Relative phosphate contents of phosphofructokinase and other soluble phosphoproteins in skeletal muscle

In vivo phosphorylation of muscle proteins has been studied by incorporation of [³²P]phosphate with emphasis placed upon the phosphorylation of glycolytic enzymes. Of the approximately 25 soluble proteins resolved by two-dimensional electrophoresis that contain significant ³²P, phosphofructokinase was the sole glycolytic enzyme identified as a phosphoprotein. The extent of phosphorylation found for this enzyme was the same as determined previously for purified phosphofructokinase and was about the same as the extent of phosphorylation of phosphorylase in resting muscle. Subsequent partial purification of several glycolytic enzymes confirmed the absence of significant amount of phosphate. However, phosphoglycerate mutase contained small amounts of covalently bound ³²P that was exchangeable with 3-phosphoglycerate and therefore, most likely was incorporated during the catalytic reaction cycle. Analogous results were obtained for phosphoglucomutase. Both mutases were also phosphorylated at the same sites by the catalytic subunit of cyclic AMP-dependent protein kinase.     

Differences in the allosteric properties of pure low and high phosphate forms of phosphofructokinase from rat liver

Low phosphate and high phosphate forms of phosphofructokinase (Furuya, E., and Uyeda, K. (1980) J. Biol. Chem. 255, 11656-11659) from rat liver were purified to homogeneity and various properties were compared. The specific activities of these enzymes and their electrophoretic mobilities on polyacrylamide in sodium dodecyl sulfate are the same. A limited tryptic digestion yields products with no change in the enzyme activity but with a reduction in the molecular weight of about 2000. Both low and high phosphate enzymes can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, and approximately twice as much [32P]phosphate is incorporated into the low phosphate than the high phosphate enzyme. A comparison of their allosteric kinetic properties reveal that the high phosphate enzyme is much more sensitive to inhibition by ATP and citrate and shows a higher K0.5 for fructose 6-phosphate than the low phosphate enzyme, and the difference in the K0.5 values becomes greater at lower pH values. Furthermore, the high phosphate phosphofructokinase is less sensitive to activation by AMP and fructose 2,6-bisphosphate. Moreover, when the low phosphate enzyme is phosphorylated by protein kinase, the resulting phosphorylated enzyme exhibits a higher K0.5 for fructose 2,6-bisphosphate than does the untreated enzyme. These results demonstrate that the phosphorylation affects the allosteric kinetic properties of the enzyme and results in a less active form of phosphofructokinase.     

Hormone-stimulated phosphorylation of liver phosphofructokinase in vivo.

The effect of glucagon on the phosphorylation and the enzymic activity of phosphofructokinase in rat liver in vivo was investigated. Glucagon stimulated the phosphorylation of liver phosphofructokinase approximately 3- to 5-fold and increased cAMP levels 5-fold and blood glucose levels 2-fold over the values obtained for control animals. The specific radioactivity of ATP isolated from liver was the same in both control and hormone-treated animals. During the purification of the 32P-labeled enzyme from both animals, no difference was observed in the total or specific enzyme activities of the enzymes from the various fractions. Thus, phosphofructokinase appears to be phosphorylated in vivo by a cyclic AMP-dependent protein kinase. Although phosphorylation does not affect the maximum catalytic activity of the enzyme, it does render the enzyme significantly more sensitive to ATP inhibition. Thus, at a given concentration of ATP, the phosphorylated phosphofructokinase exhibits considerably lower activity than the unphosphorylated enzyme. The possible relationship between our observations and glucagon-mediated control of glycolysis is discussed.     

Phosphorylation of ATP citrate lyase in response to glucagon.

