Characterization of rat muscle fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphatase has been purified from rat muscle. Although the specific activity of the enzyme in the crude extract of rat muscle was extremely low, purification by the present procedure is highly reproducible. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The subunit molecular weight of the muscle enzyme was 37,500 in contrast to 43,000 in the case of the liver enzyme. Immunoreactivity of the muscle enzyme to anti-muscle and anti-liver fructose 1,6-bisphosphatase sera was clearly distinct from that of the liver enzyme. All one-dimensional peptide mappings of the muscle enzyme with staphylococcal V8 protease, chymotrypsin, and papain showed different patterns from those of the liver enzyme. When incubated with subtilisin, the extent of activation of muscle fructose 1,6-bisphosphatase at pH 9.1 was smaller than that of the liver enzyme. The subtilisin digestion pattern of the muscle enzyme on SDS-polyacrylamide gel electrophoresis was distinct from that of the liver enzyme. The AMP-concentration giving 50% inhibition of the muscle enzyme was 0.54 microM, whereas that of the liver enzyme was 85 microM. The concentrations of fructose 2,6-bisphosphate that gave 50% inhibition of rat muscle and liver enzymes were 6.3 and 1.5 microM, respectively. Fructose 1,6-bisphosphatase protein was not detected in soleus muscle by immunoelectroblotting with anti-muscle fructose 1,6-bisphosphatase serum.     

Fructose 2,6-bisphosphate activates the cAMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase in vitro

Fructose-1,6-bisphosphatase purified from Saccharomyces cerevisiae is phosphorylated in vitro by a cAMP-dependent protein kinase. The phosphorylation reaction incorporates 1 mol of phosphate/mol of enzyme and is greatly stimulated by fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate acts upon fructose-1,6-bisphosphatase, not on the protein kinase. The phosphorylation of fructose 1,6-bisphosphatase lowers its activity by about 50%. The characteristics of the phosphorylation reaction in vitro show that this modification is responsible for the inactivation of fructose-1,6-bisphosphatase observed in vivo.     

Localization of the fructose 1,6-bisphosphatase at the nuclear periphery

The localization of fructose 1,6-bisphosphatase (D-Fru-1,6-P₂-1-phosphohydrolase, EC 3.1.3.11) in rat kidney and liver was determined immunohistochemically using a polyclonal antibody raised against the enzyme purified from pig kidney. The immunohistochemical analysis revealed that the bisphosphatase was preferentially localized in hepatocytes of the periportal region of the liver and was absent from the perivenous region. Fructose-1,6-bisphosphatase was also preferentially localized in the cortex of the kidney proximal tubules and was absent in the glomeruli, loops of Henle, collecting and distal tubules, and in the renal medulla. As indicated by immunocytochemistry using light microscopy and confirmed with the use of reflection confocal microscopy, the enzyme was preferentially localized in a perinuclear position in the liver and the renal cells. Subcellular fractionation studies followed by enzyme activity assays revealed that a majority of the cellular fructose-1,6-bisphosphatase activity was associated to subcellular particulate structures. Overall, the data support the concept of metabolic zonation in liver as well as in kidney, and establish the concept that the Fructose-1,6-bisphosphatase is a particulate enzyme that can not be considered a soluble enzyme in the classical sense. © 1996 Wiley-Liss, Inc.     

Characterization of fructose 1,6-bisphosphatase from bakers' yeast

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.     

Purification and properties of spinach leaf cytoplasmic fructose-1,6-bisphosphatase.

