Preparative Disc Gel Electrophoresis Research Articles

Pullulanase from rice endosperm.

Pullulanase (EC 3.2.1.41) in non-germinating seeds was compared with that in germinating seeds. Moreover, pullulanase from the endosperm of rice (Oryza sativa L., cv. Hinohikari) seeds was isolated and its properties investigated. The pI value of pullulanase from seeds after 8 days of germination was almost equal to that from non-germinating seeds, which shows that these two enzymes are the same protein. Therefore, the same pullulanase may play roles in both starch synthesis during ripening and starch degradation during germination in rice seeds. The enzyme was isolated by a procedure that included ammonium sulfate fractionation, DEAE-cellulofine column chromatography, preparative isoelectric focusing, and preparative disc gel electrophoresis. The enzyme was homogeneous by SDS/PAGE. The molecular weight of the enzyme was estimated to be 100 000 based on its mobility on SDS/PAGE and 105 000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 4.7. The enzyme was strongly inhibited by beta-cyclodextrin. The enzyme was not activated by thiol reagents such as dithiothreitol, 2-mercaptoethanol or glutathione. The enzyme most preferably hydrolyzed pullulan and liberated only maltotriose. The pullulan hydrolysis was strongly inhibited by the substrate at a concentration higher than 0.1%. The degree of inhibition increased with an increase in the concentration of pullulan. However, the enzyme hydrolyzed amylopectin, soluble starch and beta-limit dextrin more rapidly as their concentrations increased. The enzyme exhibited alpha-glucosyltransfer activity and produced an alpha-1,6-linked compound of two maltotriose molecules from pullulan.

Purification and properties of α-glucosidase from millet seeds

Two forms of α-glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from millet ( Panicum miliaceum L.) seeds by a procedure including fractionation with ammonium sulphate, CM-cellulofine column chromatography, Sephadex G-100 column chromatography, preparative isoelectric focusing and preparative disc gel electrophoresis. The two enzymes showed identical M r , calculated to be 85 000 on SDS-PAGE and 93 000 on gel filtration. The two enzymes readily hydrolysed maltose and malto-oligosaccharides, and native starch weakly. The two enzymes hydrolysed amylose liberating α-glucose.

Inhibition of ileal brush-border chloride conductance by specific antibody.

Antibody raised in mice was used in attempting to identify proteins responsible for the conductive chloride transport that can be measured in porcine ileal brush border membrane vesicles. Ileal brush-border membrane vesicle protein from pig was separated into five different molecular mass fractions by preparative SDS polyacrylamide disc gel electrophoresis. Separated protein fractions were used to immunize mice. Antibody was screened for reactivity with antigen by Western blotting, and for effects on conductive chloride transport in ileal brush border membrane vesicles. Immunization with brush-border protein from fraction I proteins (> 110 kDa) produced polyclonal antisera which specifically inhibited the conductive component of chloride uptake by ileal brush border vesicle preparations. Western blotting of the antigen showed the presence of several protein species of molecular mass > 100 kDa that were recognized by immune serum. Spleen cells from a mouse producing antiserum that inhibited conductive chloride transport were fused with a myeloma cell line. The resulting hybridoma colonies produced antibody that reacted with at least seven distinct protein bands by Western blot assay and inhibited chloride conductance in brush-border membrane vesicles.

Purification and properties of intracellular fructosyl transferase fromAureobasidium pullulans

Intracellular frustosyl transferase was purified fromAureobasidium pullulans C-23 by ethanol fractionation, CM-Sephadex chromatography and preparative disc gel electrophoresis. It was shown to be homogeneous on disc polyacrylamide gel electrophoresis, with a molecular size of 190kDa. The pI value of the enzyme was about 3.7. The enzyme has aK m value of 0.43 mM for sucrose and was optimally active at pH 5.0 and 60°C. The enzyme was stable from pH 2.5 to 12. It was almost completely inhibited by 5mM Hg(2+) but was not significantly affected by other cations. The transferase was inactivated by treatment with the tryptophan-specific reagentN-bromosuccinimide and the tyrosine-specific reagent, I2, suggesting that tryptophan and tyrosine residues are probably located at or near the active site of the enzyme.

