Unexpected similarity in regulation between an archaeal inositol monophosphatase/fructose bisphosphatase and chloroplast fructose bisphosphatase

Hyperthermophilic archaea have an unusual phosphatase that exhibits activity toward both inositol-1-phosphate and fructose-1,6-bisphosphate, activities carried out by separate gene products in eukaryotes and bacteria. The structures of phosphatases from Archaeoglobus fulgidus (AF2372) and Methanococcus jannaschii (MJ0109), both anaerobic organisms, resemble the dimeric unit of the tetrameric pig kidney fructose bisphosphatase (FBPase). A striking feature of AF2372, but not of MJ0109, is that the sulfhydryl groups of two cysteines, Cys150 and Cys186, are in close proximity (4 A). A similar arrangement of cysteines has been observed in chloroplast FBPases that are regulated by disulfide formation controlled by redox signaling pathways (ferredoxin/thioredoxin). This mode of regulation has not been detected in any other FBPase enzymes. Biochemical assays show that the AF2372 phosphatase activity can be abolished by incubation with O(2). Full activity is restored by incubation with thiol-containing compounds. Neither the C150S variant of AF2372 nor the equivalent phosphatase from M. jannaschii loses activity with oxidation. Oxidation experiments using Escherichia coli thioredoxin, in analogy with the chloroplast FBPase system, indicate an unexpected mode of regulation for AF2372, a key phosphatase in this anaerobic sulfate reducer.     

Isolation and partial characterization of rat gastric mucous glycoprotein

Mucus glycoproteins from the rat stomach were characterized after their isolation from homogenates of the superficial gastric mucosa by equilibrium centrifugation in CsCl density gradients. Water-soluble as well as water-insoluble glycoproteins were studied. The latter were solubilized by 2-mercaptoethanol reduction of the homogenate. From both homogenate fractions the same two glycoproteins 1 and 2 were purified, glycoprotein 1 being present in considerably higher amount than glycoprotein 2. Their respective buoyant densities in a CsCl gradient were 1.47–1.50 g/ml and 1.56–1.58 g/ml. The two glycoproteins expressed slight differences in gel electrophoresis and gel filtration. The results from column chromatographic comparisons between reduced and unreduced glycoproteins indicated strongly that both glycoproteins 1 and 2 were built from subunits kept together by SS bonds. The s_20,w values of the reduced glycoproteins 1 and 2 were 15.7 S and 11.6 S. Glycoprotein 1 contained 5% protein, 70% carbohydrate and 1–2% sulphate, whereas these percentages for glycoprotein 2 were 10% protein, 65% carbohydrate and 10% sulphate. The molar proportions of the main sugar components galactose, fucose, glucosamine and galactosamine were 4 : 2 : 4 : 1 (glycoprotein 1) and 3 : 2 : 3 : 1 (glycoprotein 2). Blood-group activity A was expressed by glycoprotein 1, whereas glycoprotein 2 showed mainly blood-group activity Le^b, some B activity and also some A activity, but to a lesser extent than glycoprotein 1.     

Isolation and partial characterization of trehalase from Ascaris muscle.

The tissues of female Ascaris suum were assayed for alpha,apha'-glucoside 1-D-glucohydrolase (trehalase) activity. A soluble from of the enzyme was isolated from muscle tissue and purified approximately 37-fold. The enzyme was specific for trehalose as substrate. The pH optimum for enzymatic activity was found to be 6.0, and the apparent Km for trehalose was estimated to be 2.1 x 10-4 M. The product of the reaction was identified as D-glucose by chemical, chromatographic and enzymatic methods.     

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.     

The synthesis and degradation of rat liver and kidney fructose bisphosphatase in vivo.

