SDS-disc Electrophoresis Research Articles

SDS disc electrophoresis of proteins in homogeneous, low‐concentrated polyacrylamide gels

In the attempt to separate in a single gel run low- and high-molecular-weight proteins, we present here a multiphasic buffer system designed for this purpose. It avoids the continuous stacking of SDS as it occurs in the 'classical' SDS-PAGE. The system allows complete stacking and destacking of proteins in the 3.5-250 kDa range at acrylamide concentrations as low as 4.5% T (total acrylamide concentration in %) and 2.6% C (degree of cross-linking in %). Taurine is used as the trailing ion in the cathode buffer and in the resolving zone of the gel, and two different counterions (Tris and imidazole) in the stacking zone. The gel system is easy to prepare and, due to the very low acrylamide concentrations, it is ideal for analytical as well as for preparative tasks.

Purification and properties of a membrane bound oxalate oxidase from Amaranthus leaves

A membrane bound oxalate oxidase has been purified to apparent homogeneity from mature leaves of the wild herb Amaranthus spinosus. The M r of the enzyme was ca. 130 kDa by Sephadex G-200 gel filtration and 65 kDa by SDS disc electrophoresis indicating two subunits of identical M r. The enzyme showed optimum activity at pH 3.5 when incubated at 40°C for 5 min. The energy of activation of the enzyme was 15.25 kcal mol −1. The K m for oxalate and V max of the enzyme reaction were 2.16×10 −3 M and 0.18 μmol min −1 ml −1, respectively. Sodium diethyl dithiocarbamate and sodium azide inhibited the enzyme while cysteine caused slight inhibition. Metals and flavins had no effect on the enzyme.

The occurrence of high-level streptothricin resistance in thermotolerant campylobacters isolated from the slurry of swine and the environment

This is the first report on the occurrence of streptothricin resistance (MIC > 400 micrograms/ml) in Campylobacter spp. The majority of resistant strains has been typed as C. coli by biotyping and SDS disc electrophoresis of bacterial whole cell proteins. The resistance to streptothricin was strongly connected with resistance to kanamycin (100%) and tetracycline (80%). As an important source of streptothricin-resistant Campylobacter strains we localized slurry of swine previously fed with feed containing streptothricins. Additionally, such strains could also be isolated from river water.

Isolation and characterization of arginine ester hydrolase from Heloderma horridum (beaded lizard) venom

1. I. An arginine ester hydrolase was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel and Q-Sepharose column chromatography, resulting in 5.4 mg of purified enzyme from 320.0 mg of crude venom. 2. 2. The enzyme was shown to be homogeneous by both SDS and non-SDS disc electrophoresis on polyacrylamide gel at pH 8.3. 3. 3. The enzyme possesses arginine ester hydrolase and transglutaminase-like activities, but did not exhibit clotting activity. 4. 4. Molecular weight was determined to be ca 29 kDa, with an isoelectric point of 4.4. 5. 5. The enzyme was stable to heat treatment (95°C, 10 min) and to pH changes over the range 2–11. 6. 6. The arginine ester hydrolase was inactivated by diisopropylfluorophosphate (DFP), β-mercaptoethanol and N-bromosuccinimide, suggesting that serine, disulfide bonds and tryptophan are involved in enzymatic activity. 7. 7. Amino terminal sequences were determined and appear to be similar to porcine pancreatic kallikrein.

Purification and Substrate Specificity ofα-Glucosidase fromPaecilomyces variotiAHU 9417

An extracellular α-glucosidase was purified from the culture broth of Paecilomyces varioti AHU 9417 by a combination of precipitation with ethanol, chromatography on DEAE-Sepharose CL-6B and Bio-Gel P-150, and preparative gel electrophoresis. The enzyme migrated as a single band on polyacrylamide gel electrophoresis. The molecular weight was estimated to be 100,000 by SDS-disc electrophoresis, and the optimum pH of activity was 4.5. The enzyme had a wide substrate specificity, as it rapidly hydrolysed maltooligosaccharides and isomaltooligosaccharides. The ratios of Vmax for maltose, isomaltose, kojibiose, nigerose, phenyl α-glucoside, maltotriose, isomaltotriose, panose, phenyl α-maltoside, and soluble starch were estimated to be 100, 72, 31, 105, 9.6, 103, 73, 47, 115, and 66, and the Km (mM of nonreducing terminal) for these substrates were 0.40, 2.4, 1.6, 2.5, 0.23, 0.33, 4.6, 0.67, 0.24, and 4.7 mM, respectively. The enzyme is characterized by a high activity toward isomaltose.