Purification and properties of α-glucosidase from suspension-cultured sugar-beet cells

The main α-glucosidase from sugar-beet cells has been isolated by a procedure including fractionation with ammonium sulphate, Sephacryl S-200 HR column chromatography, CM-cellulose column chromatography, Mono Q column chromatography, and preparative disc gel electrophoresis. The M r of the enzyme was 53 000. The enzyme hydrolysed maltooligosaccharides at a faster rate than maltose. The K m values for maltose and maltohexaose were 3.5 and 0.35 mM.

New thermostable α-amylase-like pullulanase from thermophilic Bacillus sp. 3183

The utility of extracellular pullulanase preparation from thermophilic Bacillus sp. 3183 was evaluated. It (3.2 U g −1 DS) did not increase the saccharification yield of glucose from liquefied starch (35%, w/w, maltodextrin DE 10) with glucoamylase (12.5 U g −1 DS) and also did not decrease the reaction time. However, the enzyme preparation itself was sufficient to saccharify the liquefied starch into mainly DP1 to DP4. The pullulanase and α-amylase were separated and the pullulanase was purified to homogeneity from the culture supernatant by ammonium sulfate treatment, DEAE-Sepharose CL-6B, S-Sepharose, and Octyl-Sepharose column chromatography and finally by preparative disc gel electrophoresis. The purified enzyme cleaved pullulan in α-1,6 linkages making only maltotriose, but it cleaved starch in α-1,4 linkages producing various saccharides (DP2 and higher). The pullulanase of thermophilic Bacillus sp. is a new type of enzyme having an abnormal ability to cleave starch readily at α-1,4 linkages.

Purification and Characterization of a Dipeptidase from Streptococcus cremoris Wg2

A dipeptidase was purified to homogeneity from a crude cell extract of Streptococcus cremoris Wg2 by DEAE-Sephacel column chromatography followed by preparative disc gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 49,000. The dipeptidase is capable of hydrolyzing a range of dipeptides, but not peptides with longer chains. The enzyme was shown to be a metallo-Mn enzyme with a pH optimum of 8 and a temperature optimum of 50 degrees C. The enzyme is strongly inhibited by thiol-reducing reagents but not by sulfhydryl reagents. Kinetic studies indicated that the enzyme has a relatively low affinity for leucyl-leucine and alanyl-alanine (K(m), 1.6 and 7.9 mM, respectively) but can hydrolyze these substrates at very high rates (V(max), 3,700 and 13,000 mumol/min per mg of protein, respectively).

Alkaline denaturation of bovine serum albumin. II. Analysis by isoelectric focusing in polyacrylamide gels.

Bovine serum albumins exposed to pH 11.9 for various periods (5min.-6hr.) at 20°C were analyzed by isoelectric focusing in polyacrylamide gels. Native BSA** monomers gave, in the electrofocusing pattern, three peaks with pI values 4.84, 5.15, and 5.39. After exposure to alkali, apparent shifts in the isoelectric point of some of the peaks and the appearance of new peaks with pI values 5.48, 5. 52, 5.70, 5.85, and 5.91 were observed.Components of these peaks are broadly divisible into the following three fractions: 1. The“first fraction”which consists of the main components of native BSA monomer (pI≤5.15). 2. The “second fraction”composed of SS-interchanged isomers (5.15 5.70). 3. The“third fraction” consisting of irreversibly unfolded products (pI≥5.70). The alkaline denaturation can be expressed by the following reaction diagram: the“first fraction”→the“second fraction”→the“third fraction”.When the solution of native BSA monomer is exposed to pH 11.9 at 20°C for 4hr., new components 1, 1″, 1′, 2, and 3 are formed (the“second reaction”). Each of these components was isolated in the pure state by preparative disc gel electrophoresis and analyzed by electrofocusing. The results obtained are as follows: The component 1 and the native BSA monomer are composed of only the “first and second fractions”, but differ from each other in the situation of ionizable groups. Components 1″, 1′, 2, and 3 each consists of all the three“fractions”. The components 2 and 3 are similar and 1″and 1 also resemble each other, both in the state of charged groups. It is postulated that in the“second reaction”fatty acids, the impurity bound to BSA, migrate from the component 1″ to the component 1 in the course of the reaction, 1→1″.

Purification and properties of wall-bound .ALPHA.-glucosidase from suspension-cultured rice cells.

