Transformation of neutral to alkaline fructose 1,6-bisphosphatase. Converting enzyme activity in the large-particle fraction from rabbit liver

A liver particle fraction containing lysosomes catalyzes the conversion of native rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11), having a neutral pH optimum, to a modified form with an alkaline pH optimum. The “converting enzyme” activity is partially recovered with the membranes from disrupted particles, and is also detected in “intact” particles isolated and maintained in isotonic buffered sucrose. The converting enzyme activity associated with the membrane fraction is expressed at pH 6.5, but not at pH 4.5, although activity at the lower pH appears when the enzyme is released from the membranes with Triton X-100. In contrast, proteolytic activity as measured with peptide and protein substrates is maximal at pH 5.0 or below, and is the same for the membrane-bound or solubilized proteases. The results suggest that a specific converting enzyme, at least partially associated with a particle (possibly lysosomal) membrane, is responsible for the modification of fructose bisphosphatase and the change in its catalytic properties.     

Fructose 1,6-Bisphosphatase in the Green Alga Selenastrum minutum: I. Evidence for the Presence of Isoenzymes

Two isoforms of fructose 1,6-bisphosphatase are present in the green alga Selenastrum minutum. The isoenzymes can be separated with ionexchange chromatography or acid precipitation. The stability of the two isoenzymes differ largely. The acid insoluble enzyme exhibits properties similar to that of the enzyme from the chloroplasts of higher plants, i.e. an alkaline pH optima in the absence of reductant, a lower affinity for substrate, strong inhibition by phosphate, and a low sensitivity to fructose-2,6-bisphosphate and AMP. The more abundant form of the enzyme exhibits several properties indicative of heterotrophic fructose 1,6 bisphosphatases, i.e. a high affinity for substrate and sensitivity toward fructose-2,6-bisphosphate and AMP. but is absolutely dependent on a reductant for stability and activity. Evidence is provided indicating that previously reported purification protocols cause inactivation of one of the isoenzymes which could lead to the erroneous conclusion that algae have a single fructose 1,6-bisphosphatase isoenzyme.     

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.     

Activation of rabbit muscle fructose 1,6-bisphosphatase by histidine and carnosine

Histidine and its derivatives increased rabbit muscle fructose 1,6-bisphosphatase activity at neutral pH with positive cooperativity. In the presence of histidine and carnosine the optimum pH shifted from pH 8.0 to 7.4. The cooperative response of the enzyme to AMP and fructose 1,6-bisphosphate was observed in the presence of the histidine derivatives. Of a number of divalent cations tested, only Zn2+ was found to be an effective inhibitor of enzyme activity at low concentrations. The kinetic data suggested that Zn2+ acted as inhibitor as well as activator for the enzyme activity; a high affinity binding site was associated with Ki of approximately 0.5 microM Zn2+ and a catalytic site was associated with Km of approximately 10 microM Zn2+. Rabbit muscle fructose 1,6-bisphosphatase bound 4 equivalents of Zn2+/mol, presumably 1 per subunit, in the absence of fructose 1,6-bisphosphate. Two equivalents of Zn2+/mol bound to the enzyme were readily removed by dialysis or gel filtration in the absence of a chelating agent. The other two equivalents of Zn2+/mol were removed by histidine and histidine derivatives of naturally occurring chelators with concomitant increase in activity.     

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.     

AMP makes native snake muscle fructose-1, 6-bisphosphatase to an alkaline enzyme

A substance in the crude preparation of NADP has been found, which activates snake muscle fructose-1,6-bisphosphatase at pH 9.2 and inhibits the enzyme at pH 7.5. After isolation and extensive characterization, the substance has been determined to be AMP. The activation depends on the concentrations of Mg2 and could be observed only at concentrations above 1 mmol/L. In the presence of AMP, snake muscle fructose-1,6-bisphosphatase resembles an alkaline enzyme. Kinetic studies indicate that AMP and Mg2 competitively regulate the activity of the enzyme. AMP releases the inhibition of Mg2 at high concentration at alkaline pH. It has been reported that fructose-1,6-bisphosphatase with a pH optimum in the alkaline region is caused by limited proteolysis. AMP is also able to make fructose-1,6-bisphosphatase to be an alkaline enzyme. This finding indicates that proteolysis may not be the only reason for shift of the optimum pH of fructose-1,6-bisphosphatase to alkaline side and it may imply some significanc     

