High Concentrations Of Ammonium Sulfate Research Articles

Purification of an acidic plastocyanin from MicroBiomis aeruginosa

Plastocyanin and cytochrome c-553 are two functionally equivalent electron carriers in the photosynthetic chain of cyanobacteria. Microcystis aeruginosa, a unicellular cyanobacterium which grows well at a high pH (8.6) and which was not known to possess plastocyanin, has been studied for its ability to synthesize plastocyanin in culture media with and without Cu. In the absence of Cu, an acidic cytochrome c-553 alone was isolated. With the inclusion of 2 microM Cu, cytochrome c-553 synthesis was partially suppressed and an acidic plastocyanin was isolated. A newly developed procedure, using high concentrations of ammonium sulfate to fractionate water-soluble proteins on Sephacryl S-200 was successfully used to isolate and concentrate the plastocyanin, thus allowing it to be further purified to homogeneity. This protein has an isoelectric point of 4.8 which is similar to the pI value reported for other acidic plastocyanins from higher plants and green algae. Its N-terminal sequence of the first 15 amino acids has been determined; 9 of these amino acids are identical to those in the sequence of the basic plastocyanin from Anabaena variabilis.

Purification of a novel 30,000 Da calcium-binding protein from bovine cerebellum.

A novel very acidic calcium-binding protein (CaBP) was purified from bovine cerebellum, using 45Ca autoradiography as a marker, through a preparative procedure involving salting out with a very high concentration of ammonium sulfate, DE52 column chromatography, RNAase treatment, and HPLC gel filtration. This protein showed a molecular weight of 30,0000 dalton (Da) on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and of 120,000 on in gel filtration chromatography analysis under physiological ionic strength. The calcium binding activity of this 30,000 Da CaBP was monitored on the basis of calcium-dependent changes in tyrosine fluorescence (Kd = 3.0 microM).

9] Use of antibody- sepharose columns to study hormonal activation of cAMP-dependent protein kinase isozymes

This chapter presents a procedure using immobilized isoreceptor-specific antibody to assay the amount of cAMP endogenously bound to RI and RII in intact liver tissue or in isolated liver cells. In the first step the pelleted cells are frozen by immersion in liquid N 2 or the liver freeze-clamped in situ and pulverized under liquid nitrogen. The next step is homogenization at subzero temperature. The homogenization medium contains a high concentration of ammonium sulfate to precipitate and stabilize the intracellularly formed complexes of cAMP and R isoproteins. Nucleotides (e.g., cGMP) that do not interfere with the subsequent assay of cAMP are present to block any artifactual binding of cAMP. Ammonium sulfate and glycerol were used to stabilize the endogenously formed complexes. The strategy of the assay is to preserve the intracellularly formed complex of R and cAMP. It thus differs from other assays, which aim at preserving the complex of R and the catalytic subunit of the kinase.

Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G‐250 and R‐250

An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.

A study on heterogeneity in molecular species of shark cartilage chondroitin sulfate C. Fractionation of the polysaccharide on sepharose CL-4B in the presence of high concentrations of ammonium sulfate

Shark cartilage chondroitin surfate C was fractionated by chromatography on Sepharose CL-4B-2.5 to 1.5 m ammonium sulfate in 10m m hydrochloric acid at 4°. Both unit-disaccharide composition and molecular-size distribution clearly affected the fractionation. Comparison of this fractionation with the fractionation on Sepharose 6B gel in 0.2 m sodium chloride revealed that the former is distinctly superior to the latter. The fractionation on Sepharose CL-4B in the presence of ammonium sulfate also showed that the chondroitin sulfate C molecules having a larger molecular size contain generally more chondroitin 6-sulfate units (as major constituent) and less chondroitin disulfate units (D type, as minor constituent) than those having a smaller molecular size.

