Analytical Disc Electrophoresis Research Articles

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.

Physicochemical properties of a lethal hemolysin isolated from the sea anemone Anthopleura japonica.

Hemolysins were purified from whole bodies of the sea anemone Anthopleura japonica by acetone precipitation, ion-exchange chromatography on CM-cellulose, gel filtration on Sephadex G-75 and chromatofocusing. Two hemolysins (AJH-1 and 2) were detected and only AJH-2 was obtained in pure form as judged by analytical disc electrophoresis. AJH-2 exhibited a lethal activity in mice (LD30 78μg/kg, i, v. injection) as well as a hemolytic activity (106, 500 HU/mg against sheep erythrocytes). It was stable over a wide range of pH values from 2 to 11 but rather unstable to heat treatment. The molecular weight was determined to be 17, 800 by sedimentation equilibrium or 19, 500 by SDS-polyacrylamide gel electrophoresis, indicating that AJH-2 has no subunit structure. AJH-2 was a slightly basic protein (pI 8.0) and its amino acid composition was characterized by the absence of half-cystine. It was notable that these properties of AJH-2 were closely similar to those of parasitoxin, the major lethal hemolysin isolated from the sea anemone Parasicyonis actinostoloides.

Peroxidases from Phaeophyceae II: Analytical Disc-Electrophoresis and Isoelectric Focusing of Peroxidases from Phaeophyceae

Peroxidases from Phaeophyceae II: Analytical Disc-Electrophoresis and Isoelectric Focusing of Peroxidases from Phaeophyceae

Isoenzyme studies in human leukemia — III. β-hexosaminidase (E.C. 3.2.1.30)

Enzymologic profiles of β-hexosaminidase ( N-acetyl-β-d-glucosaminidase, E.C.3.2.1.30) were studied in cells from childhood acute leukemias and lymphomas. By analytical isoelectric focusing or disc electrophoresis the β-hexosaminidase activity was separated into its components A, B, I and C. The isoenzyme patterns were correlated with immunologic cell surface marker characteristics found on the investigated leukemic cells. In all cases of T-ALL the β-hexosaminidase forms A and B were observed, an enzyme pattern similar to that found in normal lymphocytes. Seven out of 11 cases with cALL, three of six cases with AML and one case of AUL displayed the intermediate component (Hex I). Marked heterogeneity within the immunologically classified subgroup cALL was reflected in different enzyme patterns of the cALL samples. These biochemical phenotypes may indicate the different maturation and differentiation status of cells expressing the same immunologic surface markers.

F1-like ATPase from anaerobic bacterium Lactobacillus casei contains six similar subunits.