AMP makes native snake muscle fructose-1,6-bisphosphatase to an alkaline enzyme

A substance in the crude preparation of NADP+ has been found,which activates snake muscle fructose-1,6-bisphosphatase at pH 9.2 and inhibits the enzyme at pH 7.5.After isolation and extensive characterization,the substance has been determined to be AMP.The activation depends on the concentrations of Mg2+ and could be observed only at concentrations above 1 mmol/L.In the presence of AMP,snake muscle fructose-1,6-bisphosphatase resembles an alkaline enzyme.Kinetic studies indicate that AMP and Mg2+ competitively regulate the activity of the enzyme.AMP releases the inhibition of Mg2+ at high concentration at alkaline pH.It has been reported that fructose-1,6-bisphosphatase with a pH optimum in the alkaline region is caused by limited proteolysis.AMP is also able to make fructose-1,6-bisphosphatase to be an alkaline enzyme.This finding indicates that proteolysis may not be the only reason for shift of the optimum pH of fructose-1,6-bisphosphatase to alkaline side and it may imply some significance in physiological regulation.     

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.     

Cathepsin M: a lysosomal proteinase with aldolase-inactivating activity

A proteinase, designated cathepsin M, that catalyzes the limited modification and inactivation of fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) and fructose 1,6-bisphosphatase (EC 3.1.3.11) has been partially purified from rabbit liver. On the basis of its molecular size (M_r = 30,000), activation by sulfhydryl compounds and inhibition by leupeptin it has been characterized as a B-type cathepsin, but other properties distinguish it from cathepsins B, L, and H. Approximately 50% of the total cathepsin M activity is associated with membranes prepared from disrupted lysosomes; this fraction of the activity is also expressed by intact lysosomes. In the membrane-bound form the enzyme is active at neutral pH, but the soluble enzyme and the activity eluted from the membranes are maximally active at pH 5.0. Fasting increases the activity of cathepsin M; the increase is almost entirely in the membrane-bound fraction.     

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.     

Selective thiol group modification renders fructose-1,6-bisphosphatase insensitive to fructose 2,6-bisphosphate inhibition

Limited treatment of native pig kidney fructose-1,6-bisphosphatase (50 microM enzyme subunit) with [14C]N-ethylmaleimide (100 microM) at 30 degrees C, pH 7.5, in the presence of AMP (200 microM) results in the modification of 1 reactive cysteine residue/enzyme subunit. The N-ethylmaleimide-modified fructose-1,6-bisphosphatase has a functional catalytic site but is no longer inhibited by fructose 2,6-bisphosphate. The enzyme derivative also exhibits decreased affinity toward Mg2+. The presence of fructose 2,6-bisphosphate during the modification protects the enzyme against the loss of fructose 2,6-bisphosphate inhibition. Moreover, the modified enzyme is inhibited by monovalent cations, as previously reported (Reyes, A., Hubert, E., and Slebe, J.C. (1985) Biochem. Biophys. Res. Commun. 127, 373-379), and does not show inhibition by high substrate concentrations. A comparison of the kinetic properties of native and N-ethylmaleimide-modified fructose-1,6-bisphosphatase reveals differences in some properties but none is so striking as the complete loss of fructose 2,6-bisphosphate sensitivity. The results demonstrate that fructose 2,6-bisphosphate interacts with a specific allosteric site on fructose-1,6-bisphosphatase, and they also indicate that high levels of fructose 1,6-bisphosphate inhibit the enzyme by binding to this fructose 2,6-bisphosphate allosteric site.     

Effect of fructose 2,6-bisphosphate on the kinetic properties of cytoplasmic fructose 1,6-bisphosphatase from germinating castor bean endosperm

The cytoplasmic form of fructose 1,6-bisphosphatase (FBPase) was purified over 60-fold from germinating castor bean endosperm (Ricinus communis). The kinetic properties of the purified enzyme were studied. The preparation was specific for fructose 1,6-bisphosphate and exhibited optimum activity at pH 7.5. The affinity of the enzyme for fructose 1,6-bisphosphate was reduced by AMP, which was a mixed linear inhibitor. Fructose 2,6-bisphosphate also inhibited FBPase and induced a sigmoid response to fructose 1,6-bisphosphate. The effects of fructose 2,6-bisphosphate were enhanced by low levels of AMP. The latter two compounds interacted synergistically in inhibiting FBPase, and their interaction was enhanced by phosphate which, by itself, had little effect. The enzyme was also inhibited by ADP, ATP, UDP and, to a lesser extent, phosphoenolpyruvate. There was no apparent synergism between UDP, a mixed inhibitor, and fructose 2,6-bisphosphate. Similarly ADP, a predominantly competitive inhibitor, did not interact with fructose 2,6-bisphosphate. Possible roles for fructose 2,6-bisphosphate and the other effectors in regulating FBPase are discussed.     

