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.     

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.     

Hereditary hydronephrosis in C57BL/KsJ mice.

We sought to determine if the incidence of renal hydronephrosis in male C57BL/KsJ mice increased with age and if grossly normal kidneys would develop hydronephrosis over time. Spontaneous hydronephrosis was found incidentally in 32% of 234 male C57BL/KsJ mice killed as pancreas donors for islet transplantation experiments. The incidence of hydronephrosis increased with age; the incidence was 15% in 6- to 8-week-old mice, 52% in 8- to 10-week-old mice and 63% in 11- to 15-week-old mice (P less than 0.001). Additional mice received islet isografts beneath the renal capsule. Only mice with grossly normal kidneys received islet grafts. These same kidneys were then re-examined when the graft recipients were killed at the end of the experiment and the incidence of hydronephrosis was determined. The conversion of normal kidneys to hydronephrotic kidneys increased with the time since islet transplantation. Kidneys re-examined less than 4 weeks since transplantation had only 5.8% new hydronephrosis, while those re-examined later than 4 weeks after transplantation had a new hydronephrosis incidence rate of 40% (P less than 0.001). Our findings suggest that hydronephrosis is hereditary but not congenital, that it develops rapidly, and that it can complicate experiments using this strain. This may also represent a useful new animal model of progressive hydronephrosis.     

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.     

Purification and properties of Oxidized and reduced forms of Hepatic fructose 1,6-bisphosphatase

D-Fructose 1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11) was isolated from rat liver in two forms: "A," isolated in the presence, and "B," isolated in the absence of dithiothreitol. Both forms had an apparently identical molecular weight of approximately 37,000/subunit and the same Km for fructose 1,6-bisphosphate of 2 microM. However, the Ki of the AMP inhibition of form A was 140 microM and of form B, 370 microM. With form B the same inhibition as with form A was reached by incubating the enzyme with dithiothreitol. The two forms of the enzyme differed in their total, as well as in their number of fast reacting thiol groups. Form A was the more reduced form, exhibiting 22.4 thiol groups/molecule, 2.5 of them fast reacting with 5,5'-dithiobis-(2-nitrobenzoic acid). Only 0.5 fast reacting groups and a total of 19.2 were found with form B. The fast reacting thiol groups disappeared when assayed in the presence of AMP. It is suggested that a redox reaction alters a site that influences the inhibitory action of AMP, so as to regulate the activity of fructose 1,6-bisphosphatase.     

Nuclear accumulation of fructose 1,6-bisphosphatase is impaired in diabetic rat liver

Using a streptozotocin-induced type 1 diabetic rat model, we analyzed and separated the effects of hyperglycemia and hyperinsulinemia over the in vivo expression and subcellular localization of hepatic fructose 1,6-bisphosphatase (FBPase) in the multicellular context of the liver. Our data showed that FBPase subcellular localization was modulated by the nutritional state in normal but not in diabetic rats. By contrast, the liver zonation was not affected in any condition. In healthy starved rats, FBPase was localized in the cytoplasm of hepatocytes, whereas in healthy re-fed rats it was concentrated in the nucleus and the cell periphery. Interestingly, despite the hyperglycemia, FBPase was unable to accumulate in the nucleus in hepatocytes from streptozotocin-induced diabetic rats, suggesting that insulin is a critical in vivo modulator. This idea was confirmed by exogenous insulin supplementation to diabetic rats, where insulin was able to induce the rapid accumulation of FBPase within the hepatocyte nucleus. Besides, hepatic FBPase was found phosphorylated only in the cytoplasm, suggesting that the phosphorylation state is involved in the nuclear translocation. In conclusion, insulin and not hyperglycemia plays a crucial role in the nuclear accumulation of FBPase in vivo and may be an important regulatory mechanism that could account for the increased endogenous glucose production of liver of diabetic rodents.     

