DEAE-cellulose chromatography, fractionation

Three histone-specific acetyltransferases have been partially purified and characterized from rat thymus nuclei (225). The enzymes were extracted from rat thymus nuclei by sonication in the presence of 1M ammonium sulfate and separated into two active fractions (A and B) by DEAE-cellulose chromatography. Fraction B was further separated into two active fractions (Bi and B2) by gel filtration on Sephadex G-200. Each fraction was then purified further by chromatography on hydroxyapatite. The molecular weights, determined by Sephadex G-200 and by sucrose density gradient centrifugation, were 99,000, 110,000, and 92,000 for enzymes A, Bi, and B2, respectively. All three enzymes required acetyl CoA as acetate donor, and the activity of the enzymes was inhibited by p-chloromercuribenzoate. Acetyltransferase A preferentially acetylated histone I (FI) and also poly-L-lysine. Acetyltransferase Bi and B2 preferred histone H4 (other names IV, F2al) and did not acet-ylate poly-L-lysine and histone H3 (III, F3). In addition to c-N-acetyl-lysine, two other unidentified amino acid derivatives were obtained from a digest of histone H4 acetylated by the two B enzymes. 

DEAE-cellulose chromatography. The 50% ethanol solution is poured onto a column of DEAE cellulose (2.6 x 10 cm), and F adsorbed at the top is eluted with 20 mM Tris buffer, pH 7.5, containing 0.2 M NaCl. A low pressure of argon gas is applied to accelerate the flow rate. The fractions containing F are combined, concentrated, and desalted using ethanol. 

Purification of luciferin (Rudie etal., 1976). The luciferin fractions from the DEAE-cellulose chromatography of luciferase were combined and concentrated in a freeze-dryer. The concentrated solution was saturated with ammonium sulfate, and extracted with methyl acetate. The methyl acetate layer was dried with anhydrous sodium sulfate, concentrated to a small volume, then applied on a column of silica gel (2 x 18 cm). The luciferin adsorbed on the column was eluted with methyl acetate. Peak fractions of luciferin were combined, flash evaporated, and the residue was extracted with methanol. The methanol extract was concentrated (1 ml), then chromatographed on a column of SephadexLH-20 (2 x 80 cm) usingmethanol asthe solvent. The luciferin fractions eluted were combined and flash evaporated. The residue was... 

Figure 2. Anion-exchange chromatography on DEAE Cellulose of fraction IP obtained after size-exclusion separation. Fractions (2 ml each) were pooled as indicated, o - Uronic acids, A - pentoses, x - hexoses.

Figure 2. CM-cellulose chromatography of pectolytic enzymes. The activity peaks of the flow-through of a DEAE-cellulose chromatography was applied to a CM-cellulose column. The column was eluted with a NaCl (0-0.5M) continuous gradient at a flow rate of 34 ml/h. 10 ml fractions were collected and assayed for pectolytic activities Symbols (0) pectate lyase ( ) polygalacturonase (reducing sugar-releasing activity) (x) protein. Other details in Methods.

The extracellular polysaccharides of Rhizobium meliloti 201 have been examined by using enzymic degradation and chemical procedures.314 A mixture of polysaccharides produced by the bacterium, when incubated with a bacterial enzyme that hydrolyzed one of these, gave oligosaccharides that could be separated by DEAE-cellulose chromatography. The major fraction was a pentasaccharide, for which methylation analysis and Smith... 

Selective adsorption on glass Ammonium sulfate fractionation Ammonium sulfate fractionation Ethanol precipitation Sonic disintegration Selective adsorption on cellulose phosphate CM-cellulose chromatography Acetone precipitation Ammonium sulfate fractionation Ethanol precipitation Selective adsorption on cellulose phosphate Ammonium sulfate fractionation Lysozyme and trypsin digestion Ammonium sulfate fractionation DEAE-cellulose chromatography Ammonium sulfate fractionation... 

Center and Behai (49) have resolved 5 -nucleotidase from calf intestinal mucosa into three fractions using DEAE-cellulose chromatography. One of these was obtained free of nonspecific phosphatase. It had a pH optimum of 6-6.5, Mn2+, Mg2+, and Co2+ (1-10 mill) all enhanced activity and complete inactivation was produced with 1 mM EDTA. This enzyme hydrolyzes all 5 -ribonucleotides at similar rates and hydrolyzes 5 -deoxribonucleotides more slowly. These properties indicate that it is strikingly similar to the one obtained from acetone powder preparations of chicken and rat liver (32, 33) and from soluble supernatants of rat liver (36). The other two activities (which were not fully characterized) (49) could possibly have originated from particulate material or membranes because the authors employed deoxycholate in the early phase of purification. 

Elution in salts fraction during DEAE-cellulose chromatography... 

Fig. 1.4 Chromatographic separation of tryptic peptides from m-AspAT. A. Sephadex G-50 column chromatography. Fractions I—111 were applied on DEAE-cellulose chromatography (B). B. DE-52 column chromatographies of fractions, I, II and III. Data are from Kagamiyama et al.2S)...

The crude prostatic homogenate was also passed through a column of cellulose phosphate, and eluted with 0.01 M citrate, pH 6.0. The resulting single peak was then fractionated by DEAE-cellulose chromatography into two peaks. These two peaks (fractions II and IV) were further purified by gel filtration they constituted 50- and 100-fold purifications from the prostatic homogenate. 

The interaction of tilorone hydrochloride to native as well as denatured DNA encouraged us to study the template activity of the complexes in DNA- and RNA-polymerase systems from E. coli. Table 19 shows the effect of tilorone on the priming activity of denatured DNA in DNA-polymerase reaction. DNA-polymerase was isolated from 77. coli B cells according to the procedure of Richardson54, and fraction VII obtained after DEAE-cellulose chromatography was used. The reaction was carried out in the presence of denatured DNA. 

Fig. 21. Alkaline phosphatase zones after starch-gel electrophoresis of human intestinal extracts. The single pattern on the left labeled d is that of fresh butanol extract of human intestine [from Fig. 1 in Moss report (M34)]. The pattern labeled E is the same intestinal extract stored for 4 months at —20°C (before chromatography). A, B, and C are different fractions obtained by DEAE-cellulose chromatography of E (M34).

Near its isoelectric point, binding of iron to transferrin results in the introduction of a negative charge due to uptake of one bicarbonate ion by the molecule. Aisen et al. (86) first showed this effect when an increase in the anodic electrophoretic mobility of the protein was correlated with its extent of iron saturation. The observation has been corroborated by isoelectric fractionation experiments (87). It is curious that although DEAE-cellulose chromatography of partially iron-saturated transferrin yields three peaks, as would be expected from the three species present in the mixture, the 1 Fe-transferrin eluted first, followed successively by the iron-saturated molecule and the apoprotein (88). Thus the order of elution does not agree with the expectations based on the net electric... 

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