Binding of Fibrinogen Fragment D to Group A Streptococci Causes Strain Dependent Decrease in Cell Surface Hydrophobicity as Measured by the Salt Aggregation Test (SAT) and Cell Clumping in Polyethylene Glycol

Group A streptococcal reference strains of various M-types as well as three clinical isolates, and clinical isolates of group B, C and G streptococci were compared with respect to their agglutination by fibrinogen, specific binding of monovalent fibrinogen fragment D, and their hydrophobicity measured by salt aggregation in ammonium sulfate dilutions (SAT). Fibrinogen fragment D binding to M positive group A streptococci and clinical isolates of group A reduced salt aggregation of these strains from 0.06 molar to 2 molar in the ammonium sulfate dilution series. Group A, M protein negative and group B, C and G control strains used aggregated only at high ammonium sulfate concentration (2 molar) or not at all. Treatment of these strains with fibrinogen fragments did not influence their weak salt aggregation. By elution of specific retained fragment D from the type 1 M positive strain 40/58II by treatment of cells with 0.1 molar citric acid, 6 molar urea, buffer, pH 3.0, surface hydrophobicity could partially be restored (SAT titer 0.5 molar). Treatment of streptococcal cells with fibronectin or albumin at much higher protein concentrations had a moderate effect on surface hydrophobicity. These results indicate that binding of fibrinogen fragment D to the receptor can block a hydrophobic surface domain of the receptor or induce a sterical hindrance affecting surface exposure of associated surface molecules. A fibrinogen receptor — also responsible for binding of fibrinogen fragment D — is in M positive group A streptococci covalently linked to M protein. Decrease of salt aggregation tendency after mild pepsin treatment to release M protein supported these findings.

Chromatography of glycosaminoglycans on hydrophobic gel. Correlation between chromatographic behavior of glycosaminoglycans on phenyl-sepharose CL-4B and their solubility in the presence of high concentrations of ammonium sulfate

The effect of bound sulfate groups and uronic acid residues of glycosaminoglycans on their behavior in chromatography on hydrophobic gel was examined by the use of several pairs of depolymerized chondroitin, chondroitin 4- or 6-sulfate, and dermatan sulfate having comparable degree of polymerization. Chromatography on Phenyl-Sepharose CL-4B in 4.0-2.0 m ammonium sulfate containing 10m m hydrochloric acid showed that: (a) The retention of depolymerized chondroitin 4- or 6-sulfate on the gel varies with the temperature, whereas the depolymerized samples of chondroitin and dermatan sulfate does not show a temperature dependence (this is not the case for hyaluronic acid or dextrans). (b) Among depolymerized samples of chondroitin and chondroitin 4- and 6-sulfate that have a similar degree of polymerization, chondroitin 4- and 6-sulfate showed the highest retention. (c) The retention on the gel of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate decreased in this order. The solubility in ammonium sulfate solution of the polysaccharides agreed well with the chromatographic behavior, suggesting that the fractionation by the hydrophobic gel largely depends on the ability to precipitate on the gel rather than on the hydrophobic interaction between gel and polysaccharide.

The modulation of cell surface cAMP receptors from Dictyostelium disscoideum by ammonium sulfate

Dictyostelium discoideum cells contain a heterogeneous population of cell surface cAMP receptors with components possessing different affinities ( K d between 15 and 450 nM) and different off-rates of the cAMP-receptor complex ( t 1 2 between 0.7 and 150 s). The association of cAMP to the receptor and the dissociation of the cAMP-receptor complex still occur in the presence of 3.4 M ammonium sulfate. However, these processes are strongly altered. (1) Low concentrations of ammonium sulfate (≈ 50 mM) induce an approx. 2-fold increase of the number of cAMP binding sites. The same effect is induced by millimolar concentrations of CaCl 2. Ammonium sulfate and CaCl 2 are not additive, which suggests that these salts may act via the same mechanism. (2) High concentrations of ammonium sulfate (3.4 M) induce an alteration in the proportioning of the various cAMP binding sites to the components with the highest affinity. (3) High concentrations of ammonium sulfate (3.4 M) retard the dissociation of all binding sites about 3–6-fold, thus giving rise to an increase in the affinity of all cAMP-binding components.

Nuclear matrix-associated DNA methylase

Previous procedures for the extraction of DNA methylase (EC 2.1.1.37) from nuclei of mouse ascites cells have involved the use of buffers containing 0.2M NaCl. Whilst such ‘soluble’ methylase accounts for the bulk (70–80%) of DNA methylase activity a further portion of activity is detectable in a ‘bound’ form firmly associated with 2 M NaCl-resistant nuclear matrix-like structures. This association, which in part requires continuing DNA replication and protein synthesis, can, however, be disrupted in vitro with high concentrations of ammonium sulphate, and the enzymic properties of the ‘bound’ form of DNA methylase are similar to those described for the ‘soluble’ form.

