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

A simple procedure has been developed for the purification of mouse liver and kidney fructose-1,6-bisphosphatase. In addition to the conventional method, including substrate elution from phosphocellulose, Blue Sepharose column chromatography made the purification procedure highly reproducible. The enzyme from rabbit liver was also purified by this method with a small modification. The isolated preparation was electrophoretically homogeneous. The mouse liver enzyme was identical with the kidney enzyme, and different from the rabbit liver enzyme electrophoretically. The structural properties and the amino acid composition were similar to those of this enzyme from other mammalian livers; the molecular weight was 143,000, subunit size was 37,500, S20, w was 7.0, and partial specific volume was 0.74. Cysteine and methionine residues amounted to 5-6 mol per subunit. Tryptophan was not detected. The Km value for fructose-1,6-bisphosphate was 1.3 microM. The Ki value for AMP was 19 microM. EDTA strongly activated the activity of the mouse liver enzyme at neutral pH. A partial proteolytic digestion of the mouse liver enzyme decreased the activity at neutral pH, and increased it at alkaline pH.     

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.     

Two forms of fructose 1,6-bisphosphatase from immature wheat endosperm

Fructose 1,6-bisphosphatase (α-D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase) from immature wheat endosperm has been resolved into two forms, FBPase-I and FBPase-II. Their specific activities over crude homogenate increased 47- and 77-fold, respectively, by using ammonium sulfate fractionation, DEAE-cellulose chromatography and gel filtration through Sephadex G-200. The pH optimum was 7.6 for FBPase-I and 8.4 for FBPase-II. The two forms were highly specific for the substrate FBP with K_m values of 0.17 and 0.08 m M , respectively, for FBPase-I and FBPase-II at their respective pH optimum and saturating Mg²⁺ concentration. pH had no effect on the K_m value for FBPase-I, but that for FBPase-II increased below optimum pH. Neither of the forms had an absolute requirement for Mg²⁺, although it was essential for maximum activity. Mg²⁺ could not be replaced by Cu²⁺, Ca²⁺, Ba²⁺, Co²⁺ or Ni²⁺. Sulfhydryl reagents inactivated both FBPase-I and FBPase-II. Of the metabolites, only 6-phosphogluconate was inhibitory with 50% inhibition at 2 and 4 m M for FBPase-I and FBPase-II, respectively.     

Evidence for different forms of rat liver fructose 1,6-bisphosphatase

Purified liver fructose 1,6-bisphosphatase exhibits different forms upon isoelectric focusing. The enzyme focused at pH 5.75, 5.60, and 5.44. Treatment of the enzyme preparation with the catalytic subunit of cAMP-dependent protein kinase and ATP altered the isoelectric focusing profile such that the bands at 5.75 and 5.60 were diminished, the band at 5.44 increased, and two new bands appeared at 5.30, and 5.18. Fructose 1,6-bisphosphatase may be present in rat liver in different forms, one of which is phosphorylated as the enzyme is isolated.     

Chicken liver fructose 1,6-bisphosphatase:purfication and some properties.

An improved procedure is described for the purification of fructose 1,6-bisphosphatase (FbPase) from chicken liver. The purified enzyme shows a single band in gel electrophoresis either in the presence or absence of sodium dodecyl sulfate. From 200 g of frozen liver, we have obtained about 29 mg of homogeneous enzyme, with the pH profile indistinguishable from that of the enzyme in crude extracts. The overall recovery of enzyme activity is about 71%. The FbPase protein was estimated to represent approximately 0.36% of the total soluble protein of crude liver extract. Treatment of purified enzyme with papain or subtilisin results in a rapid increase in activity at pH 9.2 and a gradual decrease at pH 7.5, while digestion with trypsin or chymotrypsin results in a concomitant decrease in activities at both pH 9.2 and 7.5. The rates of hydrolysis by these four proteases are all markedly decreased in the presence of AMP. Both AMP and fructose 1,6-bisphosphate increase the thermal stability of the enzyme, and their effects are additive. Attempts were made to investigate the structural requirements for histidine activation. The results suggest that activation by this amino acid involves not only the imidazole ring but also the α-amino and α-carboxyl groups.     

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.     

Efficient purification and molecular properties of spinach chloroplast fructose 1,6-bisphosphatase.

A relatively straightforward procedure has been developed for the purification of chloroplast fructose bisphosphatase from spinach leaves to apparent homogeneity and with 80% yield. The molecular weight of the enzyme was about 160 000. Chloroplast fructosebisphosphatase consists of four possibly identical subunits and, at pH 8.8, EASILY DISSOCIATES INTO EQUAL HALVES WITH LOWered activity. Sigmoid saturation curves with Hill coefficients between 3.0 and 3.7 were obtained for fructose 1,6-bisphosphate and Mg2+. Incubation of the enzyme with 20 mM dithiothreitol slowly altered the response to pH from no activity measured at pH 7.5 and full activity at pH 8.8 to equal activity at each of these pH values; at the same time the number of freely available sulphydryl groups increased from four to twelve per molecule. These properties are considered in the context of the observed activation of this enzyme following illumination of chloroplasts.     

Evidence for an interaction between fructose 1,6-bisphosphatase and fructose 1,6-bisphosphate aldolase.