Incubation of hepatocytes with [32P]orthophosphate resulted in the incorporation of 32P into material that is precipitated by reaction with antibodies to ATP citrate lyase. The amount of radioactivity precipitated was decreased when unlabeled, purified ATP citrate lyase was added to extracts of hepatocytes that had been incubated with [32P]orthophosphate. Addition of glucagon to hepatocytes that had been preincubated with [32P]orthophosphate resulted in a 56% increase in acid-stable 32P in the trichloroacetic acid-insoluble portion of immunoprecipitates. Catalytic phosphate bound to ATP citrate lyase reaction with ATP and Mg2+ is acid-labile; thus, glucagon-dependent phosphorylation is distinguished from the catalytic phosphate. When hepatocytes were incubated in the absence of [32P]orthophosphate and extracted in a medium containing [gamma-32P]ATP, no acid-stable 32P was present in immunoprecipitates. This indicates that the incorporation into ATP citrate lyase of acid-stable phosphate occurs prior to extraction of the enzyme. Preliminary studies, using a procedure that allows for measurement of enzyme activity starting 1 min after beginning the extraction of lyase from hepatocytes, have shown no difference in lyase activity when hepatocytes are treated with or without glucagon.     

Epinephrine-stimulated phosphorylation of pyruvate kinase in hepatocytes

Incubation of rat liver parenchymal cells with 10^?5m epinephrine or norepinephrine resulted in a rapid incorporation of ³²P into pyruvate kinase. Inclusion of α-adrenergic blocking agents (phenoxybenzamine or phentolamine) in the hepatocyte incubation medium prior to addition of epinephrine suppressed the subsequent phosphorylation of pyruvate kinase. On the other hand, inclusion of the β-adrenergic antagonist, propranolol, in the hepatocyte incubation medium prior to addition of epinephrine did not suppress the epinephrine-elicited phosphorylation of pyruvate kinase. Exogenous addition of either cyclic AMP or cyclic GMP to the hepatocyte incubation medium also resulted in increased phosphorylation of pyruvate kinase. To investigate whether the same amino acid residue(s) of liver pyruvate kinase was being phosphorylated in each instance, ³²P-labeled pyruvate kinase was isolated from hepatocytes after incubation in the presence or absence of either glucagon or epinephrine. In addition, purified liver pyruvate kinase was phosphorylated in vitro with a rat liver cyclic AMP-dependent protein kinase. Each ³²P-labeled pyruvate kinase was then subjected to tryptic digestion, two-dimensional thin-layer peptide mapping, and autoradiography. Each ³²P-labeled pyruvate kinase sample yielded 44 to 48 tryptic peptides upon staining with ninhydrin and 4 peptides that contain ³²P as detected by autoradiography. Furthermore, the same 4 peptides of pyruvate kinase were radiolabeled in each instance. Thus phosphorylation of pyruvate kinase in vitro with [γ-³²P]ATP or upon addition of either glucagon or epinephrine to hepatocytes incubated with ³²P_i resulted in phosphorylation of the same amino acid residues.     

Glucagon-induced phosphorylation of pyruvate kinase (type L) in rat liver slices.

The effect of glucagon on the phosphorylation of pyruvate kinase in ³²P-labelled slices from rat liver was investigated. Pyruvate kinase was isolated by immunoadsorbent chromatography. The enzyme was partially phosphorylated in the absence of added hormone (0.2 mol of phosphate/mol of enzyme subunit). Upon incubation with 10^?7 M glucagon, the incorporation of [³²P]phosphate was 0.6–0.7 mol/mol of enzyme subunit. Concomitantly, the concentration of intracellular cyclic 3′,5′-AMP increased from 0.3 to 3.2 μM. The phosphorylation inhibited the enzyme activity at low concentrations of phosphoenolpyruvate (60% at 0.5 mM). Almost maximal phosphorylation of the enzyme was reached within 2 min after the addition of glucagon. The concentration of hormone giving half maximal effect on the pyruvate kinase phosphorylation was about 7×10^?9M. The inactivation of the enzyme paralleled the increase in phosphorylation. It is concluded that pyruvate kinase is phosphorylated in the intact liver cell.     