Cytoplasmic fructose-1,6-bisphosphatase has been purified from spinach leaves to apparent homogeneity. The enzyme is a tetramer of molecular weight about 130,000. At pH 7.5, the Km for fructose 1.6-bisphosphate was 2.5 micron, and for MgCl2 0.13 mM; the enzyme was specific for fructose 1,6-bisphosphate. Saturation with Mg2+ was achieved with lower concentrations at pH 8 than at pH 7. AMP and high concentrations of fructose 1,6-bisphosphate inhibited enzyme activity. Ammonium sulfate relieved the latter inhibition but was itself inhibitory when substrate concentrations were low. Acetylation studies demonstrated that the AMP regulatory site was distinct from the catalytic site. Cytoplasmic fructose-1,6-bisphosphatase may contribute to the regulation of sucrose biosynthesis in plant leaves.     

Competition among oxidizable substrates in brains of young and adult rats. Whole homogenates.

A new purification procedure for rat liver fructose-1,6-bisphosphatase that involves use of Procion Red-Sepharose is described. The purified enzyme was homogeneous, had a subunit Mr of 40 000-41 000 and seemed to be undegraded. The enzyme could be phosphorylated by cyclic AMP-dependent protein kinase with a stoicheiometry of one per subunit. Phosphorylation caused a 2-fold decrease in the Km of the enzyme for fructose 1,6-bisphosphate, but did not affect its allosteric responses to AMP, Mg2+ and fructose 2,6-bisphosphate.     

Development and properties of fructose 1,6-bisphosphatase in the endosperm of castor-bean seedlings.

1. The activity of fructose 1,6-bisphosphatase (EC 3.1.3.11) in the fatty endosperm of castor bean (Ricinus communis) increases 25-fold during germination and then declines. The developmental pattern follows that of catalase, a marker enzyme for gluconeogenesis in this tissue. 2. The enzyme at its peak of development was partially purified, and its properties were studied. It has an optimal activity at neutral pH (7.0-8.0). The apparent Km value for fructose 1,6-bisphosphate is 3.8 X 10(-5) M. The activity is inhibited by AMP allosterically with an apparent Ki value of 2.2 X 10(-4) M. The enzyme hydrolyses fructose 1,6-bisphosphate and not ribulose 1,5-bisphosphate or sedoehptulose 1,7-bisphosphate. 3. Treatment of the partially purified enzyme with acid leads to an 80% decrease in activity. The remaining activity is insensitive to AMP and has optimal activity at pH 6.7 and a high apparent Km value (2.5 X 10(-4) M) for fructose 1.6-bisphosphate. Enzyme extracted from the tissue with water instead of buffer has a similar modification. The effect of acid explains the discrepancies between this report and previous ones on the properties of the enzyme in this tissue. 4. The storage tissues of various fatty seedlings all contain a 'neutral' fructose 1,6-bisphosphatase. The activities of the enzyme from some of the tissues are inhibited by AMP. 5. The properties of the enzyme in fatty seedlings and in green leaves are discussed in comparison with that in animal tissues.     

Correlation of inhibition of fructose 1,6-bisphosphatase by AMP and the presence of the nucleotide-binding domain

Chicken liver fructose 1,6-bisphosphatase binds to blue dextran-Sepharose affinity columns and is eluted by AMP, an allosteric inhibitor of the enzyme. On the other hand, bumblebee fructose 1,6-bisphosphatase, which is not inhibited by AMP, does not bind to blue dextran-Sepharose. Chicken liver 1,6-bisphosphatase binds 3.6 mol of AMP/mol of enzyme, while the bumblebee enzyme binds no AMP. However, bumblebee fructose 1,6-bisphosphatase can be activated by subtilisin, indicating that it possesses a protease-sensitive region similar to that present in mammalian fructose 1,6-bisphosphatase.     

