DEAE-cellulose column

Purified by Sephadex G-200 filtration and DEAE-cellulose column chromatography. Hexosaminidase A was further purified by DEAE-cellulose column chromatography, followed by an ECTEOLA-cellulose column, Sephadex-200 filtration, electrofocusing and Sephadex G-200 filtration. Hexosaminidase B was purified by a CM-cellulose column, electrofocusing and Sephadex G-200 filtration. [Srivastava et al. 7 Biol Chem 249 2034 1974.]... 

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.]... 

Thrombin (from bovine blood plasma) [9002-04-4] Mj 32,600 [EC 3.4.4.13]. Purified by chromatography on a DEAE-cellulose column, while eluting with O.IM NaCl, pH 7.0, followed by chromatography on Sephadex G-200. Final preparation was free from plasminogen and plasmin. [Yin and Wessler J Biol Chem 243 112 796S.]... 

Figure 1 The profQe of pectinase on Figure 2 The profQe of pectinase on DEAE-cellulose column chromatograph, CM-ceUulose column chromatograph, size 3.5x40 cm, 0.05 N acetate buffer pH size 3x9 cm, 0.05 N acetate buffer pH 5.25, flow rate 60 ml/h 5.25, flow rate 60 ml/h...

These results prompted us to examine the characteristics of the extracellular pectolytic enzymes secreted in medium supplemented with glucose and galactose. Figure 2 shows the profile of elution of pectolytic activities recovered from the flow-through of a DEAE-cellulose column chromatographed on a CMC-cellulose column. 

Supernatant Apply to a 2 x 30 cm DEAE cellulose column pre-equili-brated with 50 mM Tris-HCl, pH 7.9. 

The elution profile of cytochrome P-448 (absorption at 418 nm) and epoxide hydratase activity from a sodium cholate-solubi-lized hepatic microsomal preparation (from DBA-treated male skates) applied to a DEAE-cellulose column and eluted with Buffer II is shown in Fig. 3. The void volume of the column contained significant amounts of epoxide hydratase activity. Fractions 40-70 (Fig. 3) were combined, and concentrated. The carbon monoxide difference spectrum, which had an absorption maximum at 448 nm in the induced state, is shown in Fig. 4. This form of the cytochrome (i.e.,... 

Figure 3. Elution profile of partially purified Cytochrome P-448 and epoxide hydratase activity of solubilized hepatic microsomes from DBA-treated male skates from a DEAE-cellulose column with Buffer II. Epoxide hydratase activity was determined with BP-4,5-oxide as the substrate (see Materials and Methods).

When all of the material absorbing at 418 nm (associated with the cytochrome P-448 fractions) was eluted from the DEAE-cellulose column (which in some experiments required more than 1 liter of Buffer II), elution was continued with a linear KC1 gradient (0-0.5 M) in Buffer II, as shown in Fig. 5. A different form(s) of cytochrome P-450 (fractions 130-155), having maximal absorption near 451 nm in the carbon monoxide ligated and reduced form (Fig. 6), was obtained although only 2- to 3-fold purification, relative to microsomes, was achieved. This form of cytochrome P-450 was extensively contaminated with epoxide hydratase activity. However, by combining fractions 130-150 (Fig. 5), it was possible to obtain cytochrome P-451 essentially free of cytochrome b5. The relative content of cytochrome P-448 and cytochrome P-451 Tn the DEAE-column eluates ranged from 1 1.1 to 1 1.6 in several different experiments. 

Very similar results to those described in Fig. 3-6 were obtained when sodium cholate solubilized hepatic microsomes from DBA-treated female little skates were subjected to chromatography on DEAE-cellulose as described above (data not shown). Also not shown are the results obtained with hepatic microsomes from untreated male and female little skates. With untreated animals, 80-90% of the cytochrome P-450 eluted from the DEAE-cellulose column only at higher ionic strength (i.e., with the KC1 gradient). However, in all preparations studied, an appreciable amount of cytochrome P-450 (10-20%), having its absorption maximum in the carbon monoxide-ligated and reduced state at 450 nm, was eluted from the column with buffer II, as was observed with cytochrome P-448 of hepatic microsomes from DBA-treated skates. The further purification of the various forms of cytochrome P-450 from control and DBA-pretreated little skate livers is currently in progress in our laboratory. 