Phosphofructokinase from Fasciola hepatica: activation by phosphorylation and other regulatory properties distinct from the mammalian enzyme

Phosphofructokinase from the liver fluke, Fasciola hepatica, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase isolated from this organism. Phosphorylated fluke phosphofructokinase had a sevenfold lower apparent Km for its substrate, Fru-6-P, and an eightfold higher 0.5 Vopt for ATP, the enzyme's primary inhibitor, than native phosphofructokinase. Activation of fluke phosphofructokinase following phorphorylation by a mammalian protein kinase catalytic subunit was previously reported (E. S. Kamemoto and T. E. Mansour (1986) J. Biol. Chem. 261, 4346-4351). The catalytic subunit of protein kinase isolated from the liver fluke phosphorylated sites on fluke phosphofructokinase similar to those phosphorylated by the mammalian enzyme. Maximal phosphate incorporation was 0.3 mol P/mol of protomer. The native enzyme was found to contain 1.3 mol P/mol of protomer. In contrast to fluke phosphofructokinase, activity of the mammalian heart enzyme was slightly decreased following phosphorylation. The dependence of allosteric interaction on an acidic pH observed with the mammalian phosphofructokinase was not observed with the fluke enzyme. Unlike mammalian phosphofructokinase, allosteric kinetics of the fluke enzyme was observed at alkaline pH (8.0). Fluke phosphofructokinase was found to be relatively insensitive to inhibition by citrate, a known potent inhibitor of the mammalian enzyme. Fru-2,6-P2, a potent modifier of phosphofructokinase from a variety of sources, was found to activate both native and phosphorylated fluke phosphofructokinase. The most potent activators of fluke phosphofructokinase were found to be Fru-2,6-P2, AMP, and phosphorylation. The endogenous level of Fru-2,6-P2 in the flukes was determined to be 29 +/- 1.3 nmol/g wet wt, a level that may well modulate enzyme activity. Fru-6-P,2-kinase, the enzyme responsible for synthesis of Fru-2,6-P2, was found to be present in the flukes. Our results suggest physiological roles for phosphorylation and Fru-2,6-P2 in regulation of fluke phosphofructokinase.     

Phosphorylation of phosphofructokinase from skeletal muscle: correlations between phosphorylation and muscle function.

The specific radioactivity of [³²P]-phosphate incorporated into muscle phosphofructokinase was in equilibrium with the specific radioactivity of the γ-phosphate group of ATP. The incorporation was independent of the presence of cycloheximide. The total content of covalently bound phosphate in phosphofructokinase was correlated with the functional state of the muscle from which the enzyme was purified. Muscle dissected post mortem led to phosphofructokinase containing less than 2 phosphate groups per tetramer. Muscle dissected in vivo gave phosphofructokinase with 4 phosphates per tetramer when kept at rest and 8 phosphates per tetramer when stimulated to contract.     

Purification and properties of phosphofructokinase 2/fructose 2,6-bisphosphatase from chicken liver and from pigeon muscle

Phosphofructokinase 2 and fructose 2,6-bisphosphatase extracted from either chicken liver or pigeon muscle co-purified up to homogeneity. The two homogeneous proteins were found to be dimers of relative molecular mass (Mr) close to 110,000 with subunits of Mr 54,000 for the chicken liver enzyme and 53,000 for the pigeon muscle enzyme. The latter also contained a minor constituent of Mr 54,000. Incubation of the chicken liver enzyme with the catalytic subunit of cyclic-AMP-dependent protein kinase in the presence of [gamma-32P]ATP resulted in the incorporation of about 0.8 mol phosphate/mol enzyme. Under similar conditions, the pigeon muscle enzyme was phosphorylated to an extent of only 0.05 mol phosphate/mol enzyme and all the incorporated phosphate was found in the minor 54,000-Mr constituent. The maximal activity of the native avian liver phosphofructokinase 2 was little affected by changes of pH between 6 and 10. Its phosphorylation by cyclic-AMP-dependent protein kinase resulted in a more than 90% inactivation at pH values below 7.5 and in no or little change in activity at pH 10. Intermediary values of inactivation were observed at pH values between 8 and 10. Muscle phosphofructokinase 2 had little activity at pH below 7 and was maximally active at pH 10. Its partial phosphorylation resulted in a further 25% decrease of its already low activity measured at pH 7.1 and in a negligible inactivation at pH 8.5. Phosphoenolpyruvate and citrate inhibited phosphofructokinase 2 from both origins non-competitively. The muscle enzyme and the phosphorylated liver enzyme displayed much more affinity for these inhibitors than the native liver enzyme. Fructose 2,6-bisphosphatase from both sources had about the same specific activity but only the chicken liver enzyme was activated about twofold upon incubation with ATP and cyclic-AMP-dependent protein kinase. All enzyme forms were inhibited by fructose 6-phosphate and this inhibition was released by inorganic phosphate and by glycerol 3-phosphate. Both liver and muscle fructose 2,6-bisphosphatases formed a 32P-labeled enzyme intermediate when incubated in the presence of fructose 2,6-[2-32P]bisphosphate.     

A rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts and its application to the study of 32Pi uptake in perfused rat heart

(i) A new, rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts in the presence of other ³²P-containing compounds is described. The deproteinized extract is incubated with phosphorylase b and phosphorylase kinase, and the incorporation of ³²P into protein from [γ-³²P]ATP is measured by precipitation on filter paper in trichloroacetic acid. No separation of ATP or other treatment of the extracts is required for the assay. (ii) ³²P_i uptake in perfused rat heart was found to be a relatively slow process, with a K_m of 0.084 mm, whereas equilibration between intracellular ³²P_i and [γ-³²P]ATP occurred rapidly.     

Rat liver phosphofructokinase isozymes

The labile phosphofructokinase activity of rat liver was found to be stabilized and efficiently extracted in 50 mm Tris-HCl, pH 8.0, 50 mm NaF, 10 mm dithiothreitol, and 1.0 mm ATP. By the method of DEAE-cellulose chromatography liver phosphofructokinase activity could be resolved into two isozymes. The major isozyme which was 85% of the total isolated activity was purified to homogeneity. This 15,000-fold purified isozyme had a specific activity of about 90 IU/mg protein with 25–30% recovery of the total activity. Sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis of the sodium dodecyl sulfate-treated isozyme indicated a subunit molecular weight of 65,000. Antiserum to the major isozyme was obtained from rabbits, and immunotitration of the two isozymes indicated that they were immunologically different. Kinetic properties of the two isozymes indicated that the major isozyme was more susceptible to ATP and citrate inhibition as well as relief of ATP and citrate inhibition by fructose-6-P, AMP, and ammonia. With the use of DEAE-cellulose chromatography and antiserum titration of 100,000g supernatant fluids, it was shown that the two hepatic isozymes were always found together in adult, embryonic, and neoplastic liver and in kidney.     

Effects of insulin and work on fructose 2,6-bisphosphate content and phosphofructokinase activity in perfused rat hearts

The effects of insulin and increased cardiac work on glycolytic rate, metabolite content, and fructose 2,6-bisphosphate (Fru-2,6-P2) content were studied in isolated perfused rat hearts. Steady-state rates of glycolysis increased 5-fold with the addition of insulin to the perfusate or by increasing cardiac pressure-volume work and correlated well in most conditions with changes in substrate concentration (Fru-6-P) and with concentration of the activator, Fru-2,6-P2. There was no correlation with changes in other well known regulators including citrate, ATP, AMP, Pi, or cytosolic phosphorylation potential. Using phosphofructokinase purified from hearts perfused under identical conditions, allosteric kinetic experiments were performed using the metabolite and effector concentrations determined from in vivo experiments. Reaction rates for phosphofructokinase calculated in vitro agreed well with the glycolytic rates measured in vivo and correlated with changes in Fru-6-P but not with other effectors. However, higher Fru-2,6-P2 levels were more effective in maintaining phosphofructokinase activity at high ATP and citrate levels. Kinetic experiments did not indicate a covalent modification of phosphofructokinase. These data indicate that control of cardiac phosphofructokinase and glycolysis may be accomplished by changes in the availability of substrate, Fru-6-P, and activator, Fru-2,6-P2, rather than by citrate, adenine nucleotides, or cytosolic phosphorylation potential as previously suggested.     

