Fractionating with ammonium sulfate

Dibydropteridine reductase (from sbeep liver) [9074-11-7] Mr 52,000 [EC 1.6.99.7]. Purified by fractionation with ammonium sulfate, dialysed versus tris buffer, adsorbed and eluted from hydroxylapatite gel. Then run through a DEAE-cellulose column and also subjected to Sephadex G-lOO filtration. [Craine et al. J Biol Chem 247 6082 1972.]... 

Phosphoribosyl pyrophosphate synthetase (from human erythrocytes, or pigeon or chicken liver) [9015-83-2] Mr 60,000, [EC 2.7.6.1]. Purified 5100-fold by elution from DEAE-cellulose, fractionation with ammonium sulfate, filtration on Sepharose 4B and ultrafiltration. [Fox and Kelley J Biol Chem 246 5739 197h, Flaks Methods Enzymol6 158 1963 Kornberg et al. J Biol Chem 15 389 7955.]... 

Since the discovery and partial purification of FDPase by Gomori (2), a number of purification procedures have been described (4, 22, 24-27). Among these, the most widely employed are based on the procedure of Pontremoli et al. (22), using acetone powders from freshly collected rabbit livers. The steps include precipitation of inactive protein at pH 4.5, fractionation with ammonium sulfate, heating to 50°, and chromatography on carboxymethyl cellulose columns, from which the enzyme... 

Fructosediphosphatase has been purified from the supernatant fraction of swine kidney homogenates by a procedure which included chromatography on phosphocellulose and fractionation with ammonium sulfate (20, 65). This preparation was free of other phosphatases, showed high affinity for fructose 6-phosphate, and was inhibited at concentrations of substrate above 10 4 M. The inhibition by higher concentrations of substrate was greater at lower pH this caused the apparent pH optimum to shift from pH 8.5 to 9.5 when the substrate concentration was increased from 5 X 10"5 M to 5 X 10"4 M. The molecular weight was estimated to be approximately 130,000 (65). 

The enzyme was purified from Candida utilis in 1965 by Rosen et al. (8Q). Dried yeast was allowed to autolyze in phosphate buffer at pH 7.5 for 48 hr, and the enzyme was isolated in crystalline form from these autolysates by a procedure which included heating to 55° at pH 5.0, fractionation with ammonium sulfate, and purification on phospho-cellulose columns from which the enzyme was specifically eluted with malonate buffer containing 2.0 mM FDP. Crystallization was carried out by addition of ammonium sulfate in the presence of mM magnesium chloride. The Candida enzyme was more active than the mammalian FDPases at room temperature and pH 9.5 the crystalline protein catalyzed the hydrolysis of 83 /nnoles of FDP per minute per milligram of protein. The enzyme was completely inactive with other phosphate esters, including sedoheptulose diphosphate, ribulose diphosphate, and fructose 1- or fructose 6-phosphates. Nor was the activity of the enzyme inhibited by any of these compounds. Optimum activity was observed at concentrations of FDP between 0.05 and 0.5 mM higher concentrations of FDP (5 mM) were inhibitory. 

As Streptomyces contain high proportions of proteases, a protease inhibitor, diphenylcarbamoyl chloride, was added to the buffer during purification of the dTDP-dihydrostreptose synthase. A partially purified, enzyme preparation from S. griseus could be obtained by removal of nucleic acids with streptomycin and fractionation with ammonium sulfate.40 However, when this enzyme preparation was subjected to gel filtration on a column of Sephadex G-100, enzyme activity was completely lost. By combining certain fractions of the column eluate, enzyme activity could be partially restored. 

There have been only a few kinetic studies on plant /3-D-manno-sidases, because of the apparently limited distribution of this enzyme in various species, and also because of the paucity of attempts at purification. The enzyme from malted barley has been purified 41-fold by fractionation with ammonium sulfate, and chromatography302 on Biogel P-100, DEAE-cellulose, and CM-cellulose. Some properties of /3-D-mannosidases from various tissues are summarized in Table II. 