Single active site mechanism of rabbit liver acid alpha-glucosidase.

An acid alpha-glucosidase was purified from rabbit liver by fractionation with ammonium sulfate, and chromatographies on Sephadex G-100, CM-Toyopearl, Toyopearl HW-55F, and Toyopearl HW-65F column. The resulting preparation showed a single band on polyacrylamide disc gel electrophoresis. The molecular weight was estimated to be 1.03 X 10(4) by SDS-disc electrophoresis. The optimum pH was found to be 4.7. The alpha-glucosidase showed relatively high activity not only toward maltose but also toward alpha-glucans, such as shellfish glycogen, soluble starch, beta-limit dextrin, amylopectin, and amylose. The Km values for maltose and shellfish glycogen were 2.1 and 16 mM (the concentration of non-reducing glucose units), respectively, and the ratio of maximum velocities of hydrolysis of the two substrates was 100:133. The nature of the active site catalyzing the hydrolyses of maltose and shellfish glycogen was investigated by electrophoresis in the presence of urea and by kinetic methods. The purified enzyme was not separated into two components, maltase and glycogen hydrolase, in the electrophoretic gel containing 3 M urea, contrary to the report by Belenki and Rosenfeld ((1972) Biochem. Biophys. Res. Commun. 46, 443-448). In experiments with mixed substrates of maltose and glycogen, the kinetic features agreed very closely with those theoretically predicted for a single site mechanism. The essential ionizable groups, 1 (on the acidic side) and 2 (on the alkaline side), were identified as -COO- and -COOH for the hydrolysis of both substrates. Cations, Na+, K+, Mg2+, were about equally effective for the stimulation of enzyme action on maltose and glycogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and some properties of two .ALPHA.-glucosidases in glutinous rice seed.

Two α-glucosidases were highly purified from glutinous rice seed by fractionation with am monium sulfate, CM-Sepharose CL-6B ion-exchange column chromatography, gel filtration, and preparative disc electrophoresis. The molecular weights were estimated to be 9.3×104 for α-glucosidase I and 8.6×104 for α-glucosidase II by SDS disc electrophoresis. The pH optima were 4.5 for α-glucosidase I and 4.3 for α-glucosidase II, and the pH stabilities of both enzymes were in the range of pH 3.5-10. α-Glucosidase I and α-glucosidase II were stable up to 40 and 50°C, respectively. The both α-glucosidases attacked not only maltose but also soluble starch. The Km values for maltose were 4.8 mM for α-glucosidase I and 2.0 mM for α-glucosidase II, and those for soluble starch were 1.7 mM for α-glucosidase I and 1.1 mM for α-glucosidase II, in the concentration of non-reducing terminal glucose. The ratios of the V values of hydrolyses of maltose to those of soluble starch were 100 : 118 for α-glucosidase I and 100 : 148 for α-glucosidase II.

Purification and crystallization of hemorrhagic factor, HR2b, from the venom of Trimeresurus flavoviridis (habu)

K. Yonaha, M. Iha, Y. Tomihara, M. Nozaki, M. Yamakawa, T. Kamura and S. Toyama. Purification and crystallization of hemorrhagic factor, HR2b, from the venom of Trimeresurus flavoviridis (habu). Toxicon 26, 1205–1208. — The hemorrhagic factor, HR2b, was purified from the venom of Trimeresurus flavoviridis (habu) by a combination of gel filtration, cation exchange column chromatography and high performance liquid chromatography. The purified HR2b was homogeneous by the criteria of ultracentrifugation and SDS-disc electrophoresis. The mol. wt of HR2b was 18,000 and 18,500 by gel filtration on Sephadex G-50 and by SDS-disc electrophoresis, respectively, indicating a monomer structure for the hemorrhagic factor. Crystals of HR2b, taking the form of thin plates, were obtained in the presence of ammonium sulfate.