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 one-step procedure for the isolation of fructose 1,6-bisphosphatase and fructose 1,6-bisphosphate aldolase from rabbit liver

Human ceruloplasmin, which is usually cleaved by limited proteolysis into three major fragments during preparation ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{? 18,650}$, 50,000, and 70,000) was isolated in good yield as an undegraded single-chain protein ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{? 135,00}$). The cryosupernatant from fresh frozen plasma (100 liters) was fractionated with polyethylene glycol (PEG 4000) at + 5°C yielding a ceruloplasmin-enriched fraction in the 20% PEG supernatant. Three steps of chromatography on DEAE-Sephacel, hydroxyapatite, and Sephadex G-200 produced a homogeneous protein with maximal enzymatic activity and the $\text{A}{}_{610}\text{A}{}_{280}$ ratio of 0.046 corresponding to 98–100% purity. Two forms of ceruloplasmin having this absorbance ratio were obtained; Form I was predominant and was studied further. The procedure separated both forms from apoceruloplasmin and degraded ceruloplasmin. The single-chain ceruloplasmin (Form I) had an NH₂-terminal sequence of Lys-Glu-Lys-His-Tyr-Tyr-Ile-, the same as for the 70,000 fragment, and is suitable for structural study by sequence analysis and physicochemical methods.     

Mouse (C57BL/KsJ) liver phosphofructokinase. Allosteric kinetics and age-related changes in the genetically diabetic state

The regulatory kinetic properties of phosphofructokinase partially purified from the livers of C57BL/KsJ mice were studied. The fructose 6-phosphate saturation curves were highly pH dependent. At a fixed MgATP concentration (1 mM), allosteric kinetics was observed in the range of pH studied (7.3 to 8.3) and the S0.5 values for fructose 6-phosphate decreased by about 0.2 to 0.3 mM for each 0.1-unit increment in pH. Allosteric effects on the sigmoidal response to fructose 6-phosphate: activation by AMP, NH4+, and glucose 1,6-bisphosphate, inhibition by MgATP2-, and synergistic inhibition between ATP and citrate, were all present at pH 8.0 to 8.2. Comparative kinetic studies with liver phosphofructokinase isolated from both the normal (C57BL/KsJ) and the genetically diabetic (C57BL/KsJ-db) mice of 9 to 10 and 15 to 16 weeks of age showed that the enzyme from the livers of diabetic mice exhibited decreased activity at subsaturating concentrations of fructose 6-phosphate. However, phosphofructokinase isolated from the livers of normal and genetically diabetic mice of 4 to 5 weeks of age showed no difference in kinetic properties. Thus, there appears to be a correlation between the change in properties of liver phosphofructokinase and the expression of hyperglycemia and obesity in the genetically diabetic mice. The decreased activity of liver phosphofructokinase in the older diabetic animals may well be one of the causes of the increased blood glucose levels. The results are also discussed in a general context with regard to the possible role of phosphofructokinase in the regulation of hepatic gluconeogenesis.     

The effects of estradiol and progesterone on reproductive tract atrophy and tissue adrenergic indices in diabetic C57BL/KsJ mice