Sedoheptulose 1,7-bisphosphate has been shown to be present in extracts of normal rat liver. Its concentration in this tissue, estimated by colorimetric and enzymatic assays, is in the range of 5–7 nmol/g tissue. The concentration of sedoheptulose 7-phosphate in these extracts was 110 nmol/g tissue. Also present were mono- and bisphosphate esters of d-glycero-d-ido-octulose and d-glycero-d-altro-octulose, in concentrations ranging from 1–10 nmol/g tissue. Sedoheptulose 1,7-bisphosphate may function as a reservoir for erythrose 4-phosphate. The possible origin of the eight-carbon sugars and their function are discussed.     

Computer simulation of the fructose bisphosphatase/phosphofructokinase couple in rat liver.

Recycling of fructose 6-phosphate and fructose 1,6-bisphosphate in the rat liver under gluconeogenic and glycolytic conditions was investigated with a computer model containing representations of the kinetic properties of phosphofructokinase and fructose 1,6-bisphosphatase under realistic physiological conditions. The two enzyme submodels were constructed from data for the isolated enzymes in vitro by formal optimization. Tissue metabolite concentrations were corrected for cytosolic/mitochondrial compartmentation and effects of chelation and protonation equilibria. This model, which mostly considers the behavior of livers from starved rats, predicts negligible recycling under physiologically realistic conditions. Metabolic regulation of fructose 6-phosphate, the magnesium ion concentration and the distribution of adenine nucleotides appear to prevent operation of a 'futile cycle' in vivo. Rate-limiting chemical species were identified by sensitivity analysis.     

Analytical ultracentrifugation studies on chloroplastic fructose bisphosphatase

Analytical ultracentrifugation studies performed on spinach chloroplast fructose bisphosphatase show that the tetrameric oxidized (inactive) or reduced (active) enzyme dissociates into inactive dimers and monomers at alkaline pH. The dissociation process is, at least, partially reversible if the enzyme is dimeric. Moreover, the oxidized inactive tetrameric enzyme is less prone to dissociation into dimers and monomers than the reduced active tetramer. The irreversibility of the dissociation process may be explained by a sulfhydryl-disulfide interchange. Together with the findings from previously published sulfhydryl group titration experiments (J. Pradel et al., Eur. J. Biochem., 113 (1981) 507), the above results suggest that the activation of the oxidized tetramer involves the reduction of two inter-protomeric disulfide bonds.     

Isolation and partial characterization of rat urinary esterase A2

An enzyme, esterase A2, which hydrolyzes tosyl-arginine methyl ester was isolated from the urine of female, inbred, Dahl-salt-resistant rats using DEAE-Sephadex ion-exchange, aprotinin-agarose affinity and molecular sieve column chromatography. The purest preparation obtained showed four closely migrating bands on polyacrylamide gel electrophoresis. All four bands of the esterase A2 preparation had enzyme activity since all were stainable on zymograms using N-acetyl-L-methionine alpha-naphthyl ester as substrate. Three of these four bands showed decreased electrophoretic mobility following treatment with neuraminidase, indicating that variable sialic acid content accounts for part of the microheterogeneity. The preparation of esterase A2 used was free of rat urinary kallikrein as shown by radioimmunoassay, electrophoretic and isoelectric focusing experiments. The relative kinin-generating ability of rat urinary kallikrein and esterase A2 was highly dependent on the assay used. Using canine plasma as a source of kininogen and the rat uterus to bioassay kinins, esterase A2 was 47% as active as kallikrein; using pure bovine low-molecular-weight kininogen and a radioimmunoassay to measure generated kinins, esterase A2 was only 6% as active as kallikrein. Esterase activity of A2 was activated non-specifically by proteins and detergents. Esterase A2 was 50% inhibited by an 8-fold molar excess of aprotinin and by a 26.5-fold molar excess of soybean trypsin inhibitor, but ovomucoid inhibitor was not inhibitory.     

Isolation and partial characterization of proteoglycans from rat incisors.