Purification of an antihemorrhagic factor from the serum of the non-venomous snake Dinodon semicarinatus

Y. Tomihara, K. Yonaha, M. Nozaki, M. Yamakawa, Y. Kawamura, T. Kamura and S. Toyama. Purification of an antihemorrhagic factor from the serum of the non-venomous snake Dinodon semicarinatus. Toxicon 26, 420 – 423, 1988. — An antihemorrhagic factor was purified from the serum of Dinodon semicarinatus, a non-venomous snake ( Akamata), by a series of high performance liquid chromatographies with a TSK gel DEAE-5PW column. The purified antihemorrhagic factor showed a single band on polyacrylamide gel disc electrophoresis and inhibited the hemorrhagic activity of HR1 and HR2, the hemorrhagic factors of Trimeresurus flavoviridis Okinawa. The antihemorrhagic factor was stable from 0° to 60° and at pH values 2.0 – 11.0. The molecular weight of the factor was estimated to be 59,000 and 52,000 by a gel filtration and SDS-disc electrophoresis, respectively, suggesting that it consists of a single subunit, as we also found for the antihemorrhagic factors of the mongoose Herpestes edwardsii.

Purification and characterization of a lethal factor in venom from the crown-of-thorns starfish ( Acanthaster planci)

K. Shiomi, S. Yamamoto, H. Yamanaka and T. Kikuchi. Purification and characterization of a lethal factor in venom from the crown-of-thorns starfish ( Acanthaster planci). Toxicon 26, 1077–1083, 1988. — A lethal factor in venom of the crown-of-thorns starfish ( Acanthaster planci) was obtained in an electrophoretically pure state by chromatography on CM-cellulose and Sephadex G-100. The purified lethal factor is a basic (pI 10.6) glycoprotein (carbohydrate content 3.5%). The mol. wt was estimated to be 20,000 by gel filtration or 25,000 by SDS-disc electrophoresis, suggesting that the lethal factor has no subunit structure. Despite its basicity, the lethal factor was richer in acidic amino acids than in basic amino acids. The lethal factor had an ld 50 of 0.43 mg/kg (i.p. injection into mice). Hemolytic, edema-forming and capillary permeability-increasing activities, though very weak, were also exhibited by the lethal factor, while hemorrhagic and phospholipase A activities were not present.

Purification and substrate specificity of endo-type inulinase from Penicillium purpurogenum.

An inulinase was highly purified from the culture broth of Penicillium purpurogenum by chromatographies on DEAE-Sepharose CL-6B, Toyopearl HW-65, and Bio-Gel P-100. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was 6.4 × 104 by SDS-disc electrophoresis and gel filtration on Bio-Gel P-150. The isoelectric point was pH 3.6 by isoelectric focusing. The enzyme hydrolyzed inulin rapidly, but did not affect sucrose. By paper chromatography analysis, the major products from inulin were tri-, tetra-, penta-, and hexa-saccharides. The substrate specificity of the enzyme on hydrolyses of fructo-oligosaccharides[1F(1-β-d-fructofuranosyl)n sucrose (n = 1 to 6 and n (average of polymerization degree) = 8)] were examined. The Km values and relative maximum velocities for the hydrolyses of inulin and fructo-oligosaccharides (GFn, n = 2 to 7 and n = 9) were as follows: inulin, (DP = 35) 0.21 mM and 100; GF9, 0.24 mM and 86.5; GF7, 0.33 mM and 132; GF6, 0.85 mM and 71.2; GF5, 3.8 mM and...

β-Fibrinogenase from the venom of Agkistrodon p. piscivorus

1. 1. β-Fibrinogenase was isolated from the venom of Agkistrodon p. piscivorus by column chromatography on Sephadex G-100, DEAE-Sephacel and by chromatofocusing, with a yield of 2.5 mg of purified enzyme from 1 g of crude venom. 2. 2. The enzyme was homogeneous by SDS and non-SDS disc electrophoresis on polyacrylamide gel at pH 8.3. 3. 3. β-Fibrinogenase is a glycoprotein possessing both TAME hydrolase and kinin-releasing activities. 4. 4. A mol. wt of approximately 33,500 and an isoelectric point 4.5 was determined. 5. 5. The enzyme is stable to heat treatment and to a pH range of 2–10. 6. 6. β-Fibrinogenase activity is inactivated by DFP, suggesting that serine is involved in the enzymatic activity. 7. 7. The Michaelis constant ( K m) of this enzyme for TAME and inhibition constant ( K i) for DFP were found to be 7.04 × 10 −3 and 4.13 × 10 −3 M, respectively.

