Preparative isoelectric focusing in ampholine electrofocusing columns versus immobiline polyacrylamide gel for the purification of biologically active leukoregulin

Preparative vertical and rotating horizontal (Rotofor) ampholine column and immobiline flat bed polyacrylamide gel isoelectric focusing were evaluated for the isolation of the biologically active acidic form of leukoregulin, a 50,000-Da glycoprotein lymphokine with tumor growth inhibitory activity. Leukoregulin secreted by normal human lymphocytes was concentrated by 10,000 nominal molecular weight size exclusion ultrafiltration and by DEAE anion exchange chromatography using step elution with 0.02 M Tris-HCl: 0.1 M NaCl, pH 7.4. Preparative isoelectric focusing was carried out in a 110-ml vertical column containing 1% ampholines in a pH 4-6 gradient at 15 W constant power for 16-18 h, in a Rotofor 55-ml horizontal column containing 2% ampholines in a pH 4-6 gradient at 12 W constant power for 4-6 h, or in an immobiline pH 4.5-6.5 gradient within a 5% polyacrylamide 120 X 110 X 5-mm flat bed gel at 3 W constant power for 16-18 h. Recovery of biologically active leukoregulin from the vertical and horizontal ampholine columns was similar. The pH 4.9-5.2 fractions from the Rotofor ampholine column contained 4-7% and the fractions from the immobiline gel contained 4% of the leukoregulin activity applied to the electrofocusing column or gel, respectively. Analytical immobiline isoelectric focusing of the leukoregulin in the pH 4.9-5.2 fractions from the Rotofor column demonstrated that a single silver staining band with a pI of 5.1 can be obtained by this rapid method of preparative isoelectric focusing.     

Polyacrylamide gel electrophoresis: recovery of non-stained and stained proteins from gel slices

Following electrophoresis or isoelectric focusing in gels of polyacrylamide the protein band of interest is cut out and placed above a sucrose gradient column, containing carrier ampholytes (Pharmalyte). By electrophoresis, isoelectric focusing or displacement electrophoresis the proteins migrate out of the gel slice and into the isoelectric focusing column for concentration and further purification. From this column, the proteins can be withdrawn and their isoelectric points determined. Even after staining with Coomassie Brilliant Blue at least some proteins can be recovered by this technique and used for further analyses, for instance amino acid determinations. The focusing in a pH gradient by carrier ampholytes can be replaced by an electrophoresis in a conductivity gradient column. However, in comparison with isoelectric focusing, this concentration technique has the drawback of not permitting further purification of the eluted protein.     

Association of thymidylate kinase activity with pyrimidine deoxyribonucleoside kinase induced by herpes simplex virus.

Thymidine kinase derived from LMTK+ does not exhibit thymidylate kinase activity. However, protein isolated by affinity column chromatography from thymidine kinase-deficient mouse cells (LMTK-) infected by herpes simplex virus type 1 shows thymidylate kinase activity in addition to thymidine kinase and deoxycytidine kinase activities. The virus-induced multifunctional enzyme has a molecular weight of 85,000, whereas the molecular weight of thymidylate kinase from uninfected LMTK- mouse cells is 71,000. The virus-induced enzyme has a Km for thymidine of 0.8 micromolar, and for thymidylate of 25 micromolar, and for thymidylate of 25 micromolar; the ratio of Vmax for thymidylate kinase to thymidine kinase is 1.7. When subjected to isoelectric focusing, thymidylate kinase activity is not separated from thymidine kinase activity, and even though four peaks of activity are observed they have a constant ratio of thymidylate kinase to thymidine kinase activity. The isoelectric points (pI) of these four peaks are 4.8, 5.8, 6.2, and 6.6, respectively. Thymidylate kinase, derived from uninfected cells when subjected to isoelectric focusing, separates into a major component with an isoelectric point at pH 8.2 and a minor component at pH 7.7. Although thymidine and thymidylate kinase activities derived from the virus-infected cells cannot be separated either by affinity column chromatography, glycerol density gradient centrifugation, or isoelectric focusing, there is a differential rate of inactivation when the enzyme is subjected to incubation at 37 degrees, with thymidylate kinase activity being more labile than thymidine kinase activity.     

