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Describes the circumstances guiding the Sephadex group of products for gel filtration with reference to the importance of electrophoresis. Description of dextran-based gels; Information on Jerker Porath's experience from zone electrophoresis; Details on Porath's research of electrophoresis.

The disulphide linkages of the 16 half-cystine residues in the sweet-tasting protein thaumatin have been investigated by enzymatic hydrolysis of the intact molecule. The peptides obtained after proteolytic cleavage with trypsin and pepsin, and in one case with chymotrypsin have been purified by gel filtration, high-performance liquid chromatography and peptide mapping by paper high-voltage electrophoresis in one direction and paper chromatography in the second dimension. Disulphide bonds appeared to be formed by cysteine residues in positions 9–204, 56–66, 71–77, 121–193, 126–177, 134–149, 145–158 and 159–164. The labile disulphide bond responsible for the enzymatic properties of the sweet tasting protein thaumatin appeared to be between Cys-145 and Cys-158. [ABSTRACT FROM AUTHOR]

α-Lactalbumin was isolated from human milk in a yield of 1.8 mg/ml of milk. The purification procedure involved ammonium sulphate fractionation (30% to 80% saturation) and pH-4.0 precipitation, followed by gel filtration with Sephadex G-100. A final purification stage using DEAE-cellulose was necessary in some preparations. Peptides derived from the reduced, S-aminoethylated protein by treatment with cyanogen bromide and digestion of these CNBr fragments with trypain, chymotrypsin or thermolysin, were purified by gel filtration, ion exchange chromatography and high-voltage paper electrophoresis. From the sequences of these peptides it has proved possible to deduce unambiguously the complete primary structure of the protein. Comparison with bovine α-laotaIbumin shows an identity in 72% of the residues with a further 6% being chemically similar amino acids. The corresponding figures for the human α-Iactalbumin/human lysozyme comparison, are 39% and 12%, respectively. The significance of some of the amino acid replacements is discussed. [ABSTRACT FROM AUTHOR]

An immunoglobulin M from Waldenström's disease (IgM Ga) was hydrolysed by papain and a glycopeptide fraction was isolated by gel filtration on Sephadex G-100. This fraction was then successively resolved into fourteen glycopeptides by gel filtration on Sephadex G-50 and by preparative paper electrophoresis at pH 1.9. The amino acid and carbohydrate content of these glycopeptides was determined by ionexchange chromatography in an autoanalyzer. All of them contain aspartic acid and serine, but they are quite different from one another, in amino acid composition and sequence about, the carbohydrate-peptide linkage, especially with respect to histidine, tyrosine, threonine, valine, glutamic acid, lysine and proline. Furthermore, a number of glycopeptides contain only mannose and glucosamine, while others contain in addition, galactose, fucose, and N-acetylneuraminic acid, in variable amounts. These results suggest the presence of several linkage sites of the oligosaccharide chains on the H-chain and the true heterogeneity of these oligosaccharide chains. The peptide sequence of a few glyeopeptides was reported, especially the following: r-Asn(carbohydrates)-Ser-Thr, also present, in the Fc fragment of Immunoglobulin G. [ABSTRACT FROM AUTHOR]

Seryl-tRNA synthetase was purified 1800-fold from bovine liver extract by ultracentrifugation at 150000 x g, chromatography on DEAE-cellulose, fractional precipitation with ammonium sulfate, gel chromatography on Sephacryl S-300, adsorption chromatography on hydroxyapatite, affinity chromatography on blue-Sepharose and finally on M&asline;trex gel red A. The relative molecular mass, Mr, in the denatured state was estimated as 87000 by sodium dodecyl sulfate disc gel electrophoresis; in the active state the Mr was estimated as 170000 for the dimeric native enzyme (α2 type) by chromatography on Sephacryi S-300. The amino acid composition of the enzyme was determined. The Km values for ATP and serine were 0.49 mM and 30 μM, respectively. The Km values for tNAIGASer and tRNAcmCASer were 1.40 μM and 1.25 μM, respectively. Sequences common to the two isoaccepting tRNASer molecules are discussed in relation to the recognition mechanism of the purified seryl-tRNA synthetase. [ABSTRACT FROM AUTHOR]

