Solid Ammonium Sulfate Research Articles

Production, partial purification and characterisation of lipases from Pseudomonas fragi CRDA 037

Pseudomonas fragi CRDA 037 produced extracellular (exo-) and intracellular (endo-) lipases when grown in whey media. Partially purified lipase extracts were obtained by precipitation with solid ammonium sulphate at 20–40% saturation for the exolipase and 20–60% saturation for the endolipase. Native polyacrylamide gel electrophoresis of the exolipase showed the presence of a major band with a molecular weight ( M r ) of 25·5 kDa, whereas the endolipase showed the presence of three fractions with M r of 35·5, 49 and 70 kDa. The pH optima for the exo- and endolipases were 8·75 and 9·0, respectively. Substrate specificity determinations were performed with triacetin, tributyrin, trimyristin and triolein, where exo- and endolipase demonstrated higher affinity for trimyristin, with K m values of 5·11 and 3·24 m m, respectively. Inhibition of the exo- and endolipase occurred in the presence of a range of chemicals, including sodium deoxychelate, ferrous and ferric chloride, diisopropyl fluorophosphate, N-bromosuccinimide and 5,5-dithiobis-(-2-nitrobenzoic acid).

H2SO4 vapor pressure of sulfuric acid and ammonium sulfate solutions

Few measurements of H2SO4 vapor pressure have been made for sulfuric acid in the temperature and concentration ranges of atmospheric interest because of the very low pressures involved (below 10−4 Pa, or 10−6 torr); no such measurements appear to have been made for sulfuric acid solutions neutralized with ammonia. This work presents measurements of H2SO4 vapor pressure for aqueous sulfuric acid solutions between 55 and 77 wt% H2SO4 (corresponding to about 5–25% relative humidity), ammonium sulfate solids at low humidities, and partially neutralized sulfate solutions with [NH4+]: [SO4=] ratios between 0.13 and 1.0. The vapor pressure data collected over sulfuric acid solutions generally agree with the predictions of Ayers, et al. [1980], although positive deviation was observed for the more dilute solutions. The good agreement between this measurement and previous efforts by absolute techniques suggests that the evaporative coefficient for the H2SO4‐H2O system is near unity. H2SO4 vapor pressures over solid ammonium sulfate were measured between 27°C and 60°C; the data were fitted to In p = A/T + B, with A = −5928 and B = −3.77. The H2SO4 vapor pressures of mixed H2SO4‐H2O‐(NH4)2SO4 solutions dropped significantly as the [NH4+]:[SO4=] ratio exceeded 0.5. The results suggest that ammonia could very effectively stabilize molecular clusters of sulfuric acid and water in the atmosphere against evaporation, leading to rates of new particle formation higher than those predicted by binary H2SO4‐H2O theory.

Molecular Cloning and Expression of Rat Liver Aminopeptidase B

We isolated, by immunological screening of a Uni-ZAP XR cDNA library constructed from rat liver mRNAs, a cDNA clone with 2212 base pairs encoding aminopeptidase B (EC 3.4.11.6). The open reading frame encodes a 649-amino acid protein with a theoretical molecular mass of 72,545 Da and bears the consensus sequence of the zinc metalloexopeptidases, indicating that the enzyme belongs to this family, which includes aminopeptidase A, aminopeptidase N, and leukotriene-A4 hydrolase. Escherichia coli SOLR cells infected with the pBluescript phagemid excised from the Uni-ZAP XR vector containing the aminopeptidase B cDNA had a high L-arginyl-beta-naphthylamidase activity. The recombinant protein was purified to homogeneity from the recombinant E. coli extracts. The enzyme had Cl--dependent aminopeptidase activity specifically restricted to the Arg and Lys derivatives and contained 1 mol of zinc per mol of the enzyme.

Rabbit ceruloplasmin: purification and partial characterization.

Rabbit ceruloplasmin (Cp) was purified after solid ammonium sulfate precipitation to 60% final saturation by a two-step column chromatography procedure, utilizing DEAE-Sephadex A-50 and changing the NaCl concentration in the buffer to 0.16 M to achieve the isolation of the protein. The purified Cp was used to prepare antibodies in guinea pigs that were used afterwards to determine the Cp concentration in normal rabbits and in rabbits with an experimentally induced chronic anemia. The molecular weight of rabbit Cp determined by SDS-PAGE was 125,000 and a high molecular weight Cp of 200,000 comprising 8% of the total purified protein was also found. An optical density ratio (610 nm/280 nm) of 0.0475 and a molar extinction coefficient of 7625 were obtained. Copper determinations yielded a value of 0.24% that corresponds to 5 copper atoms per molecule. The staining of Cp following discelectrophoresis in polyacrylamide gels also showed a two band pattern.