Membrane‐linked ATPase of an anaerobic bacterium Lactobacillus casei has been studied.It was found that L. casei membrane particles hydrolyzed 0.1–0.2 μmol of ATP × mg protein−1× min−1 in the presence of Mg2+ or Ca2+ ions at optimal pH 6.4. This activity was inhibited by N,N′‐dicyclohexylcarbodiimide. Oligomycin, ouabain, vanadate and hydroxylamine were ineffective.The addition of valinomycin to the intact L. casei cells in the absence of extracellular K+ and glucose resulted in an ATP synthesis which was abolished by the addition of K Cl, N,N′‐dicyclohexylcarbodiimide or carbonylcyanide m‐chlorophenylhydrazone. In the presence of glucose, L. casei cells showed an energy‐dependent accumulation of tetraphenylphosphonium cations, which was sensitive to N,N′‐dicyclohexylcarbodiimide and carbonylcyanide m‐chlorophenylhydrazone.A soluble ATPase was isolated from L. casei membranes by means of (a) washing with a low ionic strength buffer in the presence of EDTA, Mg2+ ions and a protease inhibitor ‐ diisopropylfluorophosphate or (b) a chloroform treatment. Crude ATPase extracts were purified using isotachophoresis and (or) Sepharose 6B gelfiltration. Preparations of purified ATPase had a specific activity 3.0–4.0 μmol ATP × mg protein−1× min−1 and were homogeneous when studied with analytical disc electrophoresis or analytical ultracentrifugation (s20,w= 12 ± 0.5 S). The molecular mass of the L. casei ATPase estimated with sieve chromatography on Sepharose 6B proved to be 270 kDa. Purified L. casei ATPase denaturated by 1% sodium dodecyl sulphate or 8 M urea and then studied with dodecyl sulphate or urea polyacrylamide gel electrophoresis showed a single 43‐kDa band. L. casei ATPase subunits could not be separated by isoelectrofocusing in 8 M urea/polyacrylamide gel (a pH gradient of 0.2–0.3 cm was used) and migrated as a single band of isoelectric point at pH 4.2.An electron microscopic study of the purified L. casei ATPase negatively stained with uranyl acetate showed particles of very regular structure. The predominant type of image was found to be a hexagonal particle 9.0–10.0 nm in size with 6 n point symmetry. An analysis of five different projections indicated that six similar subunits are arranged in two layers (three subunits in each layer) at vertices of two triangles rotated at 180°. This structure has a 32 point group symmetry.It is concluded that the detachable part of membrane N,N×‐dicyclohexylcarbodiimide‐sensitive ATPase of L. casei is composed of six similar subunits. Such a simplified version of factor F1 may be regarded as an evolutionary precursor of the catalytic part of the H+ ‐ATP synthase of aerobic and photosynthetic bacteria, mitochondria, and chloroplasts, containing two types of major and 2–3 types of minor subunits. The possibility is discussed that simplicity of L. casei ATPase is due to the fact that in the cell it operates as an ATP‐splitting, rather than a synthesizing, mechanism.

Binding of juvenile hormone, methoprene and hydroprene to haemolymph proteins of larvae of the southwestern corn borer, Diatraea grandiosella

The binding of tritiated juvenile hormone I, and the juvenile hormone mimics, methoprene and hydroprene (alkyl-3,7,11-trimethyl-2,4-dodecadienoates), to proteins of the haemolymph of larvae of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. The techniques of analytical isoelectric focusing, disc electrophoresis and gel filtration were employed. Juvenile hormone was shown to bind to a high affinity low molecular weight protein in the haemolymph of diapausing larvae. This protein has an apparent dissociation constant of about 3.1 × 10 −7 M at 4°C, an isoelectric point of about 4.85, and a molecular weight of about 3 × 10 4 . Juvenile hormone was also bound to at least one low affinity high molecular weight protein fraction. High juvenile hormone binding activity was found in the haemolymph of newly diapaused larvae. Methoprene and hydroprene bound selectively to a different protein fraction present in the haemolymph of pre-diapausing larvae. This protein fraction had a low relative mobility when subjected to electrophoresis at pH 8.9. A preliminary study of the binding of tritiated juvenile hormone I to proteins of the haemolymph of pre-diapausing larvae of the southern cornstalk borer, Diatraea crambidoides, was also conducted in vitro. A low molecular weight binding protein with identical electrophoretic properties to that of D. grandiosella was present. The high molecular weight binding protein fraction of the two species, however, had different electrophoretic properties.

Isolation and characterization of riboflavin-binding protein from pregnant-rat serum.

A high-affinity riboflavin -binding protein was isolated and characterized for the first time from pregnant-rat sera by affinity chromatography on a lumiflavin-agarose column. The purified protein was homogeneous by the criteria of analytical polyacrylamide-gel disc electrophoresis, gel-filtration chromatography on Sephadex G-100 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It had a molecular weight of 90000+/-5000 and interacted with [14C]riboflavin with a 1:1 molar ratio with a dissociation constant (Kd) of 0.42 micron.

Galactokinase of Bifidobacterium bifidum.