Comparison of phospho- and dephospho-ATP citrate lyase

The dephospho- form of rat liver citrate lyase has been prepared by treating purified [³²P]-ATP citrate lyase with a partially purified phosphatase. A comparison of the properties of the phospho- and dephosphoenzyme has been performed. The pH optima were the same for both forms of the enzyme in four different buffer systems although the optimum values varied identically for both enzyme forms with the buffer. Both the phospho- and dephosphoenzymes show the same kinetic properties except for the K_m observed for ATP in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer system where it was 54 μm for the phosphoenzyme and 292 μm for the dephosphoenzyme. The present study also indicates that both enzymes are cleaved by trypsin and lysosomal proteases in a similar manner. Both forms of the enzyme tend to associate with mitochondria to the same extent and both enzymes have identical temperature stability curves.     

Cyclic AMP stimulated phosphorylation of liver pyruvate kinase in hepatocytes.

The addition of glucagon (10^?6 M) to an incubation mixture containing ³²Pi and hepatocytes isolated from livers of rats fed $\text{ad libitum}$ results in both a 3-fold increased incorporation of ³²P into L-type pyruvate kinase and a decreased catalytic activity. The ³²P incorporated into pyruvate kinase was covalently bound to the enzyme as evidenced by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In addition, exogenous cyclic AMP (10^?3 M) stimulated the phosphorylation and the suppression of catalytic activity to a similar extent. On the other hand, insulin (10^?7 M) had essentially no effect on the incorporation of ³²P into pyruvate kinase or on its catalytic activity under the conditions used in this study. These results suggest that phosphorylation of pyruvate kinase $\text{invivo}$ is stimulated by glucagon via cyclic AMP and cyclic AMP-dependent protein kinase and that the activity of the enzyme is, at least in part, regulated by a phosphorylation-dephosphorylation mechanism.     

Differences in phosphofructokinase regulation in normal and tumor rat thyroid cells.

The kinetic and molecular properties of a phosphofructokinase derived from a transplantable rat thyroid tumor lacking regulatory control on the glycolytic pathway were studied. The properties of the near-purified enzyme (specific activity 140 units/mg) were compared with those of phosphofructokinase from normal rat thyroid (specific activity 134 units/mg). The electrophoretic mobilities and gel elution behavior of these two enzymes were almost similar. The thyroid tumor phosphofructokinase showed, however, a greater degree of size and/or shape heterogeneity in the presence of ATP than the normal thyroid enzyme, as determined by gel filtration and sucrose density gradient centrifugation. Kinetic studies below pH 7.4 showed a sigmoid response curve for both enzymes when the velocity was determined at 1 mM ATP with varying levels of fructose-6-P. The interaction coefficient, however, was 4.2 and 2.6 for normal and tumor thyroid phosphofructokinase, respectively. Ammonium sulfate decreased the cooperative interactions with the substrate fructose-6-P in both enzymes. The thyroid tumor enzyme, however, was less sensitive to the inhibition by ATP and by citrate. The reversal of citrate inhibition by cyclic 3':5'-adenosine monophosphate was also less effective with the thyroid tumor phosphofructokinase, while the protective effect of fructose-6-P was stronger. The difference in citrate inhibition between tumor and normal thyroid enzyme was not strongly affected by varying the MgCl2 concentration up to 10 mM. It is concluded that the complex allosteric regulation typical of the normal thyroid phosphofructokinase is still present in the enzyme isolated from the thyroid tumor tissue. The latter, however, is more loosely controlled by its physiological effectors, such as ATP, citrate, and cyclic AMP.     