A novel phospholipase C inhibitor, S-PLI produced by Streptomyces sp. strain no. A-6288.

S-PLI, an inhibitor of phospholipase C (PLC) produced by Streptomyces sp. strain No. 6288, was purified from the culture filtrate by salting-out with solid ammonium sulfate, column chromatography on CM-cellulose and gel filtration on Sephadex G-75. The molecular weight of S-PLI was estimated to be 65,000 by SDS-polyacrylamide gel electrophoresis. The inhibitor was found to be a glycoprotein with a composition of 609 amino acids and 19 glucose residues having an isoelectric point at 7.8. S-PLI was stable from pH 3 to 10 at 37 degrees C and up to 40 degrees at pH 6.0. The inhibitory activity showed pH- and temperature-dependence with a maximum around pH 7.0 at 50 degrees C. S-PLI inhibited phospholipase C in a competitive manner (Ki value; 9.5 x 10(-6) mM), but did not inhibit S-Hemolysin, phospholipase A2; phospholipase B, phospholipase D and phosphatases. S-PLI is the first reported example of a glycoproteinaceous inhibitor of microbial origin which is able to specifically inhibit phospholipase C.

Activation of Cell Growth Inhibitor by Ectoprotein Kinase-mediated Phosphorylation in Transformed Mouse Fibroblasts

Our previous studies have shown that exogenous ATP induces cell growth inhibition in transformed mouse fibroblasts, 3T6 cells, whereas the growth of their nontransformed counterparts, Swiss 3T3 cells, is only slightly affected. In this study a similar selective, ATP-induced growth inhibition was found in Balb/c SV40-3T3 cells and in primary cultures of adenovirus-transformed murine fibroblasts. The inhibitory activity was found in the conditioned media of ATP-treated cultures. Several lines of evidence have shown that ectoprotein kinase (ecto-PK) plays a major role in the ATP-induced growth inhibition. (a) There is a good correlation between the activity of ecto-PK and the ability of ATP to induce cell growth inhibition. (b) The removal of the ecto-PK from the cell surface prevents the ATP-induced growth inhibition. (c) Addition of the removed enzyme to the cell culture reconstitutes the ability of ATP to induced growth inhibition. (d) Serum-containing, or serum-free, conditioned media from untreated cultures gain an inhibitory activity after their phosphorylation, and dephosphorylation of conditioned media from ATP-treated cultures results in the loss of the inhibitory activity. (e) Growth medium by itself does not inhibit cell proliferation after its phosphorylation. The findings described in d and e indicate, as well, that the ATP-induced growth inhibitor is produced by the cells. The putative inhibitor was found to be a protein, with an apparent molecular mass of 13 kDa. The selectivity of the inhibition for transformed cells is due to the higher level of ecto-PK in these cells, as well as to their higher susceptibility to the inhibitor, as compared with their non-transformed counterparts.

Thermodynamic Properties of Aqueous (NH4)2SO4 to High Supersaturation as a Function of Temperature

Ammonium sulfate is an important component of aqueous atmospheric aerosols in both remote and urban regions. Equilibrium water vapor pressures over supersaturated aqueous ammonium sulfate have been determined from 278.15 to 313.15 K using an electrodynamic balance. Partial molar enthalpies of water (L{sub 1} to {approximately}25 mol/kg) and partial molar heat capacities (J{sub 1}, to {approximately}8 mol/kg) have been calculated from the measurements, together with enthalpies of solution, heat capacities, and boiling point elevations. The results have been used to parameterize a mole-fraction-based activity coefficient model. Equilibrium constants for the reaction (NH{sub 4}){sub 2}SO{sub 4}{sub (cr)} {l_reversible} 2NH{sub 4}{sup +} (aq) + SO{sub 4}{sup 2{minus}} aq have been evaluated from 373.15 K to the eutectic point (5.00 mol/kg, 254.21 K). The model satisfactorily represent water vapor pressures and saturations with respect to solid phases ice and ammonium sulfate from 253 to 373.15K and up to 8--10 mol/kg, and water vapor pressures from 278.15 to 313.15 K and up to {approximately}25 mol/kg.