Isoelectric focusing of oat root membranes

The feasibility of purifying subcellular membranes, especially plasma membranes, from oat roots using isoelectric focusing has been examined. Membranes from oat (Avena sativa L. cv Garry) root homogenates were fractionated using discontinuous sucrose density gradient centrifugation and then electrofocused using a microanalytical isoelectric focusing column. The column contained either a broad-range (pH 3-10) or narrow-range (pH 3-6) pH gradient stabilized by a 5 to 15% Ficoll gradient. Results from the broad-range columns confirmed that the isoelectric pH (pI) values of the membranes were in the acidic range, with pI values ranging from 3.9 to 5.2. Using narrow-range pH gradients, it was possible to fractionate further plasma membrane-enriched material obtained from a sucrose density gradient. We had no success at fractionating crude membrane preparations from oat roots. Narrow-range pH gradients generated by commercial ampholytes were more successful than those generated by acetate/acetic acid mixtures.     

Purification of recombinant human growth hormone by isoelectric focusing in a multicompartment electrolyzer with Immobiline membranes.

Recombinant human growth hormone (r-hGH) expressed in Escherichia coli, was 70-80% purified by a combination of ion-exchange chromatography and metal ion affinity chromatography. For the last purification step, a multicompartment electrolyzer was used, containing three compartments delimited by isoelectric membranes and two additional anodic and cathodic chambers. The central compartment was situated between two membranes having isoelectric points (pI) of 5.08 (anodic) and of 5.16 (cathodic), i.e. equidistant from the pI value of hGH (pI 5.12). r-hGH was isoelectric between these two membranes and could not leave the central chamber, while more acidic and more cathodic impurities collected in the two lateral chambers under the influence of the electric field. The r-hGH, thus purified, exhibited a single band by isoelectric focusing (IEF) in immobilized pH gradients (IPG) and gave recoveries greater than 90%. The problem of isoelectric precipitation in a practically ion-free environment was alleviated by focusing in 30% glycerol added with 1% neutral detergent (Nonidet-P40). The latter was eliminated by passage through a Q-Sepharose column after collecting the pI 5.12 band from the electrolyzer. Also the pre-hormone (pre-hGH) can be purified in a similar manner (30% glycerol, 1% Nonidet P-40) between two membranes having pIs 4.77 (anodic) and 4.87 (cathodic) (pre-hGH pI 4.82). This paper demonstrates the possibility of purifying by a focusing process also poorly soluble proteins at the pI.     

The isolation, purification and properties of the cellobiohydrolase component of Penicillium funiculosum cellulase.

1. A cellobiohydrolase component was isolated from a Penicillium funiculosum cellulase preparation by chromatography on DEAE-Sephadex, and purified by isoelectric focusing. 2. Purified in this way, the enzyme was homogeneous as judged by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels and isoelectric focusing in polyacrylamide gels. 3. Acting in isolation, the enzyme had little hydrolytic activity to highly ordered celluloses such as cotton fibre, but, when recombined in the original proportions with the other components [endo-(1 leads to 4)-beta-D-glucanase and beta-D-glucosidase] of the complex, 98% of the original activity was recovered. 4. Synergistic effects were also observed when the enzyme was acting in concert with endo-(1 leads to 4)-beta-D-glucanase from other fungal sources. 5. Less-well-ordered celluloses, such as that swollen in H3PO4, were extensively hydrolysed, the principal product being cellobiose. 6. Attack on carboxymethyl-cellulose (CM-cellulose), which is the substrate normally used to assay for endo-(1 leads to 4)-beta-D-glucanase activity, was minimal. 7. The enzyme was associated with 9% of neutral sugar, 88% of which was mannose. It was isoelectric at pH 4.36 (4 degrees C) and had a mol.wt. of 46 300 (determined by gel chromatography on a calibrated column of Ultrogel). 8. The enzyme was specific for the beta-(1 leads to 4)-linkage.     