Heterogeneity in the carbohydrate structure of rat angiotensinogen

Angiotensinogen (Ao), the precursor of the peptide hormone angiotensin, exists in two different forms in plasma, Ao-1 and Ao-2. The completely separated and purified molecules show distinct differences in sodium dodecyl sulfate (SDS)-disc electrophoresis and in analytical isoelectric focusing. Ao-1 is about 3500 Da heavier and contains more acid isoelectric points than Ao-2. In analytical isoelectric focusing Ao-1 displays five bands which overlap with two of the three bands of Ao-2. This difference in charge was eliminated by treatment with neuraminidase for 92 h at 37°C. Thereafter both molecules display an identical-pattern two bands, in isoelectric focusing. Treatment of these N-acetylneuraminic acid (NeuNAc)-free forms with renin leads to a shift of these double bands to a more acid pH, indicating heterogeneity within the protein structure. By affinity chromatography on concanavalin A (ConA)-Sepharose, both forms can be separated in three fractions. The analysis of these fractions and of their NeuNAc-free derivatives by SDS-disc electrophoresis, as well as by analytical isoelectric focusing, leads to the conclusion that Ao-1 contains two Asn-linked N-glycan residues with 4–8 mol of NeuNAc, while the smaller form, Ao-2, contains one carbohydrate residue with 2–4 mol of NeuNAc.

Purification of a lethal factor in the skin secretion from the oriental catfish Plotosus lineatus.

One lethal factor in the skin secretion from the oriental catfish Plotosus lineatus was purified successively by DEAE-cellulose, CM-cellulose and Sephadex G-75 and its homogeneity was sup-ported by analytical disc electrophoresis. The purified lethal factor had a LD50 of 0.71mg/kg (intravenous injection into mice). In addition, it exhibited a strong edema-forming activity; the minimum edema dose inducing 130% edema ratio was as small as 0.89μg. The molecular weight was estimated to be 11, 000 by gel filtration on Sephadex G-75. Unexpectedly the molecular weight determination by SDS disc electrophoresis was unsuccessful because no band was detected under the various tried conditions. Although the purified lethal factor was a basic protein, it was richer in acidic amino acids than basic amino acids, indicating that the acidic amino acids exist in the molecule as an amide form.

Precocious expression of the final larval instar developmental pattern in larvae of trichoplusia ni pseudoparasitized by chelonus spp.

1. 1. The present study has used a number of electrophoretic approaches to analyze the proteins in normal Trichoplusia ni larvae and those pseudoparasitized by Chelonus spp. 2. 2. A number of feeding-stage, hemolymph proteins appear or increase dramatically only during the final larval stadium. 3. 3. Other proteins highly abundant only during the penultimate stadium disappear or decrease dramatically during the final stadium. 4. 4. The comparative protein profiles of penultimate instar, pseudoparasitized larvae are very similar to those of last instar larvae. 5. 5. These changes in hemolymph proteins are seen on gels resulting from electrophoresis, isoelectric focusing (wide range and narrow range Ampholine® and very narrow range Immobiline® gels) and SDS-disc electrophoresis. 6. 6. It is concluded that the entire last instar developmental pattern of protein gene products is occurring precociously in pseudoparasitized larvae.

Purification and Properties of Bovine Kidney Ribonucleases

Two RNases (RNases K1 and K2) were purified from bovine kidney by means of column chromatography on phospho-cellulose, Sephadex G-50, CM-cellulose, heparin-Sepharose, nd agarose-APUP. They were named RNase K1 and RNase K2 in order of elution from the heparin-Sepharose column. The purity of RNase K1 thus obtained was about 90% by SDS-disc electrophoresis. RNase K2 was purified to homogeneity by SDS- and pH 4.3 disc electrophoresis. The yield of RNase K2 was 3.4 mg from 11 kg of kidneys. The antigenic properties of the two bovine renal RNases were studied by Ouchterlony's double diffusion analysis. RNase K1 and RNase A were serologically indistinguishable. RNase K2 did not cross-react immunologically with RNase K1 or RNase A. The molecular weights of these RNases determined by gel-filtration on Sephadex G-50 were 13,400 and 14,600 for RNase K1 and RNase K2, respectively. The pH optima for RNase K1 and RNase K2 were 8.5 and 6.5, respectively. Both RNase K1 and RNase K2 were as acid stable as RNase A. RNase K2 was less heat-stable than RNase K1 and RNase A. Although both renal RNases were pyrimidine nucleotide-specific enzymes, RNase K1 and RNase A were more preferential or cytidylic acid than RNase K2. The chemical composition of RNase K2 was determined. RNase K2, like human urinary RNase Us, contained one tryptophan residue. The N-terminal sequences of RNase K2 and RNase Us were determined by Edman degradation. Rnase K2 had a homologous sequence of about 10 amino acid residues with the sequence of RNase Us, a typical non-secretory RNase, within the N-terminal 30 residues.