Crystalline galactokinase was obtained in good yield from Bifidobacterium bifidum grown on galactose medium. This preparation moved as a single protein band in analytical disc electrophoresis and sedimented as a single symmetrical peak under ultracentrifugation. The enzyme exhibited similar physicochemical properties to galactokinase purified from glucose-grown cells of B. bifidum. The enzyme has a molecular weight of 47,000. Only galactose and ATP were effective as substrate. Km values, optimal pH, cation requirement, inhibition by SH-reagent, heat stability and product inhibition were also investigated.

Partial purification and immunological aspects of carboxylesterase from rat liver microsomes

Carboxylesterase (CEase) was solubilized from rat liver microsomes by autolysis followed by cholate treatment and then purified by the combination of ammonium sulfate fractionation, gel filtration, chromatography on DEAE Sephadex A-50 and hydroxyapatite and preparative Disc electrophoresis. The overall purification was 25-fold with a yield of 6 % of the original enzyme activity. Analytical Disc electrophoresis of the final enzyme preparation showed a single band. However, SDS polyacrylamide gel electrophoresis revealed one main band of 93 % and three other minor bands. To investigate the interaction between CEases of rat, monkey, pig and rabbit liver microsomes, rabbit antibody to the above enzyme preparation was prepared and immunological analyses, i.e., Ouchterlony’s test and immunoelectrophoresis, were performed. In the comparative double diffusion test, the partial fusion of precipitation line between anti-rat CEase and the enzymes of other species was observed. In the second analysis, sharp arc precipitation lines also could be seen in all specimens and, furthermore, mobilities of each enzyme were different. These observations suggest that rat liver CEase seems to be immunologically related in part but not completely identical with the CEases of other species and the charge difference may exist in these specimens.

Purification and some properties of endo-β-(1-3)-glucanase from the marine bivalve Chlamys abbidus

1. 1. β-(1-3)-glucanase (E.C. 3.2.1.6.) has been isolated from the marine bivalve Chlamys abbidus and purified by gel chromatography on a biogel P-30 followed by ion-exchange chromatography on SE-Sephadex C-50. The enzyme was homogeneous according to the data of analytical disc electrophoresis. 2. 2. Maximum activity was exhibited at pH 4.4 and a temperature of 35°C. The enzyme was stable within a pH range of 3.5 to 7.5 and heatstable to 45°C. pI was 7.5, mol. wt 20,000. 3. 3. The purified glucanase belonged to the endo-type enzyme action.

Experimental Allergic Uveitis. Isolation, Characterization, and Localization of a Soluble Uveitopathogenic Antigen from Bovine Retina

Abstract The soluble antigen involved in the pathogenesis of EAU was isolated from bovine retinal extract by precipitation with ammonium sulfate followed by molecular sieve and ion exchange chromatography. The purified fraction formed a double precipitin band by immunodiffusion and immunoelectrophoresis, and a doublet by analytical disc electrophoresis. By immunodiffusion one of these components formed a reaction of identity with guinea pig S antigen and the other a reaction of partial identity. Because the two components had the same m.w. and similar electric mobility, they are presumed to be minor structural variants of the same molecule. The purified antigen had a m.w. of approximately 102,000 by analytical ultracentrifugation, 50,500 by SDS electrophoresis, and 56,000 by gel filtration chromatography. Sedimentation velocity studies with increasing concentrations of antigen indicated self-association. An S20 value of 3.6 was obtained for the monomer, corresponding to an approximate m.w. of 50,000. The antigen was identified as a protein with a very small amount of lipid. Data are presented on the absorption spectrum, extinction coefficient, and amino acid composition. The molecule was lacking in cysteine and tryptophan, but contained a relatively high proportion of nonpolar amino acids corresponding with a lipophilic protein. It was localized by immunofluorescence to the area surrounding the entire photoreceptor cell in a pattern indicating association with the plasma membrane. The antigen was not rhodopsin, which has been implicated as a probable antigen in the pathogenesis of EAU, but has some properties resembling a recently described photoreceptor cell retinol-binding protein tentatively implicated in the transport of vitamin A from the circulation to the retina. Assay of the purified fraction for production of EAU in the guinea pig indicated it was highly pathogenic in the microgram range. In agreement with immunofluorescent findings, the photoreceptors were specifically destroyed after cellular infiltration of the uveal tract.