Etude de quelques propriétés de la phosphofructokinase érythrocytaire de l'homme normal

Human erythrocyte phosphofructokinase was purified 150 fold by DEAE cellulose adsorption and ammonium sulfate precipitation.At pH 7,5 the enzyme exhibits allosteric kinetics with respect to ATP, fructose 6 phosphate, and Mg²⁺.ATP at high concentration acted as an inhibitor and ADP, 5′AMP, 3′,5′, AMP, acted as activators. Both effectors seemed to decrease the homotropic interactions beetween the fructose 6 phosphate molecules.The activators increased the affinity of phosphofructokinase for the substrate (F6P), the inhibitor decreased it.These ligands had no effect on the maximum velocity of the reaction except in the case of ADP.Interactions between the substrates and the effector ligands on the enzyme were considered in terms of the Monod - Changeux - Wyman model for allosteric proteins.With GTP and ITP, no inhibition was observed. At saturing concentration of GTP, ATP still inhibited phosphofructokinase.Both 3′5′ AMP and fructose 6 phosphate increased the concentration of ATP required to produce an inhibition of 50 %.Citrate, like ATP, inhibited phosphofructokinase by binding most likely at the same allosteric site. Erythrocyte phosphofructokinase is inhibited by 2–3 DPG.The study of the relation log V max = f (pH) suggested, that the active center contains at least one imidazole and one sulfhydryl group.     

Catecholamine-induced phosphorylation of cardiac muscle proteins

The catecholamine-induced phosphorylation of cardiac muscle protein was investigated using a rat ventricular muscle slice preparation. Slices 0.5 mm thick and weighing 40–50 mg were incubated for 40 min in oxygenated bathing medium containing ³²P to partially label intracellular ATP. Subsequent addition of 10^?5 M isoproterenol for 10 min resulted in a 44–63% (based on protein) or a 63–70% (based on inorganic phosphate) increase in ³²P incorporation into 100 000 × g particulate and 100 000 × g supernatant (soluble) fractions without an increase into homogenates, 1000 and 29 000 × g particulate fractions prepared from the slices. The catecholamines also produced a 93% increase in ³²P incorporation ans a 27% increase in inorganic phosphate in trichloroacetic acid-insoluble protein that was obtained from ventricular slice homogenates. A significant increase in the incorporation of ³²P occurred in the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein within 2 and 1 min, respectively. While the β-adrenergic blocking agent propanolol had no effect by itself on ³²P incorporation, it prevented the isoproterenol-induced incorporation of ³²P into the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein. Removal of isoproterenol from the bathing medium eliminated the differences in ³²P incorporation, indicating that the effects of the catecholamine were reversible. Norepinephrine and ipinephrine at 10^?5 M caused phosphorylation effects similar to that of isoproterenol. When the slices were bathed under anoxic conditions isoproterenol failed to enhance the incorporation of ³²P into proteins of the 100 000 ×g particulate and supernatant fractions or acid-insoluble protein. SDS gel eloectrophoresis of ventricular slice homogenates revealed that isoproterenol enhanced the ³²P incorporation into several myocardial proteins having molecular weights of 155, 94 (glycogen phosphorylase), 79, 68–77, and 54–59 · 10³ and decreased the incorporation into a 30 · 10³ dalton protein(s). These results are consistent with the notion that catecholamines may increase the phosphorylation of myocardial proteins in the intact myocardium which in turn may play a role in catecholamine-induced glycogenolysis and augmentation of contractility.     

Protein phosphorylation in a dopamine-sensitive homogenate of rat caudate: Effect of adenosine 3′,5′-cyclic monophosphate and putative neurotransmitters

Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and its 8-methylthio derivative stimulate the incorporation of ³²P into proteins endogenous to a homogenate of rat caudate nucleus when 4 μM [γ?³²P] ATP is usedas substrate. Higher concentrations of ATP reduced the effect of the cyclic nucleotide until at 400 μM no significant increase in protein phosphorylation was seen.Incubation of the homogenate with 400 μM ATP and 100 μM dopamine resulted in an approx. 2-fold increase in cyclic AMP but did not alter caudate protein phosphorylation suggesting that the catecholamine could not stimulate protein phosphorylation under the experimental conditions used in the present study.     