Identification of the in vivo and in vitro phosphorylation sites of rat liver fructose 1,6-bisphosphatase

Rat liver fructose 1,6-bisphosphatase appears to be unique in that it extends 24-26 residues beyond the COOH-terminal amino acid of other mammalian fructose 1,6-bisphosphatases and this extension contains phosphorylation sites. Using as a frame of reference the 335-residue sequence of pig kidney fructose 1,6-bisphosphatase (Marcus, F., Edelstein, I., Reardon, I., and Heinrikson, R. L. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7161-7165), the rat liver enzyme would extend to residue 361. Limited proteolysis in the COOH-terminal region of the molecule with chymotrypsin, trypsin, or both sequentially, led us to establish that the phosphorylation sites are located at Ser residues 341 and 356. The in vitro phosphorylation of purified rat liver fructose 1,6-bisphosphatase by the catalytic subunit of cyclic AMP-dependent protein kinase results in modification at both residues, although the major site of phosphorylation (61%) is at Ser-341. In contrast, rat liver fructose 1,6-bisphosphatase purified from animals that had been injected with [32P] phosphate contains most of the label (81%) at Ser-356.     

Specific, reversible inactivation of phosphofructokinase by fructose-1,6-bisphosphatase. Involvement of adenosine 5'-triphosphate, oleate, and 3-phosphoglycerate.

Optimal conditions necessary for the reversible inactivation of crystalline rabbit muscle phosphofructokinase by homogeneous rabbit liver fructose-1,6-bisphosphatase have been studied. At higher enzyme levels (to 530 mug/ml of phosphofructokinase) the two proteins were mixed and incubated in a pH 7.5 buffer composed of 50 mM Tris-HC1, 2 mM potassium phosphate, and 0.2 mM dithiothreitol. Aliquots were removed at various times and assayed for enzyme activity. A time dependent inactivation of phosphofructokinase caused by 1-2.3 times its weight of fructose-1,6-bisphosphatase was observed at 30, 23, and 0 degree C. This inactivation did not require the presence of adenosine 5'-triphosphate or Mg2+ in the incubation mixture, but an adenosine 5'-triphosphate concentration of 2.7 mM or greater was required in the assay to keep phosphofructokinase in an inactive form. A mixture of activators (inorganic phosphate, (NH4)2SO4, and adenosine 5'-monophosphate), when added to the assay cuvette, restored nearly all of the expected enzyme activity. Incubations with other proteins, including aldolase, at concentrations equal to or greater than the effective quantity of fructose-1,6-bisphosphatase had no inhibitory effect on phosphofructokinase activity. Removal of tightly bound fructose 1,6-bisphosphate from phosphofructokinase could not explain this inactivation, since several analyses of crystalline phosphofructokinase averaged less than 0.1 mol of fructose 1,6-bisphosphate/320 000 g of enzyme. Furthermore, the inactivation occurred in the absence of Mg2+ where the complete lack of fructose-1-6-bisphosphatase activity was confirmed directly. At lower phosphofructokinase concentrations (0.2-2 mug/ml) the inactivation was studied directly in the assay cuvette. Higher ratios of fructose-1,6-bisphosphatase to phosphofructokinase were necessary in these cases, but oleate and 3-phosphoglycerate acted synergistically with lower amounts of fructose-1,6-bisphosphatase to cause inactivation. The inactivation did not occur when high concentrations of fructose 6-phosphate were present in the assay, or when the level of adenosine 5'-triphosphate was decreased. However, the inactivation was found at pH 8, where the effects of allosteric regulators on phosphofructokinase are greatly reduced. Experiments with rat liver phosphofructokinase showed that this enzyme was also subject to inhibition by rabbit liver fructose 1,6-bisphosphatase under conditions similar to those used in the muscle enzyme studies. Attempts to demonstrate direct interaction between phosphofructokinase and fructose-1,6-bisphosphate by physical methods were unsuccessful. Nevertheless, our results suggest that, under conditions which approximate the physiological state, the presence of fructose-1,6bisphosphatase can cause phosphofructokinase to assume an inactive conformation. This interaction may have a significant role in vivo in controlling the interrelationship between glycolysis and gluconeogenesis.     