Highly purified neurophysin proteins: Method of isolation based on their acidic properties

The isolation of highly purified bovine neurophysins I and II from freshly frozen posterior pituitaries is reported. The method can also be used for the isolation of neurophysins from other species, and acetone-desiccated preparations may serve as starting material as well. Crude posterior pituitary extract was obtained as described by Hollenberg and Hope (1967, Biochem. J., 104, 122–127). Basic and neutral proteins were then separated from the acidic neurophysins by cation-exchange chromatography on Cellex-CM (carboxymethyl). Neurophysin I was separated from neurophysin II by anion-exchange chromatography on DEAE-(diethylaminoethyl)-Sephadex with a continuous sodium chloride gradient (0 to 0.4 m). Highly purified bovine neurophysin I was also secured with a stepwise sodium chloride gradient (0.22 m starting gradient followed by a steep gradient from 0.22 to 0.4 m). The current method yields neurophysin proteins in a higher overall yield than previous procedures, as determined by single radial immunodiffusion and concentration-dependent absorption after disc electrophoresis. The method also gives neurophysins of greater purity than standard procedures currently in use. The proteins are characterized by a single, sharp precipitation band on immunodiffusion and immunoelectrophoretic analysis against antiporcine neurophysin antibody, by single bands on analytical gel disc electrophoresis at a running pH of either 8.8, 5.9, or 4.0. Isoelectric focusing on polyacrylamide gel gave an apparent p I value of 4.31 ± 0.07 for neurophysin I and a value of 4.79 ± 0.11 for neurophysin II. Radioimmunoassay revealed barely detectable levels of adrenocorticotropin-like material in neurophysin I (12 pg/100 μg of neurophysin) and no detectable levels in neurophysin II. Both proteins were devoid of avian vasodepressor activity in the conscious chicken, melanotropic activity in vitro in frog skin, and did not effect electrolyte excretion in hydropenic rats.

Isolation of the high molecular form of bovine factor X and some of its physical properties

A high molecular form of bovine factor X has been isolated from freshly collected bovine blood by BaSO 4 absorption, exhaustive washing with 0.001 M BaCl 2 and chromatographed on DEAE-cellulose column employing a linear salt gradient. This isolated factor X showed a single protein band on analytical polyacrylamide gel disc electrophoresis. Only one single protein peak was observed in the chromatogram of DEAE-Sephadex A-50 chromatography conducted at 3 °C. Sedimentation equilibrium analysis of this bovine factor X revealed no apparent heterogeneity or self association-dissociation phenomena. It yielded a weight-average molecular weight of 74 000 for the native factor X. In the absence of any reducing agent, factor X migrated in dodecyl sulfate gel electrophoresis as a single component with an estimated molecular weight of 74 300. Both dodecyl sulfate gel electrophoresis in the presence of 2-mercaptoethanol and agarose gel chromatography in 6 M guanidinium chloride revealed that this native factor X is composed of two polypeptide chains of molecular weights of 56 000 and 22 100. Factor X can be converted to the enzymatically active factor X a by Russell's viper venom and in the presence of Ca 2+. Factor X a was purified by DEAE-cellulose chromatography. This Russell's viper venom activated factor X a also showed a single protein band upon analytical polyacrylamide gel disc electrophoresis. Sedimentation equilibrium analysis of this factor X a yields a weight-average molecular weight of 59 000 with no apparent heterogeneity or self-association phenomena. In the absence of any reducing agent, factor X a migrated as a single component in dodecyl sulfate gel electrophoresis with an estimated molecular weight of 58 500. From the results of dodecyl sulfate gel electrophoresis in the presence of 2-mercaptoethanol as well as agarose gel chromatography in 6 M guanidinium chloride, factor X a is also composed of two polypeptide chains of molecular weights of 36 700 and 22 800. Therefore, the heavy and light chains of both native factor X and factor X a are linked together by disulfides.