Rapid and Efficient Purification Method for Small, Hydrophobic, Cationic Bacteriocins: Purification of Lactococcin B and Pediocin PA-1

The bacteriocins lactococcin B and pediocin PA-1 were purified by ethanol precipitation, preparative isoelectric focusing, and ultrafiltration. The procedure reproducibly leads to high final yields in comparison to the generally low yields obtained by column chromatography. Specifically, during isoelectric focusing no loss of activity occurs. The method, in general, should be applicable to small, hydrophobic, cationic bacteriocins.     

Utilization of Synthetic Compounds with Juvenile Hormone Activity for the Silkworm Rearing

A proteinous inhibitor of lipase was purified from soybean cotyledons by the procedures of ammonium sulfate fractionation, gel-filtration on Sephadex G–150 column, DEAE-cellulose column chromatography and isoelectric focusing. The inhibitor obtained by gelfiltration was separated into three active components, D–1, D–2 and D–3, by DEAE-cellulose column chromatography. The D–3 fraction after isoelectric focusing was homogeneous judged from disc electrophoresis. The inhibitory activity was more stable against treatments of heating and Pronase when the D–3 fraction was preincubated with substrate than without substrate. The extent of inhibition was varied by changing the order of addition of reactants and condition of substrate. From these results, the mode of inhibition is discussed.     

Nucleotide catabolism and nucleoside cycles in human thymocytes. Role of orthophosphate.

Phosphatidylinositol 4-phosphate (PtdIns4P) kinase was purified from cytosolic and particulate material of rat brain. The purification procedure of the enzyme from cytosol consisted of (NH4)2SO4 precipitation. DEAE-cellulose column chromatography and preparative isoelectric focusing. Other methods after DEAE-cellulose column chromatography failed to achieve further purification of the PtdIns4P kinase, probably caused by the tendency of the enzyme to aggregate with contaminating proteins. The final purification was 67-fold, and the recovery was 0.6%. After isoelectric focusing the fraction containing the highest PtdIns4P kinase activity showed only one protein as visualized by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and silver staining. The apparent Mr of this protein was 45 kDa and the isoelectric point about 5.8. The activity of PtdIns4P kinase was dependent on the concentration of divalent cations in the incubation medium. PtdIns4P kinase activity was found to be optimal at 10-30 mM-Mg2+. In an attempt to compare the cytosolic with the membrane-derived kinase activity, a Triton/KCl extract from synaptic membranes was subjected to the same purification procedure as the cytosolic enzyme. A difference in isoelectric focusing was observed, possibly due to a higher tendency to form aggregates. However, we tend to conclude that also in the membranes the PtdIns4P kinase activity is present as a 45 kDa protein, identical with that found in the cytosol.     

Isolation of L-dynurenine 3-hydroxylase from the mitochondrial outer membrane of rat liver.

Outer membrane preparations of rat liver mitochondria were isolated, after the mitochondria had been prepared by mild digitonin treatment under isotonic conditions. L-Kynurenine 3-hydroxylase [EC 1.14.13.9] was solubilized on a large scale from outer membrane by mixing with 1% digitonin or 1% Triton X-100, followed by fractionation into a minor fraction I and a major fraction II by DEAE-cellulose column chromatography. The distribution of total L-Dynurenine 3-hydroxylase was roughly 20 and 80% in fraction I and II, respectively. Fraction I consisted of crude enzyme loosely bound to anion exchanger. In the present investigation, fraction I was not used because of its low activity and rapid inactivation. In contrast, fraction II consisted of crude enzyme with high activity, excluded from DEAE-cellulose column chromatography in the presence of 1 M KC1. In addition, fraction II was purified by Sephadex G-200 gel filtration and DEAE-Sephadex A-50 column chromatography with linear gradient elution, adding 1 M KC1 and 1% Triton X-100 to 0.05 M Tris-acetate buffer, pH 8.1. After isoelectric focusing, the purified enzyme preparation was proved to be homogeneous, since the L-kynurenine 3-hydroxylase fraction gave a single band on disc gel electrophoresis. The molecular weight of this enzyme was estimated to be approximately 200,000 or more by SDS-polyacrylamide gel electrophoresis and from the elution pattern on Sephadex G-200 gel filtration. A 16-Fold increase of the enzyme activity was obtained compared with that of the mitochondrial outer membrane. The isoelectric point of the enzyme was determined to be pH 5.4 by Ampholine isoelectric focusing.     