Studies on the hysteretic properties of chloroplast fructose-1,6-bisphosphatase

Chloroplast fructose-1,6-bisphosphatase hysteresis in response to modifiers was uncovered by carrying out the enzyme assays in two consecutive steps. The activity of chloroplast fructose-1,6-bisphosphatase, assayed at low concentrations of both fructose-1,6-bisphosphatase and Mg2+, was enhanced by preincubating the enzyme with dithiothreitol, thioredoxin f, fructose 1,6-bisphosphate, and Ca2+. In the time-dependent activation process, fructose 1,6-bisphosphate and Ca2+ could be replaced by other sugar biphosphates and Mn2+, respectively. Once activated, chloroplast fructose-1,6-bisphosphatase hydrolyzed fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate in the presence of Mg2+, Mn2+, or Fe2+. The A0.5 for fructose 1,6-bisphosphate (activator) was lowered by reduced thioredoxin f and remained unchanged when Mg2+ was varied during the assay of activity. On the contrary, the S0.5 for fructose 1,6-bisphosphate (substrate) was unaffected by reduced thioredoxin f and depended on the concentration of Mg2+. Ca2+ played a dual role on the activity of chloroplast fructose-1,6-bisphosphatase; it was a component of the concerted activation and an inhibitor in the catalytic step. Provided dithiothreitol was present, the activating effectors were not required to maintain the enzyme in the active form. Considered together these results strongly suggest that the regulation of fructose-1,6-bisphosphatase in chloroplast occurs at two different levels, the activation of the enzyme and the catalysis.     

Studies on the regulation of chloroplast fructose-1,6-bisphosphatase. Activation by fructose 1,6-bisphosphate

Chloroplast fructose-1,6-bisphosphatase (D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) isolated from spinach leaves, was activated by preincubation with fructose 1,6-bisphosphate. The rate of activation was slower than the rate of catalysis, and dependent upon the temperature and the concentration of fructose 1,6-bisphosphate. The addition of other sugar diphosphates, sugar monophosphates or intermediates of the reductive pentose phosphate cycle neither replaced fructose 1,6-bisphosphate nor modified the activation process. Upon activation with the effector the enzyme was less sensitive to trypsin digestion and insensitive to mercurials. The activity of chloroplast fructose-1,6-bisphosphatase, preincubated with fructose 1,6-bisphosphate, returned to its basal activity after the concentration of the effector was lowered in the preincubation mixture. The results provide evidence that fructose-1,6-bisphosphatase resembles other regulatory enzymes involved in photosynthetic CO2 assimilation in its activation by chloroplast metabolites.     

Relationship between thiol group modification and the binding site for fructose 2,6-bisphosphate on rabbit liver fructose-1,6-bisphosphatase

A thiol group present in rabbit liver fructose-1,6-bisphosphatase is capable of reacting rapidly with N-ethylmaleimide (NEM) with a stoichiometry of one per monomer. Either fructose 1,6-bisphosphate or fructose 2,6-bisphosphate at 500 microM protected against the loss of fructose 2,6-bisphosphate inhibition potential when fructose-1,6-bisphosphatase was treated with NEM in the presence of AMP for up to 20 min. Fructose 2,6-bisphosphate proved more effective than fructose 1,6-bisphosphate when fructose-1,6-bisphosphatase was treated with NEM for 90-120 min. The NEM-modified enzyme exhibited a significant loss of catalytic activity. Fructose 2,6-bisphosphate was more effective than the substrate in protecting against the thiol group modification when the ligands are present with the enzyme and NEM. 100 microM fructose 2,6-bisphosphate, a level that should almost saturate the inhibitory binding site of the enzyme under our experimental conditions, affords only partial protection against the loss of activity of the enzyme caused by the NEM modification. In addition, the inhibition pattern for fructose 2,6-bisphosphate of the NEM-derivatized enzyme was found to be linear competitive, identical to the type of inhibition observed with the native enzyme. The KD for the modified enzyme was significantly greater than that of untreated fructose-1,6-bisphosphatase. Examination of space-filling models of the two bisphosphates suggest that they are very similar in conformation. On the basis of these observations, we suggest that fructose 1,6-bisphosphate and fructose 2,6-bisphosphate occupy overlapping sites within the active site domain of fructose-1,6-bisphosphatase. Fructose 2,6-bisphosphate affords better shielding against thiol-NEM modification than fructose 1,6-bisphosphate; however, the difference between the two ligands is quantitative rather than qualitative.     