Purification of antibodies using ammonium sulfate fractionation or gel filtration.

In this chapter, two commonly used techniques that are utilized in many immunoglobulin purification schemes are described. The first procedure, ammonium sulfate fractionation, is generally employed as the initial step in the isolation of crude antibodies from serum or ascitic fluid (#x2013;). Ammonium sulfate precipitation, in many instances, is still the method of choice because it offers a number of advantages. Ammonium sulfate fractionation provides a rapid and inexpensive method for concentrating large starting volumes. “Salting out” of polypeptides occurs at high salt concentrations where the salt competes with the polar side chains of the protein for ion pairing with the water molecules, and where the salt reduces the effective volume of solvent. As expected from these observations, the amount of ammonium sulfate required to precipitate a given protein will depend mainly on the surface charge, the surface distribution of polar side chains, and the size of the polypeptide, as well as the pH and temperature of the solution. Immunoglobulins precipitate at 40–50% ammonium sulfate saturation depending somewhat on the species and subclass (). The desired saturation is brought about either by addition of solid ammonium sulfate or by addition of a saturated solution. Although the use of solid salt reduces the final volume, this method has a number of disadvantages.

Cerebral metabolism of oxidized ascorbate

The brain has a high level of ascorbic acid which is thought to act as a reducing agent, e.g. in protecting tissues against oxidative stress. The mechanisms by which ascorbate is maintained in the useful, reduced state in the CNS is evaluated herein. Cerebrum from rat or calf was minced and homogenized in buffer. The endogenous levels of ascorbic acid, dehydro- l-ascorbic acid (DHAA) and reduced glutathione (GSH) were determined by HPLC with coulometric electrochemical detection. We also quantitated tissue capacity to regenerate ascorbic acid from DHAA, which is a product of electron transfer reactions of ascorbic acid. The homogenate was fractionated by centrifugation in steps up to110,000 × g and dialyzed free of low molecular weight components. The activity for reducing DHAA was approximately equal in the various supernatants; resuspended pellets had little activity. The active component has several properties of a protein, including being precipitated by solid ammonium sulfate addition to the tissue extract; most activity appeared in the 40–80% saturated fraction. The activity was stable up to a temperature of 80°C, but was lost at 95°C. The protein was digested by trypsin. The results suggest that a cytosolic component of cerebrum regenerates ascorbic acid in a step that preferentially uses GSH and NADPH as reducing cofactors. At least one form of DHAA reductase exists in brain.

Identification and purification of calcium-independent phospholipase A2 from bovine brain cytosol.

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

47] Phosphatidylinositol-specific phospholipases C from Bacillus cereus and Bacillus thuringiensis

Publisher Summary Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus and Bacillus thuringiensis catalyzes the cleavage of the sn -3 phosphodiester bond of phosphatidylinositol (PI). Bacillus cereus and B. thuringiensis are closely related rod-shaped gram-positive bacteria that are nonpathogenic to man. The enzymes are excreted in relatively large quantities across the single cell membrane into the growth medium. This facilitates the purification of PI-PLC as intracellular proteins are removed with the cells during an initial centrifugation step. PI-PLC is isolated from the supernatants of B. cereus and B. thuringiensis cultures in milligram quantities. The final product is homogeneous on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), exhibits high specific activity, and is used to raise monoclonal antibodies and provide polypeptide sequence information for the cloning and DNA sequencing of PI-PLC from B. cereus . The B. cereus or B. thuringiensis culture is cooled to 4°, and the cells are removed by centrifugation for 30 min at 13,000 g. Solid ammonium sulfate is added slowly to the supernatant to 90% saturation (576 g/liter), and the solution is kept overnight at 4°. The purity of the final enzyme preparations is generally better than 95%, with similar specific activities for the B. cereus and B. thuringiensis enzymes in the range of 1200 to 1500 units/mg. During the early steps of the purification, the specific enzyme activities are affected by the presence of colored contaminants that contribute strongly in the protein determinations. These contaminants are completely removed by the final Phenyl-Sepharose step.

