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Large single crystals of rabbit liver fructose-1,6-diphosphatase suitable for a high resolution structure analysis have been grown from polyethylene glycol. The space group of these crystals is I222 with a = 75 A, b = 81 A, and c = 132 A and there are 2 tetrameric molecules in the unit cell. These crystals have one protein subunit as the crystallographic asymmetric unit and establish point group symmetry 222 as the molecular symmetry. 相似文献
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C J Marcus 《The Journal of biological chemistry》1976,251(10):2963-2966
Purified bovine hepatic fructose-1,6-diphosphatase, which exhibits maximal activity at neutral pH, is competitively inhibited by several analogs of its substrate, fructose 1,6-diphosphate. These include glucose 1,6-diphosphate (Ki = 9.4 X 10(-5) M), hexitol 1,6-diphosphate (Ki = 2.3 X 10(-4) M), and 2,5-anhydro-D-mannitol 1,6-diphosphate (Ki = 3.3 X 10(-8) M), and 2,5-anhydro-D-glucitol 1,6-diphosphate (Ki = 5.5 X 10(-7) M). The Ki values for both 2,5-anhydro-D-mannitol 1,6-diphosphate and 2,5-anhydro-D-glucitol 1,6-diphosphate are lower than the Km of 1.4 X 10(-6) M for fructose 1,6-diphosphate. Since 2,5-anhydro-D-mannitol 1,6-diphosphate is an analog of the beta anomer of fructose 1,6-diphosphate and 2,5-anhydro-D-glucitol 1,6-diphosphate is an analog of the alpha anomer, the lower Ki for the mannitol analog may indicate that the beta anomer of fructose 1,6-diphosphate, which predominates in solution, is the true substrate. The substrate analog 1,5-pentanediol diphosphate inhibits slightly (K0.5 = 5 X 10(-3) M), but 1,4-cyclohexyldiol diphosphate does not. The Ki for product inhibition by sodium phosphate is 9.4 X 10(-3) M. 2,5-Anhydro-D-mannitol 1,6-diphosphate and alpha-D-glucose 1,6-diphosphate are substrates at pH 9.0, but not at pH 6.5. 相似文献
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Concentration of liver and kidney fructose-1,6-diphosphatase determined by specific radioimmunoassay 总被引:2,自引:0,他引:2
A radioimmunoassay for liver fructose-1,6-diphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrlase, EC 3.1.3.11) has been developed based on maintenance of its tetrameric structure and immunologic integrity after iodination by the Bolton-Hunter technique. The assay detected as little as 2 ng of standard enzyme. Nonspecific interference by tissue components did not occur. Enzyme concentration (mumol/1000 g tissue wet weight) was measured in tissue extracts of 49 rabbits subjected to a variety of conditions. In animals fed a 'balanced' diet containing 50--60% carbohydrate (by weight), the concentration in liver was 3.4 microM +/- 0.3. After fasts of 48, 72, or 96 h, the concentration in liver increased approximately 1.4-fold. A high-fat diet did not alter the concentration significantly but a high-protein diet caused an increase of 2.1-fold to 7.2 microM +/- 1.4. The greatest concentrations, 8.7 microM +/- 1.9, were observed in the livers of severely diabetic rabbits. The increase paralleled the increasing severity of diabetes and provides one explanation for the augmented gluconeogenesis which occurs in the diabetic state. Changes were less marked in kidney. The greatest apparent incrase, from 2.6 microM +/- 1.1 in the normal fed rabbit to 4.7 microM +/- 2.8, occurred in the severely diabetic animal. However, variation was sufficiently great in kidney to render apparent increases during fasting, protein feefing and diabetes statistically insignificant. For the most part changes in assayable activity followed changes in enzyme concentration except in the rabbits maintained on high-protein diets. In these, liver enzyme concentration increased by 2.4-fold whereas activity increased by only 1.3-fold, and the kidney enzyme concentration increased 1.3-fold whereas activity decreased by 20%. 相似文献
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Here we describe a two-step procedure for purification of human tenascin from conditioned medium of the SK-MEL-28 human melanoma cell line. The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin--Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5-300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin-sulfate-containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3-4 mg tenascin which corresponds to a yield of about 40-50%. This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions. 相似文献
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Mastoparans are tetradecapeptides found to be the major component of wasp venoms. These peptides possess a variety of biological activities. Three related mastoparans, mastoparan from Polistes jadwagae (MP-PJ), mastoparanX (MP-X) and its carboxyl-free C-terminal form (MP-X-COO-), were crystallized. X-ray diffraction data for them were collected at resolutions of 1.2 A, 2.0 A and 3.3 A respectively. 相似文献
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R B Buchanan P Schfürmann R A Wolosiuk 《Biochemical and biophysical research communications》1976,69(4):970-978