Purification and characterization of glpX-encoded fructose 1, 6-bisphosphatase, a new enzyme of the glycerol 3-phosphate regulon of Escherichia coli

In Escherichia coli, gene products of the glp regulon mediate utilization of glycerol and sn-glycerol 3-phosphate. The glpFKX operon encodes glycerol diffusion facilitator, glycerol kinase, and as shown here, a fructose 1,6-bisphosphatase that is distinct from the previously described fbp-encoded enzyme. The purified enzyme was dimeric, dependent on Mn(2+) for activity, and exhibited an apparent K(m) of 35 microM for fructose 1,6-bisphosphate. The enzyme was inhibited by ADP and phosphate and activated by phosphoenolpyruvate.     

Yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase. Properties of phospho and dephospho forms and of two mutants in which serine 11 has been changed by site-directed mutagenesis

The properties of dephospho- and phosphofructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae and of two mutant enzymes in which the phosphorylatable Ser11 had been changed by site-directed mutagenesis (Ser----Ala and Ser----Asp) were studied to clarify the role of cyclic AMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase. The mutant enzymes and wild type Ser11 fructose-1,6-bisphosphatase were overexpressed and purified to homogeneity. Phosphofructose-1,6-bisphosphatase was prepared by in vitro phosphorylation. The comparison of the properties of the above enzymes demonstrated that all four had similar maximum activity. However, the phosphoenzyme was about 3-fold more sensitive to AMP and fructose 2,6-bisphosphate inhibition than the dephosphoenzyme, suggesting that regulation operates in vivo by this mechanism, leading to decreased enzyme activity. The purified mutant enzymes Ala11 and Asp11 exhibited properties closely similar to those of dephospho- and phosphofructose-1,6-bisphosphatase, respectively. These results indicate that the functional group at residue 11 is an important factor in the regulation of fructose-1,6-bisphosphatase activity and that Ser(P) can be functionally substituted by Asp in this enzyme.     

Selective modification of rabbit liver fructose bisphosphatase

Chemical modification of rabbit liver fructose 1,6-bisphosphatase by 5,5′-dithiobis-(2-nitrobenzoic acid) results in thiolation of four highly reactive sulfhydryl groups and a diminished sensitivity to AMP inhibition but not loss of enzyme activity. Ethoxyformylation of the histidine groups of fructose 1,6-bisphosphatase does not result in a sharp loss of activity until at least 4 or 5 of the 13 residues have reacted. Exhaustive formylation does abolish the enzyme's activity. These four most reactive sulfhydryl groups and the one or two least easily modified histidine moieties (those responsible for activity) can be protected against modification by fructose-1,6-P₂ and to a lesser extent by fructose-6-P. The binding of fructose-1,6-P₂ to fructose 1,6-bisphosphatase, however, depends on the presence of structural metal ion since EDTA which removes all endogenous Zn²⁺ from the protein prevents binding of fructose-1, 6-P₂ to the enzyme.     