45] Purification of lysophospholipase and lysophospholipase-transacylase from rabbit myocardium

Publisher Summary This chapter describes the chromatographic methods developed for the purification of rabbit myocardial lysophospholipase and lysophospholipase-transacylase to homogeneity. Lysophospholipase and lysophospholipase-transacylase activities are quantified by incubating 1-[1- 14 C]hexadecanoyl- sn -glycero-3-phosphocholine (palmitoyllysophosphatidylcholine) with 50 or 100 μl of column fractions in 75 mM potassium phosphate, pH 7.0 for 10 min at 37 ° in a final volume of 700 μl. New Zealand White rabbits (typically 10–20) are sacrificed by cervical dislocation, a left thoracotomy is performed, and hearts (∼6 g/heart) are rapidly placed in ice-cold buffer. All procedures are performed at 0°–4°. Ventricular myocardium is isolated, extensively rinsed in buffer, and homogenized utilizing three 30 sec bursts from a Polytron homogenizer at a setting of 5 to yield a 25% homogenate. Nuclei, mitochondria, and cellular debris are removed by low-speed centrifugation, and the resulting supernatant is further centrifuged at 100,000 g max for one hour to obtain myocardial cytosol. Myocardial cytosol is brought to 55% saturation by slow addition of solid ammonium sulfate, the mixture is stirred at 4° for an additional nine min, and the resulting precipitate is pelleted by centrifugation at 10,000 g max for 5 min.

Improved high-performance liquid chromatographic assay for the quantification of 5-bromo-2′-deoxyuridine and 5-bromouracil in plasma

A sensitive and specific procedure using high-performance liquid chromatography (HPLC) was developed for the quantification of 5-bromo-2′-deoxyuridine (BUdR) and 5-bromouracil (BU) in plasma. BUdR and BU were first extracted with a mixture of ethyl acetate and 2-propanol from plasma presaturated with solid ammonium sulfate. Following evaporation of the organic extract, the remaining residue was reconstituted in saturated ammonium sulfate solution, washed with a mixture of n-pentane—methylene chloride and re-extracted with the original solvent mixture. The organic extract was evaporated, reconstituted in mobile phase and chromatographed on a regular-bore ODS HPLC column using ultraviolet absorbance detection. The BUdR and BU quantification limits were both 0.1 μ M, the mean intra-assay coefficients of variation were 5.0 and 5.6%, respectively, and the mean inter-assay coefficients of variation were 5.4 and 10.7%, respectively. This method was used to determine steady-state femoral arterial and hepatic venous plasma concentrations of BUdR and BU in a patient receiving a continuous intravenous infusion of BUdR (20 mg/kg per day).

Studies on Seed Peroxidase Phaseolus vulgaris cv, Haricot

ABSTRACT Phaseolus vulgaris cv, haricot seed peroxidase was extracted and partially purified. The precipitation of an active peroxidase fraction with solid ammonium sulfate (at 35‐90% saturation) increased its activity by a factor of 3. The pH for optimum activity was 5.4. The addition of 4 μm hydrogen peroxide increased the activity 92‐fold; however, enzyme activity was decreased by higher concentrations of this reagent. Carbonyl compounds resulting from peroxidase activity were isolated as their dinitrophenylhydrazones and then purified by preparative TLC. Subsequent GC‐MS analysis revealed that acetone was the principal carbonyl compound resulting from enzyme activity.

37] Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle

Publisher Summary This chapter describes the purification of two forms of protein phosphatase- 1 (PP-I 1 and PP-l G ), and three forms of protein phosphatase-2A (PP-2A 0 , PP-2A 1 , and PP-2A 2 ) from skeletal muscle. Procedures for isolating the free catalytic subunits (termed PP-l c and PP-2A c ) are documented and the structures of these enzymes are summarized in the chapter. The free catalytic subunits are isolated by a modification of the procedure of Lee and co-workers. Skeletal muscle extracts prepared from 3000 g of muscle are adjusted to pH 7.2 with 10 M ammonium hydroxide, and 350 g of solid ammonium sulfate are added to each liter of solution to bring the degree of saturation to 55%. After standing for 30 min the suspension is centrifuged for 40 min at 4200 g and the supernatant discarded. The PP-2A c from poly(L-lysine)-Sepharose is purified in an identical manner to PP-I c .

Properties of a virus inhibitor from spinach leaves and mode of action.