Purification of Cytochrome b5 from Pig Testis Microsomes by Isoelectric Focusing in an Immobiline pH Gradient

Testicular cytochrome b5 was purified by a procedure including preparative isoelectrofocusing. The cytochrome b5 was determined to have an isoelectric point of 4.45 on analytical isoelectric focusing. The purified cytochrome b5 was found to be homogeneous and its molecular weight was estimated to be 16,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The oxidized and reduced forms of the purified preparation exhibited absorption spectra of a typical cytochrome b5. A 69-fold purification was achieved with an overall yield of 6.2%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing column chromatography and preparative isoelectric focusing.     

Solution phase isoelectric fractionation in the multi-compartment electrolyser: a divide and conquer strategy for the analysis of complex proteomes.

Sample complexity frequently interferes with the analysis of low-abundance proteins by two-dimensional gel electrophoresis (2DGE). Ideally, high abundance proteins should be removed, allowing low-abundance proteins to be applied at much higher concentrations than is possible with the unfractionated sample. One approach is to partition the sample in a manner that segregates the bulk of extraneous proteins from the protein(s) of interest. Solution phase isoelectric focusing in the multi-compartment electrolyser generates fractions of discrete isoelectric point (pI) intervals allowing isolated narrow segments of a proteome to be analysed individually by 2DGE. It is particularly useful for the isolation of low-abundance proteins of extremely basic or acidic pI.     

Purification of cytochrome P-450 from pig testis by aniline-sepharose 4B and isoelectric focusing

Testicular cytochrome P-450 was purified by a procedure including preparative isoelectrofocusing. The cytochrome P-450 was determined to have an isoelectric point of 6.47 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited absorption spectrum of a typical cytochrome P-450. 284-fold purification was achieved with an yield of 10.6%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing Aniline-Sepharose 4B column chromatography and preparative isoelectric focusing.     

Multiple bands produced by interaction of a single macromolecule with carrier ampholytes during isoelectric focusing

Equilibrium isoelectric focusing patterns have been computed for reversible, carrier ampholyte-induced macromolecular isomerization reactions. The calculations predict that an amphoteric macromolecule, interacting with n species of ampholyte located at different positions along the isoelectric focusing column, can give a pattern showing n + 1 well-resolved peaks under appropriate conditions.     

On Basic Subunits Dissociated from C (11S) Component of Soybean Proteins with Urea

Galactolipase (galactolipid acyl hydrolase, EC 3.1.1.26) was purified 147-fold in good yield (91 %) from rice bran by affinity chromatography, in which the enzyme was adsorbed on a palmitoylated gauze column at pH 5.5 and then was eluted with a buffer solution containing a detergent such as sodium deoxycholate or Triton X–100 at pH 8.0. The preparation obtained was further purified by gel filtration on a Sephadex G–100 column and isoelectric focusing. After electrophoresis, the enzyme separated into four components with different isoelectric points. It seems that galactolipase in rice bran exists in multiple forms. The major component (G–2) with isoelectric point of 7.3, one of them, was purified 268-fold and electrophoretically homogeneous. The enzyme (G–2) hydrolyzed rapidly galactolipid and also slowly phospholipid, but hardly triglyceride.     

Purification and properties of cytochrome c-556 from Agrobacterium tumefaciens B2a.