Wall-bound α-glucosidase (EC 3.2.1.20) has been solubilized from suspension-cultured rice cells with Sumyzyme C and Pectolyase Y-23 and isolated by a procedure including fractionation with ammonium sulfate, Sephadex G-100 column chromatography, CM-cellulose column chroraatography, Sephadex G-200 column chromatography, and preparative disc gel electrophoresis. The molecular weight of the enzyme was 64, 000. The enzyme readily hydrolyzed maltose, maltotriose, and amylose, but hydrolyzed isomaltose and soluble starch more slowly. The Michaelis constant for maltose of the enzyme was estimated to be 0.272 mM. The enzyme produced panose as the main α-glucosyltransferred product from maltose.

Studies on the cell wall of Neurospora crassa. Part V Purification and some properties of two wall-associated (.BETA.1,3)glucanases from Neurospora crassa cells.

Two cell wall associated (β1,3)glucanases in Neurospora crassa, 1,3-glucanase-I and II, were solubilized by autolysis of the cell walls with 2 m sodium chloride, and purified by laminarioligosaccharide AH-sepharose affinity chromatography followed by preparative disc gel electrophoresis. The 1,3-glucanase-I has a strict specificity for (β1,3)glucan and produces D-glucose by an exo-type mode of action. The 1,3-glucanase-II has specificities for β-glucoside in addition to (β1,3)glucan. The 1,3-glucanase-I is more resistant to pH change and metal ions than the 1,3-glucanase-II.

Three forms of .ALPHA.-glucosidase from suspension-cultured rice cells.

Three forms of α-glucosidase (EC 3.2.1.20), designated as I, II, and III, have been isolated from suspension-cultured rice cells by a procedure including fractionation with ammonium sulfate, CM-cellulose column chromatography, and preparative disc gel electrophoresis. The three enzymes were homogeneous by Polyacrylamide disc gel electrophoresis. α-Glucosidase I was secreted in the culture medium during growth, α-glucosidase II was readily extracted from rice cells with the buffer alone, and α-glucosidase III required NaCl to be solubilized. The molecular weights of the three enzymes were 96,000 (I), 84,000 (II), and 58,000 (III). The three enzymes readily hydrolyzed maltose, maltotriose, maltotetraose, amylose, and soluble starch. α-Glucosidase I possessed strong isomaltose-hydrolyzing activity and hydrolyzed isomaltose about three times as rapidly as α-glucosidase III. The three enzymes produced panose as the main α-glucosyltransfer product from maltose. Half the maltose-hydrolyzing activities of the thr...

Degradation of biogene oligosaccharides by β- N-acetylglucosaminidase secreted by Entamoeba histolytica

β- N-Acetylglucosaminidase secreted by Entamoeba histolytica was extracted from the growth medium by affinity chromatography on CH-Sepharose 4 B coupled to p-aminophenyl-1-thio-β-2-acetamido-2-deoxyglucopyranoside. The enzyme was further purified by isoelectric focusing, by sequential chromatography on DEAE-cellulose and Sephadex G-150, and by preparative disc gel electrophoresis. Chitobiose (βGlcNAcl-4GlcNAc) derived from chitin as well as the oligosaccharides βGlcNAc1-4βGlcUA1-3GlcNAc, βGlcNAc1-4βGlcUA1-3βGlcNAc1-4GlcUA, and βGlcNAc1-4βGlcUA1-3βGlcNAc1-4βGlcUA1-3βGlcNAc1-4GlcUA derived from hyaluronic acid were tested as potential physiological substrates. All these oligosaccharides are susceptible to action of β- N-acetylglucos-aminidase from E. histolytica. Under identical conditions chitobiose is cleaved 38–48 times faster than hyaluronate oligosaccharides. No release of N-acetylglucosamine was observed when glycopeptides from ovalbumin were used as substrate. The pH optimum of hydrolase activity was 4.5 when chitobiose was used as substrate. Optimal hydrolysis of hyaluronate oligosaccharides was observed at pH 3.0 for trisaccharide and pH 2.0 for tetra- and hexasaccharide, respectively. Estimation of molecular weight by means of gel filtration gave values of 75 000. The isoelectric point was 5.02. β- N-Acetylglucosaminidase from E. histolytica does not act on macromolecular chitin and hyaluronic acid.

Studies on microbial esterases. Part I. Purification of four esterases from Aspergillus niger NRRL 337.