A procedure was shown for obtaining inhibitor of virus infection from the sap of spinach leaves. The leaf sap was brought to 50% saturation with solid ammonium sulfate. The precipitate was suspended in neutral phosphate buffer (PB) and filtrated through a Sephadex G-100 gel column. The active fractions were combined and further purified by DEAE cellulose and CM-Sephadex C-25 column chromatographies. The purified inhibitor lost its inhibitory activity by heating at 100C for 10min. The inhibitor inhibited TMV infection on bean and tobacco, while it showed no or a little effect on Chenopodium amaranticolor. When the inhibitor was treated on the undersurface of bean leaves and TMV was inoculated on the uppersurface, the weak inhibitory activity was observed. The inhibitor adsorbed on the leaf surfaces could not be easily removed by Triton X-100 treatment. The inhibitor also showed inhibitory activity to TMV-RNA infection on bean leaves. This reaction was not due to contamination of ribonuclease in the inhibitor preparation.

8702024 Reduction of nitrogen-based pollutants through the use of urea solutions containing oxygenated hydrocarbon solvents

A process for maintaining low effluent ammonia contents while reducing nitrogen oxides in an effluent from the combustion of a carbonaceous fuel under oxygen-rich conditions which minimize the production of carbon-based pollutants. An aqueous solution of urea and an oxygenated hydrocarbon is injected into an effluent at a temperature above 1600oF, and preferably above 2000oF. The oxygenated hydrocarbon will be employed at a level effective to reduce the levels of free ammonia in the effluent. This is important when burning fuels containing significant sulfur contents because the free ammonia tends to react with the sulfur compounds to produce solid ammonium sulfate and/or bisulfate which can decrease boiler efficiency.

Characterization of Seed Lipoxygenase of Phaseolus vulgaris cv, Haricot

ABSTRACT Phaseolus vulgaris cv, haricot seed lipoxygenase was extracted and partially purified. The precipitation of an active lipoxygenase fraction with solid ammonium sulfate (at 20–50% of saturation) increased its activity by a factor of 3. The pH for optimum activity was 7.3. The addition of 40 mM potassium cyanide resulted in a doubling of the enzyme activity. Enzyme activity was completely lost on storage at 4°C for 48 hr. The activity of haricot lipoxygenase extract was considerably greater on linoleic acid than on its esters, and decreased in the order: mono‐ > di > trilinolein. The haricot lipoxygenase shares many of the characteristics of the corresponding enzyme found in several seed sources.

Spatial Variation of Stratospheric Aerosol Acidity and Model Refractive Index: Implications of Recent Results

Recent experimental results indicate that little or no solid ammonium sulfate is present in background stratospheric aerosols. Other results allow straightforward calculation of sulfuric acid/water droplet properties (acidity, specific gravity, refractive index) as functions of stratospheric temperature and humidity. These results are combined with a variety of latitudinal and seasonal temperature and humidity profiles to obtain corresponding profiles of droplet properties. These profiles are used to update a previous model of stratospheric aerosol refractive index. The new model retains the simplifying approximation of vertically constant refractive index in the inner stratosphere, but has sulfuric acid/water refractive index values that significantly exceed the previously used room temperature values. Mean conversion ratios (e.g., extinction-to-number, backscatter-to-volume) obtained using Mie scattering calculations with the new refractive indices are very similar to those obtained for the old indices, because the effects of deleting ammonium sulfate and increasing acid indices tend to cancel each other.

Mode of interaction between forskolin and manganese ion in activating catalytic unit of adenylate cyclase from rat brain.

Rat brain adenylate cyclase was solubilized with a combination of 0.7% sodium cholate and 0.6 M ammonium sulfate, and fractionated by addition of solid ammonium sulfate. The precipitate at 35% ammonium sulfate saturation contained neither guanine nucleotide-binding regulatory protein (G protein) nor calmodulin, and was used as the catalytic unit of the enzyme system. This catalytic unit was activated synergistically by forskolin and Mn2+. An apparent Km value for Mg-adenosine triphosphate (ATP) of the catalytic unit was about 80 microM in the basal state, while it increased in concurrence with the increase in the enzyme activity when forskolin was added to the assay system. The increase in the Km value depended on the forskolin concentration up to 1 microM, above which the value converged on ca. 200 microM. Furthermore, activation of the catalytic unit by forskolin was more marked at higher concentration of Mg-ATP. On the other hand, Mn2+ suppressed the increase in the Km value for Mg-ATP by forskolin, though the value in the basal state was not changed by Mn2+ alone. These findings indicate that the activation of the catalytic unit by forskolin is accompanied by the change in the affinity for Mg-ATP and Mn2+ modifies the change.