Newly synthesized proteoglycans of rat incisors were labelled in vivo for 6h with [35S]-sulphate in order to facilitate their detection during purification and characterization. Proteoglycans were extracted from non-mineralized portions (predentine) of rat incisors with 4M-guanidinium chloride and subsequently from dentine by demineralization with a 0.4M-EDTA solution containing 4M-guanidinium chloride. Both extractions were performed at 4 degrees C in the presence of proteinase inhibitors. Purification of proteoglycans was achieved with a procedure involving gel-filtration chromatography, selective precipitation of phosphoproteins, affinity chromatography and ion-exchange chromatography. Two proteoglycan populations were found in the initial extract (Pd-PG I and Pd-PG II), whereas only one fraction (D-PG) was obtained after demineralization. The minor proteoglycan fraction from the first extract, Pd-PG I, although not totally characterized, differed sharply from the other proteoglycans in that it had a larger molecular size with larger glycosaminoglycan chains composed of chondroitin 4- and 6-sulphate isomers. In contrast, the major proteoglycans Pd-PG II and D-PG had smaller hydrodynamic sizes with smaller glycosaminoglycan chains (but larger than those from bovine nasal cartilage proteoglycans) composed exclusively of chondroitin 4-sulphate. The major proteoglycans were incapable of interacting with hyaluronic acid. In general, the amino acid compositions of the major proteoglycans of rat incisors resembled that of bovine nasal cartilage proteoglycans, but the former had lower proline, valine, isoleucine, leucine, and higher aspartic acid, contents.     

Isolation and partial characterization of rat brain synaptic plasma membranes

Abstract— Synaptic plasma membranes from the cortices of adult rat brain were isolated from synaptosomes prepared by flotation of a washed mitochondrial pellet (P2) in a discontinuous Ficoll-sucrose gradient. Contamination of the synaptosome fraction by microsomes was estimated by enzymic and chemical analysis to be less than 15 per cent. (2) The purified synaptosome fraction was subjected to osmotic shock, subfractionated on a discontinuous sucrose gradient and the distribution of enzymic and chemical markers for synaptic plasma membranes, microsomal membranes and mitochondria was determined. (3) Comparison of synaptosome subfractions prepared in the presence and absence of 1 mM NaH₂ PO₄/0.1 mM EDTA buffer pH 7.5, indicated that the ionic composition of the isolation medium markedly affected the distribution and enzymic composition of the subfractions. (4) Synaptic plasma membranes prepared in the presence of PO₄/EDTA exhibited a 10-fold enrichment in [Na⁺+ K⁺] ATPase and were characterized by less than 15 and 10 per cent contamination by microsomes and mitochondria respectively. (5) The polypeptide composition of the purified synaptic plasma membranes was compared with the microsomes and mitochondria by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. No differences between the protein and glycoprotein composition of the synaptic plasma membranes and microsomes were detected. The mitochondria, in contrast, possessed a unique protein composition.     

Isolation and partial characterization of the rat liver ligandosome fraction

The subcellular distribution in rat liver of non-latent and latent NADH pyrophosphatase was determined by analytical sucrose density gradient centrifugation. Non-latent NADH pyrophosphatase activity was distributed similarly to the plasma membrane marker, 5′-nucleotidase. However, latent NADH pyrophosphatase was found at the low density region of the gradient, similar to the distribution of galactosyl transferase, a Golgi marker. A population of membranes, corresponding to those from the low density region, was prepared by discontinuous sucrose gradient centrifugation. Radiolabelled insulin was used, to monitor the involvement of these membranes in ligand internalization. The membrane perturbant, digitonin, was used to effect a partial separation between membranes bearing NADH pyrophosphatase and those bearing galactosyl transferase. The mechanism by which this separation is effected has been investigated and it was shown that, although digitonin caused a loss of enzyme latency, the density shift was not due to this effect. The partially purified ligandosome-rich fraction was characterized by enzymic and ultrastructural analysis. A novel EM cytochemical stain for NADH pyrophosphatase identified a vesicular fraction distinct from Golgi lamellae.     

The allosteric properties of beef-liver fructose bisphosphatase.

