to 0.4 at mature age (greater than 200 days). Epinephrine (10 microM) treatment of the heart for 5 min increased the ratio at all ages but the proportional increase diminished with age. For mature-age lean Zucker rats carrying the genetic determinant for obesity the results were similar to those obtained for comparable non-obese colony rats. For fat-fed obese rats the activity ratio of phosphofructokinase at 200 days of age was 0.2 and was increased to 0.6 by epinephrine treatment. For mature-age obese Zucker rats the activity ratio was 0.2 and no significant response to epinephrine occurred. The activity ratio of glycogen phosphorylase and its response to epinephrine (beta-adrenergic receptor mediated) in heart was unaffected by age, diet or the gene for obesity. The present findings indicate a specific defect in the adrenergic regulatory mechanism for phosphofructokinase in genetically obese rats.

The apparent insensitivity of phosphofructokinase to activation by adrenaline in hearts from genetically obese Zucker (fa/fa) rats [Patten, Filsell & Clark (1982) Metab. Clin. Exp. 31, 1137-1141] was examined in detail. Perfusion of hearts from obese (fa/fa) animals with medium containing 1nM-100 microM-adrenaline for 4 min did not significantly activate phosphofructokinase, but fully activated glycogen phosphorylase. Activation of phosphofructokinase occurred in hearts from lean (Fa/?) animals, with half-maximal activation at 10nM-adrenaline. Binding characteristics of cardiac alpha 1-adrenergic receptors were comparable between lean and obese animals. Properties of phosphofructokinase from obese and lean hearts were similar. High extracellular Ca2+ concentration, which activated phosphofructokinase in hearts from lean animals and phosphorylase in all hearts, failed to activate phosphofructokinase in hearts from obese rats. The data indicate that the failure of adrenaline to activate phosphofructokinase in hearts from obese (fa/fa) rats results from a post-receptor defect in the as-yet unidentified Ca2+-dependent phosphofructokinase-activating reactions.