High resolution phosphorus NMR spectroscopy of transfer ribonucleic acids

Summary A new activator of phosphofructokinase, which is bound to the enzyme and released during its purification, has been discovered. Its structure has been determined as -D Fructose-2,6-P₂ by chemical synthesis, analysis of various degradation products and NMR. D-Fructose-2,6-P₂ is the most potent activator of phosphofructokinase and relieves inhibition of the enzyme by ATP and citrate. It lowers the K_m for fructose-6-P from 6 mM to 0.1 mM.Fructose-6-P,2-kinase catalyzes the synthesis of fructose-2,6-P₂ from fructose-6-P and ATP, and the enzyme has been partially purified. The degradation of fructose-2,6-P₂ is catalyzed by fructose-2,6-bisphosphatase. Thus a metabolic cycle could occur between fructose-6-P and fructose-2,6-P₂, which are catalyzed by these two opposing enzymes. The activities of these enzymes can be controlled by phosphorylation. Fructose-6-P,2-kinase is inactivated by phosphorylation catalyzed by either cAMP dependent protein kinase or phosphorylase kinase. The inactive, phospho-fructose-6-P,2-kinase is activated by dephosphorylation catalyzed by phosphorylase phosphatase. On the other hand, fructose-2,6-bisphosphatase is activated by phosphorylation catalyzed by cAMP dependent protein kinase.Investigation into the hormonal regulation of phosphofructokinase reveals that glucagon stimulates phosphorylation of phosphofructokinase which results in decreased affinity for fructose-2,6-P₂, and decreases the fructose-2,6-P₂ levels. This decreased level in fructose-2,6-P₂ appears to be due to the decreased synthesis by inactivation of fructose-2,6-P₂,2-kinase and increased degradation as a result of activation of fructose-2,6-bisphosphatase. Such a reciprocal change in these two enzymes has been demonstrated in the hepatocytes treated by glucagon and epinephrine. The implications of these observations in respect to possible coordinated controls of glycolysis and glycogen metabolism are discussed.     

Phenylalanine-induced phosphorylation and activation of rat hepatic phenylalanine hydroxylase in vivo.

Rats were given intraperitoneal injections of 2 mCi of carrier-free 32Pi and substances known to activate liver phenylalanine hydroxylase. After 30 min, these animals were anesthetized and their livers removed for analysis of enzyme activity, 32Pi incorporation into immunoprecipitated phenylalanine hydroxylase and [gamma-32P]ATP specific activity. Following glucagon treatment, rat liver phenylalanine hydroxylase activity was stimulated more than 6-fold when assayed in the presence of the natural cofactor, tetrahydrobiopterin (BH4). Glucagon injection also resulted in an incorporation of 0.41 mol of 32Pi/mol of hydroxylase subunit (approximately 50,000 Da). In vivo stimulation of phenylalanine hydroxylase activity and 32Pi incorporation by glucagon had been previously observed in this laboratory (Donlon, J., and Kaufman, S. (1978) J. Biol. Chem. 253, 6657-6659). However, we show for the first time in the present study that in vivo treatment with phenylalanine alone results in a 4-fold increase in the BH4-dependent activity of phenylalanine hydroxylase concomitant with a significant incorporation of phosphate into phenylalanine hydroxylase (0.51 mol of 32Pi/mol of hydroxylase subunit). It is further demonstrated in vivo that the combined treatment with phenylalanine and glucagon results in a greater than 10-fold stimulation of BH4-dependent activity and the greatest level of 32Pi incorporation (0.75 mol of 32Pi/mol of hydroxylase subunit). Phenylalanine did not produce an elevation in plasma glucagon in these animals. A model is, thereby, proposed with respect to the ligand binding effects of phenylalanine on the state of phosphorylation and activation of phenylalanine hydroxylase. The significance of these regulatory roles are considered in light of the probable physiological environment of the enzyme.