Three distinct lines of evidence suggest interaction and possible complex formation between fructose 1,6-biphosphate aldolase (EC 4.1.2.13) and fructose 1,6-biphosphatase (EC 3.1.3.11) from rabbit liver. (1) Fructose 1,6-biphosphatase, which does not contain tryptophan, causes changes in the fluorescence emission spectrum of tryptophan in rabbit liver aldolase. (2) Aldolase reduces the affinity of binding of Zn²⁺ to the two high-affinity sites of fructose 1,6-biphosphatase. (3) Gel penetration coefficients are decreased for both enzymes when they are tested together, as compared to the coefficients observed when each is tested separately. These interactions were not observed when either liver enzyme was replaced by the corresponding enzyme purified from rabbit muscle; this specificity for enzymes purified from the same tissue excludes effects attributable to the catalytic activities of the enzyme. Maximum interaction was observed in the pH range between 8.0 and 8.5 and appeared to require the presence of two fructose 1,6-biphosphatase tetramers per tetramer of aldolase. The change in fluorescence emission spectrum was also observed, to a smaller extent, when muscle fructose 1,6-biphosphatase was added to a solution of muscle aldolase.     

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.     

Catabolite inactivation of fructose 1,6-bisphosphatase inKluyveromyces fragilis

Catabolite inactivation of fructose 1,6-bisphosphatase inKluyveromyces fragilis was found to occur as a one-step process with a half-life of approximately 90 min in contrast to the two-step process previously reported forSaccharomyces cerevisiae. No rapid initial 50% loss of activity immediately after a glucose-induced catabolite inactivation was found; nevertheless, fructose 1,6-bisphosphatase was rapidly phosphorylated within 5 min of glucose addition. This result supports the hypothesis that protein phosphorylation serves as a signal for the specific degradation of fructose 1,6-bisphosphatase during catabolite inactivation.     

Purification and properties of fructose-1,6-bisphosphatase of Bacillus subtilis.

Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrase, EC 3.1.3.11) of Bacillus subtilis is a constitutive enzyme that was purified 1000-fold (30% yield) to 80% purity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis where it exhibits a band corresponding to 72,000 daltons. It sediments at 15 S in sucrose density gradients indicating a molecular weight of 380,000, but apparently is very asymmetric. Its activity is irreversibly inactivated in the absence of Mn2+. The enzyme specifically catalyzes dephosphorylation of D-fructose 1,6-bisphosphate with a pH optimum of 8.0. It has 40 to 60% of full activity in the absence of P-enolpyruvate; 20 microM P-enolpyruvate activates it maximally. High concentrations of monovalent cations also activate, NH4+ being most effective. Inhibitors fall into two groups. 1) Nucleoside monophosphates, phosphorylated coenzymes, and polynucleotides inhibit competitively with P-enolpyruvate (AMP (Ki = 2 microM) and dAMP are most effective). 2) The inhibition by nucleoside di- and triphosphates, PPi, and highly phosphorylated nucleotides (guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and adenosine 5'-triphosphate 3'-diphosphate are most effective) is not competed by P-enolpyruvate but is partially overcome by fructose 1,6-bisphosphate (2 microM). Therefore, highly phosphorylated nucleotides (pppGpp and others), produced in over 0.2 mM concentrations upon step down from fast to slow growth rates (Gallant, J., and Lazzarini, R.A. (1976) in Protein Synthesis (McConkey, E.H., ed) Vol. 2, pp. 309-349, Marcel Dekker, Inc., New York), can reduce the conversion rate of fructose 1,6-bisphosphate to fructose 6-phosphate during gluconeogenesis. Comparing glycolytic growth on D-glucose and gluconeogenic growth on L-malate, the intracellular concentrations of fructose 1,6-bisphosphate differ but are both above the Km (13 microM) of the enzyme, those of AMP are similar, whereas those of P-enolpyruvate (0.18 mM versus 1.3 mM) indicate that the enzyme has only 40% of its full activity during glycolysis; nucleotides other than AMP may inhibit additionally. Thus, the futile cycle of fructose 1,6-bisphosphate synthesis and degradation during glycolysis is partially avoided, but the cells are poised for rapid adaptation upon change to gluconeogenic growth conditions.     

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.     

Effects of pH and fructose 2,6-bisphosphate on oxidized and reduced spinach chloroplastic fructose-1,6-bisphosphatase

This report describes the effects of pH and fructose 2,6-bisphosphate (an analog of fructose 1,6-bisphosphate) on the activity of oxidized and reduced fructose-1,6-bisphosphatase from spinach chloroplasts. Studies were carried out with either fructose 1,6-bisphosphate, the usual substrate, or sedoheptulose 1,7-bisphosphate, an alternative substrate. The reduction of the oxidized enzyme is achieved by a thiol/disulfide interchange. The pK values relative to each redox form for the same substrate (either fructose 1,6-bisphosphate or sedoheptulose 1,7-bisphosphate) are identical, suggesting the same site for both substrates on the active molecule. The finding that the analog (fructose 2,6-bisphosphate) behaves like a competitive inhibitor for both substrates also favours this hypothesis. The inhibitory effect of this sugar is more important when the enzyme is reduced than when it is oxidized. The shift in the optimum pH observed when [Mg2+] was raised is interpreted as a conformational change of oxidized enzyme demonstrated by a change in fluorescence. The reduced and oxidized forms have the same theoretical rates relative to both substrates, but the reduced form has an observed Vmax which is 60% of the theoretical Vmax while that of the oxidized form is only 37% of the theoretical Vmax. The reduced enzyme appears more efficient than the oxidized one in catalysis.     

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.