Relative hydrophobicities of Actinomyces viscosus and Actinomyces naeslundii strains and their adsorption to saliva-treated hydroxyapatite.

The present study examined 42 strains of Actinomyces spp. to determine whether adsorption to saliva-treated hydroxyapatite (SHA) of the selected strains of this prominent group of dental-plaque bacteria correlated with hydrophobicity. The relative hydrophobicity of the strains was determined by their adsorption to hydrophobic gels (i.e., phenyl-Sepharose) and their aggregation in ammonium sulfate. Within serogroups the relative hydrophobicity for the strains was similar. The relative adsorption of strains to SHA was also similar within the respective serogroups. Strains which were relatively hydrophobic, as judged by their binding to the hydrophobic gel and aggregation in low concentrations of ammonium sulfate, adsorbed well to SHA. Strains which adsorbed poorly to SHA were relatively hydrophilic since they did not bind well to the hydrophobic gel and were only aggregated in relatively high concentrations of ammonium sulfate. Tween 80, a nonionic detergent known to inhibit hydrophobic interactions, blocked binding of cells to the hydrophobic gel, suggesting that hydrophobic interactions had been inhibited. However, Tween 80 exhibited no influence on the adsorption of cells to SHA. Thus, although there was a strong statistical correlation between the relative hydrophobicity of a strain and its adsorption to SHA, the data were consistent with the view that other interactions, such as ionic bonds and interactions between complimentary macromolecules, are involved in adsorption of the Actinomyces strains to SHA.

Adsorption chromatography on toyopearl: A single-step alternative to salt fractionation of protein mixtures

Toyopearl media for gel filtration tend to adsorb proteins at high ionic strength, presumably by hydrogen bonding. This is used in the technique proposed here for resolution of crude protein mixtures and initial purification of their components. Proteins can be selectively adsorbed on a column of Toyopearl at high ammonium sulfate concentration and then eluted by decreasing the salt concentration. This single-step procedure can replace the usual salt fractionation of protein mixtures, which is demonstrated with yeast cytochrome oxidase.

Novel approaches to the purification of penicillin acylase.

Penicillin acylase of E. coli NCIM 2400 has been purified to homogeneity using a combination of hydrophobic interaction chromatography and DEAE-cellulose treatment. A variety of substituted matrices were synthesized using D- or DL-phenylglycine, norleucine, ampicillin, or amoxycillin as ligands, all of which retained penicillin acylase at high concentrations of ammonium sulfate or sodium sulfate. The enzyme could be eluted nonbiospecifically by buffer of lower ionic strength with over 95% recovery of the activity. Ammonium chloride, ammonium nitrate, sodium chloride, sodium nitrate, and potassium chloride were ineffective in either adsorption or elution of the enzyme on these columns. Further purification of this partially pure enzyme with DEAE-cellulose at pH 7.0-7.2 yielded an enzyme preparation of very high purity according to electrophoretic and ultracentrifugal analyses, its specific activity being as high as 37 U/mg protein. The purified enzyme has a molecular weight of 67,000 a sedimentation coefficient of 4.0S, and resolves into two forms upon isoelectric focusing. Overall recoveries ranged between 75 and 85%. Ease of operation, high recoveries, high purity of the enzyme and prolonged reuse of the conjugates make the process economically feasible and possibly of great commercial importance.