The diabetes-associated changes in tissue norepinephrine (NE) concentrations and related adrenergic receptor types were correlated with changes in blood glucose and serum insulin levels in 8- to 16-week-old C57BL/KsJ-db/db mice relative to corresponding age-matched control (+/?) parameters. In addition, the ability of estradiol and progesterone treatments to modify the diabetes-related adrenergic imbalance was investigated. Tissue (i.e., ovarian, uterine, pancreatic, and adrenal) NE levels were determined by high-performance liquid chromatography and compared with the associated changes in tissue alpha 1,2 and beta-adrenergic membrane receptor populations. All db/db mice exhibited overt hyperglycemia, hyperinsulinemia, and obesity relative to controls between 8 and 16 weeks of age. Tissue NE levels in diabetics were either similar to, or elevated, as compared with those of age-matched controls. Although the alpha 1 and beta receptor populations (except liver) were similar in 16-week-old groups, alpha 2 receptor populations in db/db mice were elevated relative to controls. Chronic estradiol therapy effectively counteracted the diabetes-induced elevations in tissue NE and influenced all adrenergic receptor populations, normalizing both parameters to control levels as well as modifying the hyperglycemia, but not the hyperinsulinemic component, of the diabetes-obesity syndrome in this species. Chronic progesterone treatment was found to be less effective in modulating these systemic and adrenergic parameters in diabetics relative to oil- or estradiol-treated mice. These data demonstrate that a marked modification in tissue adrenergic parameters occur in association with the overt expression of the diabetes mutation in this species. The ability of estradiol treatment to normalize both blood glucose levels and tissue adrenergic parameters in C57BL/KsJ-db/db mice indicates that a direct association between systemic and cellular counter-regulating influences, relative to the severity of the Type II diabetic condition, exists in this species. The therapeutic correction of these metabolic problems by ovarian steroid hormones suggests the existence of a causal relationship between cellular glucose homeostasis and steroid action in the diabetic model.     

Depressed progesterone accumulation by the brain and peripheral tissues of diabetic C57BL/KsJ mice: normalization by estrogen therapy

The effects of diabetes on the uptake and incorporation of 3H-progesterone (P) in various brain areas and peripheral tissues were analyzed in C57BL/KsJ mice. Littermate control (+/?) and diabetic (db/db) mice were pulse-treated (60) min with 10 mu Ci of 3H-P at either 8 or 16 weeks of age. Subsequently, the peripheral tissues and brains were dissected, weighed, digested and the amount of incorporated 3H-P assessed. The pituitary, amygdala, septum and hypothalamus were found to concentrate the highest levels of 3H-P of all the brain areas examined. The brain areas of 16-week-old (+/?) mice accumulated higher concentrations of 3H-P than the comparable 8-week-old brain tissues. In addition, all the brain areas of 8-week-old (db/db) mice accumulated equal or more 3H-P than the comparable (+/?) brains. By 16 weeks of age, however, the 3H-P accumulation rate was significantly higher in all brain areas of (+/?), as compared to (db/db), mice. Of the peripheral tissues examined, the ovary, uterus, pancreas, kidney and mesometrial fat pad consistently exhibited the highest rates of 3H-P accumulation in both (+/?) and (db/db) mice. By 16 weeks of age, all peripheral tissues of (+/?) mice exhibited greater accumulation rates of 3H-P than the comparable (db/db) tissues. Following 4 days of estradiol treatment (10 micrograms/day s.c.), the 3H-P accumulation rates in 16-week-old (+/?) and (db/db) brain were essentially equal. Only the hypothalamus, septum and amygdala of the (db/db) mice failed to normalize to (+/?) levels following estradiol treatment. In a similar manner, the pancreas, uterus, ovary and fat of (db/db) mice exhibited 3H-P accumulation rates similar to those of (+/?) mice following estradiol treatment. These data indicate that estradiol therapy effectively normalizes the normal age- and diabetes-related declines in brain and peripheral tissue sensitivity and/or responsitivity to progesterone in C57BL/KsJ mice.     

Phosphate content of muscle phosphofructokinase in the genetically diabetic mouse (C57BL/KsJ)

The amount of phosphofructokinase based on total soluble protein in extracts of skeletal muscle from genetically diabetic mice C57BL/KsJ (db/db) was about 30% lower than that of normal controls (db/m). Organic phosphate content of five control animals varied between 0.11 and 0.19 mol/mol protomer. On the other hand, the phosphate content of diabetic mice had a much broader range (0.11 to 0.39) with a mean content for five animals of 0.24 mol/mol enzyme protomer. Partial resolution of high- and low-phosphate forms can be achieved by ion-exchange chromatography. The more highly phosphorylated enzyme is slightly more sensitive to ATP inhibition than the low-phosphate enzyme.     