Isolation and partial characterization of a low molecular weight acid stable protease inhibitor from human bronchial secretion.

An acid stable protease inhibitor was isolated from human bronchial secretion. Two important stages of the purification procedure were affinity chromatography on trypsin bound to Affi-Gel 10 and ion-exchange chromatography on SP-Sephadex C-50. The isolated inhibitor appeared as a single band on analytical disc electrophoresis and eluted as a homogeneous protein peak on gel filtration on Sephadex G-75 corresponding to a molecular weight of about 10500. Amino acid analyses showed no tryptophan or histidine and as N-terminal amino acid tyrosine. No glucosamine or galactosamine was detected. The results of the analyses suggest that the purified inhibitor is identical to the low molecular weight trypsin-chymotrypsin inhibitor of human seminal plasma (HUSI-I).

Isolation and Partial Characterization of Elastase from Dog Granulocytes

1. An elastolytic enzyme has been isolated from dog granulocyte leukocytes. The purification procedure included preparation of the granula fraction, chromatography on Sephadex G-75 and ion-exchange chromatography on SP-Sephadex C-50 at pH 6.0. 2. The elastase isolated was homogeneous in analytical disc electrophoresis and showed in sodium dodecylsulfate electrophoresis a single protein component with the molecular weight of 24800. The enzyme lacked tyrosine and lysine and the N-terminal amino acid was phenylalanine. No carbohydrate or sialic acid were detected. 3. The dog granulocyte elastase showed similar activities as human granulocyte elastase on elastin and fibrin. The Km value for 3-carboxypropionyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide was 2.50 mM and the pH optimum 8.5. The elastase preparation obtained was 99.5% active as judged from active-site titration. 4. The enzyme is a cationic protein and shows pronounced trailing on agarose gel electrophoresis. 5. A monospecific antiserum against the purified enzyme was produced in rabbits.

Plasmodium berghei: I. Immunochemical properties of fractions of a soluble extract

Soluble extracts of Plasmodium berghei were separated into 12 fractions following preparative disc electrophoresis in polyacrylamide gel. One or two protein bands were detected in each fraction by analytical disc electrophoresis. Similarly, one or two precipitinogens were generally detected in each of Fractions 1 through 11 by immunoelectrophoresis and by double immunodiffusion in agar gel, while the unfractionated extract contained 10 precipitinogens. Antisera produced in rabbits against each fraction each contained two or three (sometimes five) antiplasmodial precipitins demonstrable by immunoelectrophoresis. Serial fractions obtained in separate runs were closely similar to each other, although some degree of overlapping sometimes occurred between neighboring fractions. Glycoproteins were detected in all the fractions, but chiefly in Fractions 4 and 12. The bulk of the RNA in the extract was located in Fraction 4, while hemoglobin was usually confined to Fraction 6. The molecular weights of the soluble components of P. berghei range between 8000 and 130,000.

Purification and Characterization ofN-Acetylneuraminate Lyase fromClostridium perfringens