Growth and ectomycorrhizal development of Pinus caribaea seedlings inoculated with basidiospores of Scleroderma dictyosporum in fertilized nursery soil in Tanzania

Seeds of Pinus caribaea were sown in plastic pots containing nursery soil with or without a basidiospore inoculum of Scleroderma dictyosporum. After seedling establishment, five fertilizer treatments involving 0, 250, 500, 750 and 1000 g NPK per m 3 of nursery soil were administered. The ratio of N:P:K in NPK was 3:3:1. Basidiospores of S. dictyosporum were an effective inoculum for P. caribaea seedlings. Inoculated seedlings showed superior growth in height, root collar area, length of stem with needles and dry matter production. Inoculation also increased the concentration of P in the plant tissues but had no effect on other nutrients studied. Fertilizer response was evident, but there were generally no benefits of using fertilizer levels above 250 g NPK per m 3 soil. In fact, mycorrhizal development was progressively reduced with each increase in the level of NPK fertilizer. Tissue Mg concentrations were also deficient, a reflection of nutrient uptake imbalance due to high concentrations of ammonium sulphate and potassium chloride in the soil. Basidiospores of S. dictyosporum can therefore be used for routine inoculation of P. caribaea seedlings in nursery soils at low levels of NPK. Coincidentally such a reduction in the amount of NPK applied in nursery soil will have both biological as well as economic benefits.

Isozyme composition and phosphorylation of brain phosphofructokinase

Rabbit brain phosphofructokinase was purified to homogeneity by a rapid procedure involving affinity chromatography and gel filtration. The enzyme consists of hybrids of the three phosphofructokinase subunit types C, A, and B. The molecular weights of these subunits are 86,000, 84,000, and 80,000, respectively; they are present in brain phosphofructokinase in a ratio of approximately 5:4:1.5. The enzyme as isolated from rabbit brain contains 0.16–0.18 mol phosphate per mole of subunit; another 0.4–0.5 mol phosphate per mole subunit can be incorporated in vitro in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase. The initial rate of phosphorylation is increased by fructose 2,6-bisphosphate or AMP and decreased by citrate or high concentrations of ammonium sulfate. All three subunit types are phosphorylated in vitro, and the phosphorylation site on each subunit is sensitive to cleavage by trypsin at a terminal region of each subunit. However, these sites show different relative rates of phosphorylation in vitro in the presence of ammonium sulfate. In vitro phosphorylation of brain phosphofructokinase had no affect on specific activity, inhibition by ATP, or activation by fructose 2,6-bisphosphate.

Separation of heparin on sepharose CL-4B in the presence of high concentrations of ammonium sulphate

Heparin was fractionated by chromatography on Sepharose CL-4B/3.8–2.0 M ammonium sulphate in 0.01 M hydrochloric acid at 4°C based on a principle different from that of gel filtration —possibly due to multiple interaction mechanisms including those based on hydrophobic bonds. The results of the fractionation were very similar to those of chromatography on Phenyl-Sepharose CL-4B/3.8–2.0 M ammonium sulphate in 0.01 M hydrochloric acid at room temperature. That is, the separation was related to the molecular size distribution, N-acetyl content and anticoagulant activities. As the result of studies on a set of Sepharose 4B gels with different degrees of cross-linking, it has been shown that the introduction of the cross-linked structure, OCH 2CH(OH)CH 2O, into the gel matrix enhances the interaction between the Sepharose CL-4B gel and heparin, indicating that heparin retention by the highly cross-linked Sepharose 4B gel surpasses that by Phenyl-Sepharose CL-4B.

Fetal calf ligament fibroblasts in culture secrete a low molecular weight collagen with a unique resistance to proteolytic degradation.

A highly unusual collagen was secreted by fibroblasts cultured from 150- and 270-d-old fetal calf nuchal ligaments. Purification revealed that this protein (which may be synthesized in a higher molecular weight form) was precipitated at unusually high concentrations of ammonium sulfate and was also eluted from DEAE-cellulose at greater salt concentrations than were types I and III procollagens. On SDS PAGE, the collagenous protein exhibited an Mr of approximately 12,750 that was not altered in the presence of reducing agent. The low molecular weight collagen (FCL-1) was sensitive to bacterial collagenase and had a [3H]glycine content comparable to that found in type I procollagen, although the [3H]Hyp to [3H]Pro ratio was 0.43. FCL-1 was not cleaved by human skin collagenase, mast cell protease, trypsin, Staphylococcal V8 protease, or proteinase K at 37 degrees C. The collagen was susceptible to trypsin, but not to V8 protease, only after heating at 80 degrees C for 30 min. Preliminary structural studies indicate that FCL-1 was resistant to cleavage by CNBr but exhibited limited proteolysis with pepsin. Both 150- and 270-d-old fibroblasts produced comparable levels of interstitial (types I and III) procollagens, which comprised approximately 70% of the total protein secreted into the culture medium. However, 270-d-old (term) fibroblasts secreted approximately 50% more FCL-1, as percent of total culture medium protein, in comparison to the cells from the earlier gestational stage. This collagen may therefore play a role in the development of the nuchal ligament.