Purification of human liver fructose-1,6-bisphosphatase

Human liver fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) has been purified 1200-fold using a heat treatment step followed by absorption on phosphocellulose at pH 8 and specific elution with buffer containing the substrate (fructose 1,6-bisphosphate) and allosteric effector (AMP). The enzyme is homogeneous in electrophoresis in polyacrylamide gel, in the presence and absence of denaturing agent. It has a molecular weight of 144 000 and is composed of four identical or nearly identical subunits. Fluorescence spectra indicate that the enzyme does not contain tryptophan residues. The pH optimum is 7.5 and the Km is determined as 0.8 microM. The enzyme is inhibited by AMP in cooperative manner with a K0 x 5 of 6 microM.     

Hepatic adrenergic receptors in the genetically diabetic C57 BL/KsJ (db/db) mouse

1. At 30 weeks of age, homozygote diabetic C57 BL KsJ (db/db) mice were grossly obese, lethargic and displayed moderate hair loss relative to heterozygote control C 57 BL KsJ (db/+) mice. 2. In diabetic mice, compared to control, the total body weights, liver weight: body weight ratios, and blood glucose levels were increased 2.3 fold, 20% and 3.1 fold, respectively. 3. Analysis of plasma membranes isolated from control and diabetic mouse liver established that comparable purity levels were achieved since relative specific activities of the plasma membrane markers 5'-nucleotidase and gamma-glutamyltranspeptidase were similar: 10.2 and 11.4 fold with respect to 5'-nucleotidase in control and diabetic states respectively; and 8.0 and 8.3 fold with respect to gamma-glutamyltranspeptidase in control and diabetic states respectively. 4. A select effect of diabetes on gamma-glutamyltranspepetidase, however, was observed. The activity of this enzyme was found to be reduced 16% in diabetic liver compared to control liver. 5. Assessment of [3H]prazosin and [3H]dihydrolalprenolol binding to mouse liver plasma membranes indicated that although there was no difference in beta-adrenergic receptor binding in control and diabetic states, alpha 1-adrenergic receptor binding was found to be reduced 43% in diabetic mouse liver plasma membranes. 6. Scatchard analyses of kinetic studies indicate that the reduction is a reflection of decreases in alpha 1-adrenergic receptor numbers with no change in alpha 1 receptor affinity in the diabetic state: since for diabetic and control liver plasma membranes, Kd values were 3.41 +/- 0.02 nM and 3.40 +/- 0.01 nM respectively; and Bmax were 650.12 +/- 16.44 fmol mg-1 and 380.76 +/- 12.92 fmol mg-1, respectively.     

Purification of the Ah receptor from C57BL/6J mouse liver

The photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, previously has been shown to selectively label two peptides in the cytosol fraction of C57BL/6J mouse liver: a 95-kDa peptide, the ligand binding moiety of the Ah receptor, and a 70-kDa proteolytic fragment formed from the larger peptide (Poland, A., Glover, E., Ebetino, F. H., and Kende, A.S. (1986) J. Biol. Chem. 261, 6352-6365). These two peptides were partially purified to an approximately 20,000-fold enrichment with a 15-20% yield by the following scheme: 1) photoaffinity labeling of the 35-55% ammonium sulfate fraction of liver cytosol; 2) chromatography on polyethyleneimine-Sepharose coupled at low charge density and heparin/Mn2+ precipitation of the dilute column eluate; 3) DEAE-Sepharose chromatography to remove heparin; 4) chromatography on heparin-Sepharose; 5) preparative sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by electroelution of the protein and ion pair extraction to remove sodium dodecyl sulfate; and 6) high performance liquid chromatography on a reverse-phase C-4 column. Following initial chromatography on polyethyleneimine Sepharose, it was found that substantial subsequent purification could only be achieved under denaturing 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.     

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.