Aspergillus rager NRRL 337, a significant lipase producer, also produced esterases in a semisolid medium. From the culture extract, four esterases (Esterase-I, -II, -III, -IV) were purified by means of ion-exchange chromatography, gel nitration, gel permeation with high performance liquid chromatography, and preparative disc gel electrophoresis. Homogeneity of each esterase was confirmed using disc gel electrophoresis. The four esterases were found to exhibit low isoelectric points (pH 3.63.9). The molecular weights determined by gel permeation chromatography of Esterase-I, Esterase-II, Esterase-III and Esterase-IV were 23000, 127000, 29500 and 27000, respectively.

Porcine pancreatic prokallikrein. II. Purification and some properties of prokallikrein A.

The presence of two main components of porcine pancreatic prokallikrein (prokallikreins A and B) was observed on anion-exchange chromatography. One of them, prokallikrein A, was purified by water extraction, followed by a combination of ammonium sulfate fractionation, ion-exchange chromatographies, preparative disc gel electrophoresis and gel filtration in the presence of various protease inhibitors. Prokallikrein A reacted immunologically with the antibody against kallikrein A obtained from autolyzed porcine pancreas. It migrated slightly more slowly than kallikreins A and B on immunoelectrophoresis. The molecular weight of prokallikrein A was estimated to be 3.6-3.9×104 by high performance liquid chromatography. On the other hand, those of kallikreins A and B were estimated to be 3.3-3.4×104 by the same method. Thus, it was speculated that at least large fragment would not be liberated from the prokallikrein molecule during the activation by protease (s).

Multiple forms of human intestinal alkaline phosphatase: chemical and enzymatic properties, and circulating clearances of the fast- and slow-moving enzymes

Two forms of alkaline phosphatase (orthophosphoric monoester phosphohydrolase (alkaline optimum, EC 3.1.3.1) have been purified from human small intestine by column chromatography on DEAE-cellulose and tyraminyl derivative affinity gel, and by preparative disc gel electrophoresis. Intestinal phosphatases were electrohoretically separated into two components, fast- and slow-moving enzymes, with apparent molecular weights of 140000 and 168000 and with subunit weights of 68000 and 80000, respectively. Analyses of carbohydrate and amino acid revealed marked differences in the two enzymes. Enzymatic properties and affinities for an anti-blood group antibody were also found to differ. Papain digestion released a hydrophobic small peptide from the slow-moving enzyme and its enzymatic properties resembled those of the fast-moving enzyme. Circulating clearance (T 1 2 ) of the slow- and fast-moving enzymes from adult intestine was found to be 7.5 h and 1.3 h, respectively; that of fetal intestinal enzyme was 2.8 h. Sialidase, sialidase/β-galactosidase, or sialidase/β-galactosidase/ N-acetyl- β-glucosaminidase treatment of the fetal enzyme reduced the value to about 40 min. Further, digestion with α-fucosidase, α-mannosidase or both restored it to nearly the original level. Organ distribution of injected 125I-labelled enzymes indicates that the desialylated hepatic enzyme was selectively distributed in liver, while the degalactosylated intestinal enzyme was incorporated into liver, lymph fluid, and small intestine. These results suggest that the pathway of circulating clearance of alkaline phosphatase has several routes.

Study of human placental estradiol-17β dehydrogenase/20α-hydroxysteroid dehydrogenase by preparative disc-gel electrophoresis

The soluble enzyme, estradiol-17β dehydrogenase from human term placenta, appears to co-purify with a second soluble enzyme, 20α-hydroxysteroid dehydrogenase. The enzyme, which had been partially purified by affinity chromatrography, fractionated on a preparative electrophoresis gel to a homogeneous preparation containing both estradiol-17β dehydrogenase and 20α-hydroxysteroid dehydrogenase activities in a ratio of ∼100:1. Analytical polyacrylamide disc-gels resolved this homogeneous preparation as a single band by both protein and activity staining techniques. Homogeneous enzyme inactivated and affinity-radioalkylated by 16α-[2′-su14C]bromoacetoxyprogesterone or 16α-[2′-su14C] bromoacetoxyestradiol 3-methyl ether, and when analyzed by SDS disc-gel electrophoresis, gave a single protein band which corresponded identically to the radioactivity peaks. These observations support the hypothesis that estradiol-17β dehydrogenase and 20α-hydroxysteroid dehydrogenase represent dual oxidoreductase activity in one enzyme. Preparative disc-gel electrophoresis, a technique which has not been previously adapted to purification of these human placental enzyme activities, was useful to rapidly (3 days) effect a 15-fold enrichment of the estradiol-17β dehydrogenase specific activity from “heat-treated cytosol”. Thus, laboratory-scale preparative disc-gel electrophoresis is useful for rapid, small-scale enrichment of this soluble enzyme.