Nuclear binding and sedimentation properties of two activated forms of glucocorticoid-receptor complexes from rat mammary glands

Mammary cytoplasmic glucocorticoid-receptor complexes, which had been exposed to a high concentration of ammonium sulfate, were found to be separated into two receptor forms, consisting of DNA-cellulose-bound and non-DNA-cellulose-bound receptors. Experiments under cell-free conditions revealed that not only the DNA-cellulose-bound receptor but also the non-DNA-cellulose-bound receptor have binding abilities to nuclei and to nuclear chromatin. In sucrose density gradient centrifugation, the non-DNA-cellulose-bound receptor sedimented at higher sedimentation coefficients than the DNA-cellulose-bound receptor under all the salt conditions tested. These data strongly suggest that in the mammary glucocorticoid receptor system there exist at least two activated receptor forms with different conformations.

Formation of a catalytically active complex between tRNA Asp and aspartyl-tRNA synthetase from yeast in high concentrations of ammonium sulphate

The interactions of yeast tRNAAsp with cognate aspartyl-tRNA synthetase have been studied in high concentrations of either sodium chloride or ammonium sulphate by fluorescence titration and small-angle neutron scattering. In solutions containing more than 1M NaCl no complex is formed and enzymatic activity is abolished. In strong contrast, however, the physical measurements showed the formation of a two-to-one tRNA-enzyme complex, with high affinity, in 1.6 M (NH4)2SO4. Aminoacylation assays under the same salt conditions showed the enzymatic fixation of aspartic acid to tRNAAsp to occur at an appreciable rate. The present study emphasizes that the effects of salts on protein-nucleic acid interactions do not depend only on ionic strength but also on the nature of the salt. This study has allowed a rational approach to the crystallisation of a functional tRNAAsp-aspartyl-tRNA synthetase complex (Giegé, Lorber, Ebel, Thierry and Moras (1980) C.R. Acad. Sci. Paris, série D, 291, 393-396).

Inhibition of germination inStriga by means of urea

In vitro seed germination percentages and radicle lengths ofStriga hermonthica markedly decreased in the presence of urea at concentrations which could be expected in the field after standard application rates. Relatively high concentrations of ammonium sulphate brought about a similar effect while sodium nitrate was ineffective.

Phosphoenolpyruvate carboxylase of Escherichia coli. Hydrophobic chromatography using specific elution with allosteric inhibitor.

The adsorption of Escherichia coli phosphoenolpyruvate carboxylase [EC 4.1.1.31] to butyl-, hexyl-, and octyl-Sepharose gels was investigated. The enzyme was nearly completely adsorbed to the latter two gels both in the absence and presence of high concentrations of ammonium sulfate. At intermediate concentrations--0.1 M in the case of hexyl-Sepharose--virtually no adsorption was observed. Upon application of an increasing or decreasing concentration gradient of the salt, the enzyme was eluted at various concentrations of the salt depending on chain length of the immobilized alkyl groups. The adsorption to hexyl-Sepharose at 0.7 M ammonium sulfate was markedly decreased by L-aspartate, the allosteric inhibitor, whereas it was increased by acetyl-CoA, one of the allosteric activators. Evidence was obtained suggesting that these changes in adsorption were due to conformational alterations of the enzyme elicited by these effectors. The enzyme seemed to have been adsorbed at its hydrophobic regions which were distinct from the allosteric site for long-chain fatty acids. The specific elution with L-aspartate in the presence of 0.82 M ammonium sulfate could successfully be applied to purification of the enzyme. By this hydrophobic interaction chromatography, the enzyme was purified about 55-fold over its partially purified preparation with a recovery of 73%. The obtained enzyme preparation was almost homogeneous as judged from sodium dodecylsulfate-polyacrylamide gel electrophoresis.