Regulation of rabbit liver fructose-1,6-bisphosphatase by metals, nucleotides, and fructose 2,6-bisphosphate as determined from fluorescence studies

The fluorescent nucleotide analogue formycin 5'-monophosphate (FMP) inhibits rabbit liver fructose-1,6-bisphosphatase (I50 = 17 microM, Hill coefficient = 1.2), as does the natural regulator AMP (I50 = 13 microM, Hill coefficient = 2.3), but exhibits little or no cooperativity of inhibition. Binding of FMP to fructose-1,6-bisphosphatase can be monitored by the increased fluorescence emission intensity (a 2.7-fold enhancement) or the increased fluorescence polarization of the probe. A single dissociation constant for FMP binding of 6.6 microM (4 sites per tetramer) was determined by monitoring fluorescence intensity. AMP displaces FMP from the enzyme as evidenced by a decrease in FMP fluorescence and polarization. The substrates, fructose 6-phosphate and fructose 1,6-bisphosphate, and inhibitors, methyl alpha-D-fructofuranoside 1,6-bisphosphate and fructose 2,6-bisphosphate, all increase the maximal fluorescence of enzyme-bound FMP but have little or no effect on FMP binding. Weak metal binding sites on rabbit liver fructose-1,6-bisphosphatase have been detected by the effect of Zn2+, Mn2+, and Mg2+ in displacing FMP from the enzyme. This is observed as a decrease in FMP fluorescence intensity and polarization in the presence of enzyme as a function of divalent cation concentration. The order of binding by divalent cations is Zn2+ = Mn2+ greater than Mg2+, and the Kd for Mn2+ displacement of FMP is 91 microM. Methyl alpha-D-fructofuranoside 1,6-bisphosphate, as well as fructose 6-phosphate and inorganic phosphate, enhances metal-mediated FMP displacement from rabbit liver fructose-1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carp (Cyprinus carpio) muscle fructose 1,6-bisphosphatase: purification and some properties.

1. Fructose 1,6-bisphosphatase from the white muscle tissue of the carp, Cyprinus carpio L. was purified. 2. The mol. wt of the enzyme was 145,000. Its subunit mol. wt was ca. 35,000. 3. The enzyme exhibited neutral pH optimum, activation by monovalent cations, and temperature-dependent allosteric AMP inhibition. 4. Carp muscle fructose 1,6-bisphosphatase was 10- to 30-fold more sensitive to AMP inhibition than the carp liver enzyme. 5. The carp muscle enzyme was less sensitive to AMP inhibition than the muscle enzyme from a homeothermic mammal. These results are interpreted as an example of temperature-adaptation of an enzyme regulatory property.     

Purification and properties of fructose 1,6-bisphosphatase from turkey liver.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) has been purified 360-fold from turkey liver. The purified enzyme appears to be homogeneous by disc gel electrophoresis and has a pH profile indistinguishable from that of the enzyme in crude extracts. Mn²⁺ is significantly more effective than Mg²⁺ as the essential metal cofactor of this enzyme. The maximal effect of histidine is equivalent to that of EDTA except that EDTA is more efficient at lower concentrations. The histidine effect is decreased with an increase in pH or if substrate is first bound to the enzyme. The enzyme activity is activated equally by d- and l-forms of histidine. Enzyme affinity for the substrate decreases with an increase in pH. The inhibition by high substrate concentrations observed at pH 7.5 is markedly reduced in the absence of chelating activator or when Mg² is replaced by Mn²⁺ as the metal cofactor. Turkeys liver fructose 1,6-bisphosphatase resembles the enzyme from mammalian sources in that the sensitivity to AMP inhibition is decreased with the increase in pH, temperature, and Mg² concentration.     

Effect of dietary excess-histidine on fructose 1,6-bisphosphatase and 6-phosphofructokinase activities, and activation of fructose 1,6-bisphosphatase by basic amino acids in rat liver.

1. Dietary excess histidine caused an increase in the total activity of fructose 1,6-bisphosphatase, and a decrease in 6-phosphofructokinase in the liver. 2. The hepatic concentrations of free histidine and lysine were higher in rats fed a histidine-excess diet. 3. The addition of histidine, lysine or arginine to the assay mixture for fructose 1,6-bisphosphatase resulted in a significant increase in its activity. The 6-phosphofructokinase activity in the liver was not enhanced by the addition of histidine to the assay mixture.