Chloroplast fructose 1,6-diphosphatase isolated at pH 5.5 as the dimer dissociated to the monomer at pH 8.5. When the pH was adjusted from 8.5 back to 5.5, the newly formed monomer partly reassociated to form the dimer. The monomer lacked the fructose diphosphatase activity characteristic of the dimer (measured in the presence of a saturating concentration of Mg++) but retained ferredoxin-dependent activity (measured in the presence of Mg++ plus protein factor and either reduced ferredoxin or dithiothreitol). In addition, the monomer acquired sedoheptulose 1,7-diphosphatase activity that was dependent on either reduced ferredoxin or dithiothreitol and the protein factor. 相似文献
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Fructose-1-phosphate-6-sulfate was prepared by direct sulfurylation of fructose, and selective phosphorylation of the 6-sulfuryl isomer by phosphofructokinase. The ketose derivative was used as a substrate for aldolase and fructose-1,6-diphosphatase. Kinetic studies with aldolase showed that the alternative substrate binds one third as well as fructose-1,6-P2 yet 900 fold greater than fructose-1-P. The Vm was intermediate between the two ketose phosphates. From kinetic studies with skeletal muscle fructose-1,6-diphosphatase at pH 7.5 a Km of 8 μM and a Vm approximately 6% that for fructose-1,6-P2 was obtained. 相似文献
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The activity of glucose-6-phosphatase (G6Pase) and fructose-1,6-bisphosphatase (FDPase) was determined in the homogenate of the liver of 69 pig fetuses during the last third of gestation (80th to 114th day), 47 piglets from birth to 4 weeks old (suckling period) and to slaughter pigs. G6Pase is evident in fetal liver at an early date and raises steadily during gestation. In newborn piglets, the enzyme activity increases rapidly during the first hours of life and remains at this high level during the first week of life. Afterwards the enzyme activity returns to birth level, which exists also in pigs at slaughtering. The activity of FDPase is constant during the fetal period. After birth enzyme activity rises at a lower rate than the G6Pase during the first week of life. This level remains constant during the suckling period and increases thereafter until the time of slaughtering of pigs. The role of hormones in the perinatal development of these enzymes is described. Probably, thyroxine causes the prenatal increase of the activity of both the enzymes. The rapid postnatal rise of G6Pase activity may be induced by the high level of hydrocortisone at parturition, and furthermore, glucagon may have a permissive effect. 相似文献
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The soluble chromatin fragments from nuclei of avian erythrocytes digested with micrococcal nuclease were fractionated by the addition of sodium phosphate to 0.1 m. The supernatant consisted predominantly of monomeric nucleosomes, while most dimeric and larger nucleosomes precipitated. A variable percentage of monomers also precipitated, the exact amount depending upon the extent of digestion. The solubility properties can be used for the simple preparation of fractions that are highly enriched for monomers either containing, or deplete in, lysine-rich histone. 相似文献
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Tyrocidine A was crystallized from 2-methyl-2,4-pentanediol and water, or methanol, to yield crystals that are large enough for X-ray diffraction studies. Four crystals were examined; three were in equilibrium with mother liquor and the fourth was air-dried. They belong to the rhombohedral space group R32. Parameters of the hexagonal cell of the fully solvated crystals vary slightly and are approximately . The asymmetric unit consists of one molecule of tyrocidine and several molecules of 2-methyl-2,4-pentanediol. Air-dried crystals appear to contain about half the number of solvent molecules. Three-dimensional X-ray diffraction data showing a maximum resolution of have been recorded. 相似文献
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A new purification procedure for spinach leaf fructose-1,6-bisphosphatase is proposed, which includes the use of affinity chromatography on mercaptoethylamine-Sepharose. A homogeneous preparation of the enzyme can be obtained in 48 hr, with a specific activity of 67 U/mg and a yield of 23%. The method may also be useful for the purification of other thioredoxin-activated chloroplast enzymes. 相似文献
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W F Fong I Hynie L Lee J B McKendry 《Biochemical and biophysical research communications》1979,88(1):222-228
The specific activity of fructose-1,6-diphosphatase in freshly isolated human peripheral lymphocytes is usually less than 10% of what can be found in normal animal livers. The enzyme activity is strongly inhibited by AMP and is also inhibited by its substrate, fructose-1,6-diphosphate, at concentrations higher than 20 μM. In cultured lymphocytes the enzyme activity gradually increases so that by day 3 of the incubation the specific enzyme activity could be 15 to 80 fold higher than the preincubation level. The increase would continue for at least 10 days if the culture medium is periodically renewed. When phytohemagglutinin is present in the culture medium, the increase of the enzyme activity is completely suppressed. No free soluble enzyme inhibitor could be detected in phytohemagglutinin-treated cells by mixing and dialysis experiments. 相似文献