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.     

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : III. Properties of the Cytosolic Fructose 1,6-Bisphosphatase

The cytosolic fructose 1,6-bisphosphatase from spinach (Spinacia oleracea U.S. hybrid 424) leaves has been partially purified and its response to fructose 2,6-bisphosphate, AMP, and fructose 1,6-bisphosphate studied, using concentrations present in the cytosol during photosynthesis. In the presence of fructose 2,6-bisphosphate, the substrate saturation kinetics for fructose 1,6-bisphosphate are sigmoidal, with half-maximal activity being attained in 0.1 to 1 millimolar concentration range. The inhibition is enhanced by AMP. Using these results, and information published elsewhere on metabolite concentrations, it is discussed how fructose 1,6-bisphosphatase activity will vary in vivo in response to alterations in the availability of triose phosphate and AMP, and the accumulation of the product, fructose 6-phosphate.     

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.     

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.     

The effect of chaotropic anions on the activation and the activity of spinach chloroplast fructose-1,6-bisphosphatase

The effect of chaotropic anions was studied on processes that constitute the chloroplast fructose-1,6-bisphosphatase reaction, i.e. enzyme activation and catalysis. The specific activity of chloroplast fructose-1,6-bisphosphatase was enhanced by preincubation with dithiothreitol, fructose 1,6-bisphosphate, Ca2+, and a chaotropic anion. When chaotropes were ranked in the order of increasing concentrations required for maximal activation they followed a lyotropic (Hofmeister) series: SCN- less than Cl3C-COO- less than ClO4- less than I- less than Br- less than Cl- less than SO4(2-). On the contrary, salts inhibited the catalytic step. The stimulation of chloroplast fructose-1,6-bisphosphatase by chaotropic anions arose from a decrease of the activation kinetic constants of both fructose 1,6-bisphosphate and Ca2+; on the other hand, in catalysis neutral salts caused a decrease of kcat because the S0.5 for both fructose 1,6-bisphosphate and Mg2+ remained unaltered. The molecular weight of chloroplast fructose-1,6-bisphosphatase did not change after the activation by incubation with dithiothreitol, fructose 1,6-bisphosphate, Ca2+, and a chaotrope; consequently, the action of these modulators altered the conformation of the enzyme. Modification in the relative position of aromatic residues of chloroplast fructose-1,6-bisphosphatase was detected by UV differential spectroscopy. In addition, the concerted action of modulators made the enzyme more sensitive to (a) trypsin attack and (b) S-carboxymethylation by iodoacetamide. These results provide a new insight on the mechanism of light-mediated regulation of chloroplast fructose-1,6-bisphosphatase; concurrently to the action of a sugar bisphosphate, a bivalent cation, and a reductant, modifications of hydrophobic interactions in the structure of chloroplast fructose-1,6-bisphosphatase play a crucial role in the enhancement of the specific activity.     

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.     

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.     

Hysteretic behavior of rat liver fructose 1,6-bisphosphatase induced by zinc ions

The measurement of the time dependency of the activity of rat liver fructose 1,6-bisphosphatase shows that the enzyme under certain conditions exhibits kinetic hysteretics. After addition of the substrate, the enzyme is initially in a state characterized by a “high” K_m of about 2 μm. During the reaction the enzyme is converted in a slow process to a low K_m form (K_m is about 0.5 μm). The transition is accompanied by a decrease in V. It is concluded that the hysteretic behavior is caused by binding of the Zn²⁺ substrate complex to the enzyme. The earlier reported effect of glucagon treatment on the activity of fructose 1,6-bisphosphate (O. D. Taunton, F. B. Stifel, H. L. Greene, and R. H. Herman (1974) J. Biol. Chem.249, 7228–7239) was reinvestigated, taking into account the hysteretic behavior. Under conditions where the pyruvate kinase activity is decreased by glucagon injection, no activity change of fructose 1,6-bisphosphatase is observed. It can be suggested that for studies concerning the effects of incubation or hormone treatment on fructose 1,6-bisphosphatase, the complex kinetics of the rat liver enzyme has to be taken into account.