Purification and substrate specificity of glucoamylase of Paecilomyces varioti AHU 9417.

A glucoamylase was purified from the culture broth of Paecilomyces varioti AHU 9417 by precipitation with ethanol, chromatography on DEAE-Sepharose CL-6B, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was estimated to be 6.9×104 by SDS-disc electrophoresis, and the optimum pH was 4.5. The enzyme preferentially hydrolyzes α-1,4-glucosidic linkages in a series of maltooligosaccharides, and is capable to slowly hydrolyzing nigerose, isomaltose, and panose, but is not active on kojibriose. Soluble starch, amylopectin, glycogen, and β-limit dextrin are rapidly degraded. Activity toward raw starches is very low, but rice and waxy corn raw starches are relatively subject to attack. The subsite affinities (Ai) of each subsite (i) in the active site of the enzyme were evaluated from the Michaelis constants (Km) and the molecular activities (k0) for a series of maltooligosaccharides according to the subsite th...

Purification and characterization of arginine ester hydrolases from the venom of the Chinese habu snake ( Trimeresurus mucrosquamatus)

An arginine ester hydrolase (ME-5) was isolated from the venom of Trimeresurus mucrosquamatus by column chromatography on Sephadex G-100, CM-Sephadex C-50, DEAE-Sephacel and by isoelectric focusing, obtaining 1.4 mg of purified enzyme from 1 g of crude venom. The enzyme was homogeneous by SDS and non SDS disc electrophoresis on polyacrylamide gel at pH 8.3. ME-5 is a glycoprotein which possesses both TAME hydrolase and capillary permeability-increasing activity, but it did not show clotting or bradykinin-releasing activities. Its molecular weight is approximately 33,000 and its isoelectric point is 6.48. The enzyme is stable to heat treatment and to pH changes between 5 and 9. Trimeresurus mucrosquamatus venom contains five arginine ester hydrolases, designated as ME-1, 2, 3, 4 and 5. Three of the five (ME-3, 4 and 5) are inactivated by DFP, suggesting that the serine hydroxyl group is involved in enzymatic activity. All five arginine ester hydrolases showed capillary permeability-increasing activity, but none of the enzymes showed clotting activity. Their amino acid compositions were determined and all appear to be unique and distinct from those of other snake venoms.

Purification and some properties of 2-alkenal reductase of Mucor griseo-cyanus AHU6044.

A 2-alkenal reductase, which catalyzes the reduction of sorbaldehyde (trans-2, trans-4- hexadienal) to trans-4-hexenal, was purified from Mucor griseo-cyanus by dialysis against an acidic buffer solution (pH 5.2), salting-out fractionation with ammoniumsulfate, chromatography on DEAE-Sephadex, preparative disc electrophoresis, and gel filtration on Bio-Gel P-75. A 2-alkenal reductase of a microorganismwas purified for the first time. The molecular weight was estimated to be 2.6 x 104 by SDS-disc electrophoresis, and the optimum pH was found to be 6.5. The enzyme was dependent on both NADPHand NADH, and was specific for cis-2-heptenal and -octenal in a series of 2-alkenals, trans-2-alkenals (C4, C6-C10 and C12), cis-2-alkenals (C7 and C8), acrolein, and trans-2-cinnamaldehyde.

Characterization of two forms of base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi.

Two forms of RNases (RNase ML and RNase MM) from Aspergillus saitoi which are base non-specific and adenylic acid preferential were separated from each other by DEAE-cellulose column chromatography. They are indistinguishable with respect to enzymatic properties such as base preferability, pH optimum, kinetic constants measured with 2',3'-cUMP and 2',3'-cCMP as substrates, and effects of ionic strength, physical properties such as heat stability, isoelectric point and circular dichroism spectra, amino acid composition and immunological property. They only differ in carbohydrate content. The apparent molecular weight determined by SDS-disc electrophoresis was 36,000 for RNase ML and 32,000 for RNase MM. Both RNases were reduced and carboxymethylated, and then digested with trypsin, separately. Glycopeptides were isolated from the both digests by gelfiltration and paper chromatography. The amino acid compositions of glycopeptides obtained from RNase ML (ML TS-IIC) and that obtained from RNase MM (MM TS-IIIC) were the same. The amino acid sequences of both glycopeptides determined by Edman degradation and carboxypeptidase digestion were also the same. The results indicated that RNase ML and RNase MM were the same protein having different sizes of carbohydrate chains at one site on the molecule.