Clostridium perfringens cells were cultivated on a large scale using an automatic system. 2) N-Acetylneuraminate lyase, which is a cytosolic enzyme, was liberated from the bacteria by cell lysis using lysozyme in hypotonic solution. The enzyme was purified 770-fold by precepitation with ammonium sulfate, filtration on Sephadex A-50 and final preparative electrophoresis in a 7.5% polyacrylamide gel. Yield: 12 mg from 1 kg wet cell paste; specific activity: 167 nkat/mg protein. 3) The enzyme preparation appeared homogeneous in analytical disc electrophoresis, in gel electrophroesis in 0.1% sodium dodecylsulfate or 8m urea and in immunoelectrophoresis. Contaminating enzyme activities were not detected. 4) The isoelectric point of pH 4.7 was found for the enzyme. At 278 nm a molar extinction coefficient of 6.4 x 10(4)M-1 Xcm-1 was determined. The enzyme exhibited a Km value for N-acetylneuraminic acid of 2.8mM at its pH optimum of pH 7.2. The pH dependence of the Km value gives evidence that an ionizing guoup in the active center of the enzyme with a pKe value of 6.4 may be involved in the catalytic reaction. Pyruvate inhibited the cleavage reaction of N-acetylneuraminic acid competitively; Ki = 2.9mM. 5) An average molecular weight of 99200 was determined for the native enzyme using different methods. After denaturation in sokium dodecylsulfate or urea, a mean molecular weight of only 50000 could be demonstrated, indicating the existence of two enzyme subunits. The lyase molecule was shown by electron microscopy, using a negative staining technique, to consist of two hemispherical parts. 6) Two active sites per native enzyme molecule, probably corresponding to one active site per subunit, were found by incubation of the enzyme with radioactive pyruvate followed by borohydride reduction. The results obtained from chemical modification of the lyase with 5-diazonium-1H-tetrazole and iodocaetamide under various conditionsare interpreted as evidence for the presence of two reactive histidine residues in the enzyme molecule. It is probable that one residue per subunit forms the nucleophilic group participating in enzyme catalysis. A model suggesting the mechanism of reversible cleavage of N-acylneuraminic acids by the lyase is presented.

Preliminary studies on splenocytes and peritoneal macrophages from mice with varying genetic resistance to visceral leishmaniasis. II. Analytical disc-electrophoresis and enzyme activity of cell lysates of normal, uninfected animals

Preliminary studies on splenocytes and peritoneal macrophages from mice with varying genetic resistance to visceral leishmaniasis. II. Analytical disc-electrophoresis and enzyme activity of cell lysates of normal, uninfected animals

A specific cephalosporin-binding protein of Citrobacter freundii

1. A cephalosporin-binding protein obtained from a strain of Citrobacter freundii was purified to the extent of a single band in analytical and sodium dodecyl sulfate-containing disc electrophoresis. 2. The molecular weight determined by disc electrophoresis was 53 000. 3. The binding protein did not show any β-lactamase activity at substrate concentrations examined: 6 mM to 100 μM of penicillins and 12 mM to 100 μM of cephalosporins. 4. In gel filtration, [ 14C]benzylpenicillin was found not to bind to the binding protein. 5. In fluorescence titration, all cephalosporins tested quenched the fluorescence of the binding protein, while penicillins did not quench the fluorescence. Association constants of cephalosporins were in the range of 0.8–12·10 3 M −1, and one binding site was calculated for all cephalosporins tested.

β- d-xylosidase from Bacillus pumilus: Molecular properties and oligomeric structure

Bacillus pumilus β- d-xylosidase, purified by affinity chromatography, seems to be homogeneous, as judged by disc electrophoresis and gel filtration. The absorption coefficient at 280 nm, A 1cm 0,1%, determined by the dry weight method, is 1.78. The complete amino acid composition is determined. Sedimentation velocity studies show the presence of two components with s 20,w values of 10.0 S and 6.6 S. After glutaraldehyde cross-linking two, enzymically active, components, with apparent molecular weights 126 000 and 243 000, can be isolated by preparative sucrose gradient ultracentrifugation. These values are confirmed by analytical disc electrophoresis at different acrylamide concentrations. The subunit molecular weight is 60 000. l-Methionine is the only N-terminal amino acid detectable. The possible presence of both dimeric and tetrameric forms of the β- d-xylosidase in solution has to be envisaged.