The light-harvesting bacteriochlorophyll-carotenoid-protein complex B800–850 has been isolated from membranes of the phototroph-negative mutant strain Y5 of Rhodopseudomonas capsulata. The three polypeptides of the complex have been found to be soluble in chloroform-methanol (1:1, v/v) in the presence of 0.1 M ammonium acetate. They were extracted from the complex and separated by gel filtration on Sephadex LH-60 in the same solvent mixture. Minimum molecular weights based on amino acid composition are 12000, 9300, and 5100. Values previously determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis are 14000, 10000 and 8000. The two smaller polypeptides (polarities 31% and 39%) are completely soluble in chloroform/methanol/ammonium acetate while the largest and most polar (41%) polypeptide is only partially soluble. The largest polypeptide contains no tryptophan. The middle polypeptide contains no cysteine and arginine, while the small polypeptide lacks cysteine. Methionine is shown to be the amino terminus for the small and middle polypeptides by two independent methods (Edman degradation and dansylation). Both methods also indicated that the N terminus of the 14000 polypeptide seems to be blocked. Partial N-terminal amino acid sequences were obtained for the two smaller polypeptides. No homology between the two proteins was observed. [ABSTRACT FROM AUTHOR]

The threonine-sensitive aspartokinase-homoserine dehydrogenase of Escherichia coli can exist either as a tetramer (Mr = 360000) or a dimer, depending on the conditions. The subunit interactions of this enzyme have been investigated by reacting-enzyme sedimentation, gel filtration, and electrophoresis. Mild proteolysis with trypsin converts the native enzyme to a dimer which is smaller (Mr = 110000) than the native dimer (Mr = 175000). The new dimer fragment possesses homoserine dehydrogenase activity, sediments as a 6-S species in reacting-enzyme sedimentation but does not show aspartokinase activity. The dehydrogenase activity is lower, however, and the inhibition by threonine of this reaction has largely been lost. L-Threonine or to a lesser extent L-aspartate plus potassium ion protect aspartokinase-homoserine dehydrogenase against proteolysis probably by stabilization of the tetrameric form of the enzyme. Conditions which promote the formation of dimer (removal of effectors) increase the susceptibility to proteolysis. Various treatments cause dissociation of tetrameric aspartokinase-homoserine dehydrogenase, namely alteration of pH, removal of effectors, proteolysis, or sulfhydryl modification. Singly or in combination these treatments always resulted in the formation of dimers (6 S to 8 S in reactingenzyme sedimentation) rather than monomers. This finding strongly indicates that the directs are formed in all cases by disruption of the same intersubunit bonding domain of the tetramer. Based upon these and other studies, we have proposed a subunit interaction model for aspartokinase-homoserine dehydrogenase. The model describes a molecule in which each of four identical subunits contains somewhat independent entities corresponding to aspartokinase and homoscrine dehydrogenase activities. The native tetramer is held together solely by isologous interactions, two between aspartokinase entities and two between regions in which aspartokinase and homoserine dehydrogenase entities overlap. Dissociation by all mild treatments is explained by cleavage of the isologous interaction between aspartokinase entities resulting in loss of aspartokinase activity and production of a homoserine dehydrogenase dimeric unit. [ABSTRACT FROM AUTHOR]

Mouse histocompatibility antigens were solubilized from lymphocyte membranes by limited papain degradation. Spleens from Balb/c mice (H-2d), enlarged by administration of lymphoma cells, were used for the membrane preparations. The solubilized protein components were purified by ion-exchange chromatography, gel filtration and discontinuous polyacrylamide-gel electrophoresis. A further characterization was achieved in a two-dimensional protein mapping system using disc-electrophoresis and isotachophoresis in the first and second dimension, respectively. The purification was monitored for alloantigens H-2.4(D) and H-3.21(K) (H-2D and H-2K represent two regions of the H-2 system). On Sephadex G-100 partially purified H-2-alloantigen chromatographed in a region of Mr = 35500. The pI of these substances showed a maximum at pH 5.5 and 5.3 for H-2.4 and at 5.2 for H-2.31. Free SH- and dithio-groups were determined with 5,5′-dithiobis(2-nitrobenzoic acid). 1.56 μmol SH/μmol alloantigenic material was found, but no SS-bond could be detected after alkaline cleavage. This suggests the absence of covalently linked protein subunits in papain-solubilized molecules bearing single private specificites. This was confirmed after reduction and alkylation in 4 M urea: 30-50% of the alloantigenic activity was retained and shown, in polyacrylamide-gel electrophoresis, to migrate in the original region of activity (RBPB 0.29 and 0.33 for H-2.4; about 0.33 for H-2.31, BPB = bromophenol blue). After reductive alkylation gel filtration resulted in a further purification of the alloantigenic material. Highly purified substances showed a diffuse staining pattern and a broad serological profile after electrophoresis. We assume this heterogenity to be a genuine property of H-2 antigens. Structural and genetic implications of these findings are discussed. [ABSTRACT FROM AUTHOR]