The resolved complex is composed of two fractions, a soluble part, which comprises about 15% of complex I proteins, and a water-insoluble part consisting of the rest of the protein and the bulk of complex I lipids. The soluble fraction is easily separated from the insoluble material by centrifugation. Upon fractionation with ammonium sulfate, it yields a soluble flavoprotein containing iron and labile sulfide and a dark brown protein, which contains large amounts of iron and labile sulfide but no flavin. The latter appears to be an iron-sulfur protein and exhibits an EPR signal which is characteristic of iron-sulfur center 2 of intact complex I (46). Its absorption spectrum is shown in Fig. 8. The insoluble fraction also contains equimolar amounts of iron and labile sulfide and little or no flavin. 

In terms of overall efficiency, chemical precipitation of the bound fraction with ammonium sulfate or polyethylene glycol is often less satisfactory than systems such as second antibody. In particular, they tend to yield high assay blank values. However, for many routine assays this defect is compensated by their high level of practicality. 

Thymosin fraction 5 (TF5) is a partially purified mixture of polypeptides prepared from calf thymus glands as starting material (Hooper et al., 1975). The crude thymus extract is purified by a heat step, acetone precipitation, and fractionation with ammonium sulfate. The 25%-50% ammonium sulfate precipitate is further subjected to ultrafiltration using an Amicon DC-2 hollow fiber system to yield fraction 5 that is lyophilized. Fraction 5 consists of 10 major and at least 30 minor polypeptides on analytical isoelectric gel focusing (Fig. 8), with molecular weights ranging from 1000 to 15,000, and is... 

Thymostimulin (TS) is an extract of calf thymus glands that has been partially purified by Falchetti et al. (1977) in Italy. Calf thymus tissue is first minced and extracted with ammonium acetate. This extract is then fractionated with ammonium sulfate precipitation. The 0-25% ammonium sulfate cut is further purified by ultrafiltration on an Amicon PM-10 membrane, desalted on Sephadex G-25, and gel filtered on Sephadex G-50. The biologically active preparation exhibits two predominant bands on polyacrylamide gels at pH 8.6. At the present time there have been no attempts to further define the constituents of this partially purified preparation. Although TS is similar to thymosin fraction 5 in its purification schema, a 0-25% ammonium sulfate precipitation step is used, whereas a 25-50% saturation fractionation cut is employed for the isolation of thymosin fraction 5. In addition, the purification procedure for TF5 includes an acetone precipitation step, whereas this procedure is not included in the preparation of TS. Thus, there... 

Myosin phosphatases are prepared from both the soluble (cytosolic) and the myofibrillar fraction of smooth muscle. If the source of phosphatase is the soluble fraction, then chromatographic procedures are usually preceded by fractionation with ammonium sulfate. Myosin phosphatase from myofibrils, crude actomyosin, or myosin is solubilized at high ionic strength by 0.6 NaCl plus detergent (Alessi et al.,... 

Partial purification of the lividomycin-inactivating enzyme was attempted by Mitsuhashi and coworkers, who briefly described their discovery that the enzyme obtained by fractionation with ammonium sulfate and column chromatography on Sephadex G-lOO inactivates lividomycins A and B, but not kanamycin A, indicating involvement of two different enzymes in the phosphorylation of lividomycin and kanamycin. However, it was definitely proved by H. Umezawa and coworkers that kanamycin-neomycin phosphate transferase I phosphorylates the 5"-hydroxyl group of lividomycins. The observation by Mitsuhashi and coworkers was probably occasioned by instability of the enzyme and the higher activity of the enzyme in phosphorylating lividomycins than in phosphorylating kanamycin A. 

Killick and Wright examined the levels of the enzyme trehalose 6-phosphate synthetase at various stages of development of the slime mold, and obtained results that contradicted those of Both and Suss-man. They found that the enzyme was not only present at preculmination, but was also present in a masked form at the beginning of differentiation. By selective fractionation with ammonium sulfate and pre-incubation at 35°, they were able to unmask the enzyme and show its activity. Some evidence was obtained for the presence of an extracellular inhibitor of the enzyme. 

---