1. The activity of beef liver fructose bisphosphatase has been shown to respond cooperatively to increasing concentrations of the activating cations Mg2+ and Mn2+. The allosteric inhibitor AMP caused an increase in this cooperativity and a decrease in the apparent affinity of the enzyme for the activating cation. 2. The cooperative response of the enzyme to AMP is similarly increased by increasing cation concentrations with a concomitant decrease in the apparent affinity. 3. Direct binding experiments indicated that in the absence of either Mg2+ or Mn2+ the enzyme bound AMP non-cooperatively up to a maximum of two molecules per molecule of enzyme, a result that is indicative of half-sites reactivity. The binding became increasingly cooperative as the concentration of the activating cation was increased. 4. The substrate fructose bisphosphate had no effect on any of these cooperative responses. 5. These results may be most simply interpreted in terms of concerted model in which the activating cation functions both as an allosteric activator and as an essential cofactor for the reaction.     

Activity and properties of fructose bisphosphatase of turbellarian Phagocata sibirica

Activity and properties of fructose bisphosphatase (FBPase) was studied in the free-living turbellarian Phagocata sibirica. All subcellular fractions of P. sibirica (12 000 g cytosol, 105 000 g cytosol, mitochondria, and microsomes) have the FBPase activity. There was studied dependence of the FBPase reaction rate on the substrate concentration. For realization of the enzyme activity, the high affinity to substrate and presence of bivalent cations (Mg2+ or Mn2+) are necessary. The was studied the effect of various effectors as well as of monovalent (Na+, K+, Li+, and NH4+) and bivalent (Zn2+ and Cu2+) cations.     

Crystallographic data for chicken liver fructose bisphosphatase.

Large, single crystals of fructose bisphosphatase have been obtained under a variety of conditions. Preliminary crystallographic analysis reveals that the space group is R3, the cell dimensions on the hexagonal axes are a = b = 304 A and c = 80.4 A, and there is one tetramer per asymmetric unit.     

Isotope-tapping experiments with rabbit liver fructose bisphosphatase.

Isotope-trapping experiments with mental-free rabbit liver fructose 1,6-bisphosphatase have shown that enzyme-bound D-fructose 1,6-bisphosphate completely dissociates prior to enzyme turnover initiated by Mn2+ as the catalytic metal. The exchange rate of the binary enzyme-D-fructose 1,6-bisphosphate complex with the substrate pool is, therefore, more rapid than its conversion to products, suggesting that structural Mn2+ is necessary for productive substarate binding. Rapid-quench isotope-trapping experiments confirm the requirement for structural Mn2+ ions for productive binding to occur. These experiments also show that an ordered formation of the enzyme-Mn2+ s-D-fructose 1,6-bisphosphate ternary complex which features metal-ion addition prior to substrate constitutes a catalytically competent pathway in the mechanism of fructose 1,6-bisphosphatase and that all four subunits are active in a single turnover event.     

Isolation and partial characterization of a rat hepatoma heparan sulfate proteoglycan

A proteoglycan was isolated from a Morris rat hepatoma by sequential precipitations with ammonium sulfate and cetyl pyridinium chloride followed by chromatography on Sepharose CL-4B and DEAE-cellulose. The proteoglycan has a molecular weight of about 1.5 × 10⁵ with 40,000 molecular weight glycosaminoglycan side chains, identified as heparan sulfate based on resistance to chondroitinase and susceptibility to nitrous acid treatment. Immunological studies showed that the protein core of this proteoglycan is immunologically distinct from a rat yolk sac tumor chondroitin sulfate proteoglycan (Å. Oldberg, E. G. Hayman, and E. Ruoslahti, 1981,J. Biol. Chem.256, 10847–10852), but resembles a heparan sulfate proteoglycan isolated from a basement membrane-producing mouse tumor (J. R. Hassell, P.M. Robey, H.-J. Barrach, J. Wilczek, S. R. Rennard, and G. R. Martin, 1980, Proc. Nat. Acad. Sci. USA77, 4494–4498).