Cytochrome c-556 from Agrobacterium mefaciens B2a was isolated in a pure, homoneous state. The best purification procedure volved ammonium sulphate fractionation, delting on Sephadex G-25, column chromatographic fractionation on DEAE- and CM-cellulose, and gel filtration on Sephadex G-75 superfine. Substitution of the CM-cellulose step by isoelectric focusing was successful. The purity of the final preparation is warranted by the purity index value, the electrophoretic patterns in the absence and presence of sodium dodecylsulphate, the sedimentation profile and the N-terminal amino acid analysis (alanine). The absorption spectrum of reduced cytochrome c-556 has maxima at 318, 419, 526 and 555.5 nm. The molar extinction coefficient for the alpha-band is 20 200M-1cm-1. The isoelectric point, determined both by preparative and analytical isoelectric focusing, is 5.55 +/- 0.10. The molecular weight of cytochrome c-556 was determined by gel filtration as 12000 and by dodecylsulphate gel electrophoresis as 11 500.     

Isoelectric focusing of interacting systems. I. Carrier ampholyte-induced macromolecular isomerization.

A phenomenological theory of isoelectric focusing is formulated for rapidly reversible, ampholyte-induced macromolecular isomerization. The calculations reveal that such interactions can give well resolved, bimodal transient and equilibrium isoelectric focusing patterns in which the two peaks correspond to different chemical equilibrium compositions and not to separated isomers. The kinetics of approach to the equilibrium pattern are characteristically biphasic: During the first phase, which is controlled by the rate of migration of the isomers in the electric field, two peaks are positioned in the region between the isoelectric points of the two isomers; one of the peaks then grows slowly at the expense of the other with a diffusion-dominated rate. The kinetics are dependent upon the initial distribution of macromolecule in the isoelectric focusing column, and in certain cases only a single peak is apparent during the first phase. These findings have practical implications for unambiguous interpretation of isoelectric focusing patterns, furnish explanations for hitherto puzzling experimental observations, and provide theoretical insights required for application of isoelectric focusing to the detection and characterization of macromolecular interactions in general.     

Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.     

Purification and characterization of a sea squirt beta-galactosidase

A beta-galactosidase was extracted from the internal organs of a sea squirt, Styela plicata, and purified 959-fold, with an 18% yield, by successive gel chromatography, anion-exchange chromatography, chromatofocusing, and affinity chromatography on a Con A-Sepharose column. The purified enzyme was fairly homogeneous, as judged on disc PAGE, SDS-PAGE, and gel chromatography on a Sephadex G-200 column. The molecular weight of the enzyme was estimated to be 77,000 and 75,000 by gel chromatography and SDS-PAGE, respectively, and its isoelectric point was determined to be 4.9 by the isoelectric focusing method. The enzyme was substantially stable in the pH range of 3.5 to 7.5, the optimum pH being 4.0. The enzyme was significantly inhibited by 9 mM HgCl2 and 9 mM DFP, while the inhibition by 0.9% PCMB was only 60% at 0 degrees C for 30 min. The purified beta-galactosidase apparently liberated galactose from a sea squirt antigen (H-antigen), two allergenically active glycopeptides (Gp-1 and Gp-2) derived from another sea squirt antigen (Gi-rep), asialo-ovomucoid glycopeptide, asialo-fetuin glycopeptide, GA1, CDH, and an ABEE-derivative (Gal beta 1----3ThrNAc-ABEE) of Gal beta 1----3GalNAc-ol isolated from bovine submaxillary gland mucin.     

Staphylococcal toxic shock exotoxin (its isolation and characteristics)

The method for obtaining the preparation of toxic shock exotoxin (TSE) has been developed. This method comprises the following operations: the sorption of the toxin from the culture fluid on Amberlite CG-50, elution, dialysis, gel chromatography in a column with biogel P-2, isoelectric focusing, and gel chromatography in a column with Sephadex G-75. TSE is a relatively thermostable protein with a molecular weight of 24,000. Its isoelectric point is 7.2. Monospecific antiserum to TSE with precipitating antibody titer equal to 1:16, identical to the reference serum (M. S. Bergdoll), has been prepared. This antiserum has shown no cross reactions with the homogeneous preparations of staphylococcal enterotoxins.