In the first step, 2.2 g of the ethanol-precipitated solid were dissolved in 55 mL of 0.05M sodium phosphate buffer at pH 7.3, the undissolved residue was removed by centrifugation, and the supernatant was added to a 50-mm i.d., 180-mm long DEAE-cellulose column held at room temperature and eluted with 3.33 mL/min of the same buffer. Since the isoelectric point of the desired xylanase was above the buffer pH, it passed through the column without being retarded, and contaminating protein was removed. 

Lipids can be identified and quantified using thin-layer chromatography (TEC) and gas chromatography (GC) (Galliard, 1968). Extraction of lipids is achieved by homogenizing potato tubers with isopropanol in a blender, followed by a series of filtrations and extractions with chloroform-methanol (2 1). Chloroform is removed by rotary evaporation and the residue is redissolved in benzene-ethanol (4 1). This extract is passed through a DEAE-cellulose column, and the fractions collected are subjected to TEC on 250 p,m layers of silica gel G, using three solvent systems. Fatty acid methyl esters for GC analysis are prepared by transmethylation of the parent lipids, or by diazomethane treatment of the free fatty acids released by acid... 

Separation of the radioactively labeled product from the labeled substrate on a DEAE cellulose column with 20 mM HC1,14C-galactose-l-phosphate is eluted and UDP-14C-galactose is retained. The latter is eluted with 100 mM HC1. Radioactivity is determined with the aid of a scintillation counter (/ -counter) [unpublished]. 

Hoffmann GF, Brendel SU, et al. (1992) Mevalonate kinase assay using DEAE-cellulose column chromatography for first-trimester prenatal diagnosis and complementation analysis in mevalonic aciduria. J Inherit Metab Dis 15 738-746... 

Creamer (1974) to separate asl-casein-A from whole casein containing both the A and B variants. The B variant was degraded with pepsin or rennet, and the A variant was isolated from the degradation products on a DEAE-cellulose column with an NaCl gradient (0.0 to 0.5 M) in 4.5 M urea buffered at pH 5.5 containing 0.1% mercaptoethanol. 

Another technique employed to facilitate the chromatographic fractionation of casein involves the reduction and subsequent alkylation of the casein prior to chromatography (Rose et al. 1969 Yaguchi and Rose 1971 Davies and Law 1977). Rose et al. (1969) reduced whole casein with mercaptoethanol, alkylated the product with iodoacetamide, and separated the components on a DEAE-cellulose column (Figure 3.18). Davies and Law (1977) modified this procedure and achieved a quantitative estimation of the major caseins. 

Nagasawa, T., Kiyosawa, I., Kuwahara, K. and Ganguly, N. C. 1973. Fractionation of buffalo milk casein by acrylamide gel electrophoresis and DEAE cellulose column chromatography. J. Dairy Sci. 56, 61-65. 

Ribadeau-Dumas, B., Maubois, J. L., Mocquot, G. and Gamier, J. 1964. A study of casein by DEAE-cellulose column chromatography in urea. Biochim. Biophys. Acta 82, 494-506. 

Tarrassuk, N. P., Yaguchi, M. and Callis, J. B. 1965. Effect of temperature on the composition of casein fractions eluted from DEAE-cellulose column. J. Dairy Sci. 48, 606-609. 

The main problem in this approach is the very low permeability of mevalonic acid to membranes, resulting in very low incorporation. Positive results have been obtained by the use of cell-free systems incubated with [14C]-mevalonic acid,26,27 [14C]isopentenyl diphosphate,28 or [32P]orthophos-phate.29 Incubation of these radioactive lipids with glycosyl nucleotides labelled in the glycosyl group with a different isotope, followed by extraction and cochromatography in different solvent systems, may indicate that both compounds are present in the same molecule. When the lipid moiety becomes labelled from mevalonic acid or isopentenyl diphosphate, chromatography on DEAE-cellulose columns should be performed, in order to avoid confusion with steryl glycosides. 

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