Methods for the rapid purification of trehalase from Dictyostelium discoideum.

A rapid and reliable method for the preparation of homogeneous trehalase from the cellular slime mold, Dictyostelium discoideum for usage in enzyme characterization studies and trehalose assays was developed. This procedure takes advantage of the fact that trehalase activity is secreted by Dictyostelium during the course of development, the major fraction being released late in fruiting body formation. Purification of trehalase to electrophoretic homogeneity was accomplished utilizing the techniques of ultrafiltration, streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephacel chromatography and preparative disc gel electrophoresis. Analysis of the purified enzyme by analytical polyacrylamide disc gel electrophoresis demonstrated the presence of a single protein band which was stainable with Coomassie blue. Assay of trehalase activity in eluates from segments of a companion gel indicated that all of the recovered trehalase activity was associated with this band of protein. Examination of the substrate specificity of the purified enzyme indicated absolute specificity for trehalose.

Two forms of ?-glucosidase from sugar-beet seeds

Two forms of α-glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from sugarbeet (Beta vulgaris L.) seeds by a procedure including fractionation with ammonium sulfate and ethanol, carboxymethyl-cellulose column chromatography, and preparative disc gel electrophoresis. The two enzymes were homogeneous by polyacrylamide disc gel electrophoresis. Their molecular weights were 98,000 (I) and 60,000 (II). α-Glucosidase I readily hydrolyzed maltose, isomaltose, kojibiose, maltotriose, panose, amylose, soluble starch, amylopectin and glycogen. α-Glucosidase II also hydrolyzed maltose, kojibiose and maltotriose but hydrolyzed the other substrates only very weakly or not at all. α-Glucosidase I hydrolyzed soluble starch at a faster rate than maltose. It produced isomaltose and panose as the main α-glucosyltransfer products from maltose, whereas maltotriose was the main product of α-glucosidase II. α-Glucosidase I hydrolyzed amylose liberating α-glucose. The neutral-sugar content was calculated to be 2.7% for α-glucosidase I and 8.8% for α-glucosidase II. The main neutral sugar was mannose in α-glucosidase I, and glucose in α-glucosidase II.

Two forms of .ALPHA.-glucosidase from rice seeds at the milky stage.

Two forms of α-glucosidase were isolated from rice seeds at the milky stage by a procedure that included fractionation with ammonium sulfate, DEAE-cellulose column chromatography, CM-cellulose column chromatography, SP-Sephadex column chromatography and preparative disc gel electrophoresis. The isolated α-glucosidases were designated as α-glucosidase I and α-glucosidase II, and were glycoproteins containing 5.5% and 3.4% carbohydrate, respectively. The molecular weight and isoelectric point were: 78,900 and pH 9.3 for α-glucosidase I, and 47,300 and pH 9.3 for α-glucosidase II. They were found to be homogeneous on polyacrylamide disc gel electrophoresis. The two enzymes hydrolyzed maltose, maltotriose, phenyl α-maltoside, amylose and soluble starch to liberate glucose, but did not act on sucrose. Their enzymes produced maltotriose and 4-α-nigerosyl-glucose from maltose as main α-glucosyltransfer products. They hydrolyzed amylose to liberate α-glucose.

Purification and some characteristics of the human coagulation factor VII.

1. A purification procedure for factor VII (proconvertin) from human plasma is described. The procedure involves barium sulphate adsorption and elution. DEAE-Sephadex column chromatography, barium sulphate adsorption and elution, heparin-Sepharose column chromatography, preparative disc gel electrophoresis and finally adsorption with antiserum to prothrombin coupled to Sepharose and antiserum to albumin coupled to Sepharose. This procedure gave an approximately 8 . 10(5)-fold purification. 2. The factor VII obtained from the electrophoresis step was mainly a single-chain protein with an apparent molecular weight of 53000 +/- 2000. 3. After the final purification step, additional forms of factor VII, resulting from a fragmentation of the factor VII molecule were detected. 4. Amino acid composition data of the purified factor VII are given. 5. Antisera were raised in two different rabbits by injection of the purified factor VII. The antisera obtained gave a good titre against the factor VII activity and were not directed against any of the three other vitamin-K-dependent coagulation factors.