Phosphorylase kinase was partially purified (530 - 970-fold) from chicken gizzard smooth muscle by a procedure involving ammonium sulfate fractionation, chromatography on 8-(6-aminohexyl)adenosine-5'-phosphate -- Sepharose 4B and glycerol density gradient ultracentrifugation. The final and most efficient purification step takes advantage of the relatively high molecular mass of gizzard phosphorylase kinase, which was found to be similar to that of rabbit skeletal muscle enzyme. The gizzard kinase, further purified to near homogeneity by calmodulin-Sepharose 4 B affinity chromatography, showed one main protein band of 61 kDa, upon dodecyl sulfate acrylamide gel electrophoresis. Four minor protein bands of higher molecular mass were also present but no protein stain was seen at the position of the y subunit. The gizzard phosphorylase kinase showed a high pH 6.8/8.2 activity ratio of 0.53, it was stimulated by Ca2+, inhibited up to 80% by EGTA and it was activated about 1.9-fold by calmodulin. The km value for ATP was 0.45 mM, while the K0.5 for rabbit muscle phosphorylase b was extremely low, more than 200-fold lower than the Km of nonactivated skeletal muscle phosphorylase kinase for its protein substrate. High concentrations of phosphorylase b were found to be inhibitory. At 10 mg/ml phosphorylase b, the maximum activity of the kinase was inhibited fivefold. No evidence has been obtained indicating autophosphorylation or the existence of active and inactive forms of gizzard phosphorylase kinase. Limited proteolysis of the smooth muscle kinase with trypsin was accompanied by a twofold activation at pH 6.8. [ABSTRACT FROM AUTHOR]

Two proteic inhibitors (I and II) of serine proteases have been purified from the parasitic worm Parascaris equorum by affinity chromatography on immobilized trypsin followed by preparative electrophoresis. They have an apparent relative molecular mass of 9000 and 7000 as determined by gel filtration, a slightly acid isoelectric point (5.5 and 6.1) and a similar amino acid composition. Both inhibitors lack serine, methionine and tyrosine. They bind bovine trypsin extremely strongly with an association constant, Ka, larger than 109 M-1 and form a 1:1 complex with this protease. The Ka values for the binding to bovine chymotrypsin are ≈ 3.3 × 108 M-1 (inhibitor I) and ≈ 2 × 106 M-1 (inhibitor II). Inhibitor I interacts also with porcine elastase (Ka ≈ 5 × 107 M-1, while inhibitor II is inactive towards this enzyme. [ABSTRACT FROM AUTHOR]

A glycoprotein fraction possessing peanut agglutinin receptors has been isolated from detergent extracts of neuraminidase-treated human peripheral blood T-lymphocyte plasma membranes with affinity matrices comprising the peanut agglutinin co-valently immobilised on Sepharose 4B. This fraction could be specifically eluted from affinity columns using buffer solutions supplemented with 0.2 M D-galactose and was shown, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (0.1 %), to contain four major glycoprotein components with apparent molecular weights of 2000003 190000, 110000 and 95000. It is suggested, from the observed reactivity of these glycoproteins with various lectins in double-diffusion experiments, that they possess both O-glycosidically and N-glycosidically linked carbohydrates. [ABSTRACT FROM AUTHOR]

1. 3-Deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) from Escherichia coli K12 has been purified to near homogeneity. The purified enzyme has a specific activity of 67 units mg which is about 1000 times that found in cell-free extracts of wild-type E. coli K12. 2. The minimum molecular weight of the enzyme was estimated by dodecylsulphate-gel electrophoresis to be 33000. Re-estimation of the native molecular weight by gel filtration confirmed the previously determined value of 110000. 3. Amino acid analysis and tryptic fingerprints indicated that the subunits of the enzyme are very similar and possibly identical. 4. The purified enzyme does not contain Co2+. [ABSTRACT FROM AUTHOR]

Lysozyme (mucopeptide N-acetylmuramylhydrolase) from Asterias rubens was obtained in a chromatographically and electrophoretically pure state by gel filtration and affinity chromatography. The quantitative amino acid composition, the molecular weight and the N-terminal sequence determined by a sequencer are reported. This new invertebrate enzyme presents important differences when compared to previously studied lysozymes. [ABSTRACT FROM AUTHOR]

The intracellular proteinase, cathepsin D, has been isolated from bovine spleen and thymus, in times as short as several hours, by affinity chromatography of partially purified and unpurified tissue extracts on haemoglobin-agarose resin. After subsequent separation from an inactive higher-molecular-weight protein by gel permeation chromatography, the enzyme from both tissues shows three dominant proteolytically active bands on gel electrophoresis at pH 4.3 and 9.5: this proteolytic activity is completely inhibited by the acid-proteinase inhibitor, pepstatin. These enzyme electrophoretic patterns were approximately constant with variation in isolation time and with various preliminary purification procedures. The enzyme shows only traces of polypeptides other than that with an apparent molecular weight of 42 000 on dodecylsulphate electrophoresis, in contrast to the enzyme prepared by conventional methods, which contains considerable amounts of smaller polypeptides. This difference in polypeptide composition is shown to be the result of degradation of the enzyme in vitro during isolation by the previously published methods. [ABSTRACT FROM AUTHOR]

A β-1,4-endoxylanase from the ericoid mycorrhizal fungus H. ericae has been purified to electrophoretic homogeneity using isoelectric focusing, ion exchange and gel permeation chromatography. The enzyme has an isoelectric point of 4.85-5.2O and a molecular weight of 58.4 kDa. The apparent S0.5 of the enzyme for soluble birchwood glucuronoxylan is 3.75 mg ml-1 and the Vmax 468.0 nkatal mg-1 protein. The pH optimum for activity is 4.5 and that for stability is 3.5-4.0; these values are discussed in the context of the pH of the mor humus. The role of wall-degrading activities in the establishment of the ericoid mycorrhizal symbiosis is considered. [ABSTRACT FROM AUTHOR]

Intracellular β‐glucosidase from Evernia prunastri has been purified to homogeneity using anion exchange on DEAE‐Seaphadex A‐50, and gel filtration chromatography on Sephadex G‐100 and Sepharose 6B. The purified β‐glucosidase showed a single protein band on native electrophoresis and its isoelectric point was at pH 3.12. The molecular mass, calculated from its partition coefficient on the Sepharose 6B column, was 311 kDa, being composed of several subunits of 60 and 70 kDa. The highest activity of this enzyme was attained at pH 4.0 and 60°C. The enzyme showed strong resistance to thermal inactivation. Its activation energy was about 15 kJ/mol. Cellobiose, salicin, and p‐nitrophenyl β‐D‐glucoside, but not carboxymethylcellulose, were hydrolyzed by the enzyme, following substrate inhibition kinetics. The purified β‐glucosidase was considered a true cellobiase because of its great affinity towards cellobiose. Cellobiose inhibition does not seem to be a physiological phenomenon. Glucose inhibited enzyme activity in a competitive way (Ki= 1.26 mM). Fe3+ and Co2+ inhibited activity notably. Hg2+, Cu2+ and EDTA were practically ineffective. Even 200 mM gluconolactone did not affect enzyme activity. [ABSTRACT FROM AUTHOR]

The hexon and fiber antigens of the adenoviruses types 2 and 5 have been extensively purified by a four step procedure comprising freon extraction, neutral salt precipitation, gel filtration chromatography and preparative liquid film electrophoresis. The physical and chemical properties of these two antigens isolated from two adenoviruses belonging to the same immunological subgroup were compared . The hexon antigen is constituted of at least four fractions. It was demonstrated that this apparent heterogeneity corresponds in fact to a series of polymers. The sedimentation coefficient of the monomer is about 11 S. The adenoviruses types 2 and 5 hexon and fiber antigens have similar chemical compositions. The similarity between the types 2 and 5 hexon antigens postulated from immunological studies and from the amino acid compositions was confirmed by peptide "finger-prints" of enzymic hydrolyzates. The results of the biochemical studies suggest that the hexon antigen is constituted of one single polypeptide chain, whereas the fiber antigen is constituted of several peptidic chains. [ABSTRACT FROM AUTHOR]

Immunochemical identification of a distinct immunoglobulin class associated with mouse reaginic antibody and designated IgE has been performed. An antiserum against mouse reaginic antibody was prepared in rats by immunization with homologous peritoneal mast cells sensitized with mouse reaginic antibody. This antiserum, after adequate absorption, recognized only one component existing in mouse serum or fractions of serum containing reaginic antibody. This component was not any of the already known γ1, γ2, IgA or IgM immunoglobulins; its immunoglobulin nature was indicated by its antigen-binding capacity in radioimmunodiffusion analysis. Fractionation experiments (zone electrophoresis, gel filtration on Sephadex G-200) showed that there was a strict association between reaginic anaphylactic activity and IgE immunoglobulin. Molecular weight of IgE was found to be about 200,000. In biological tests, anti IgE neutralized anaphylactic activity attributed to reaginic antibody, but not that attributed to γ1-antibody. Anti-IgE degranulates normal and sensitized mouse peritoneal mast cells, and rat peritoneal mast cells after they have been sensitized by mouse reaginic antibody. [ABSTRACT FROM AUTHOR]