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An apparatus for electrophoresis on cellulose coated glass plates is described. Combined with a subsequent chromatographic separation, thin-layer electrophoresis permits a rapid, convenient and complete analysis of the amino acid composition of various biological fluids and extracts. Relative mobility values and Rf-values are given for 21 of the more common acids and amines. [ABSTRACT FROM AUTHOR]

1. A β-glucosidase (cellobiase) has been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride. 2. The crude material was pre-fractionated by chromatography on Bio-Gel P-10 and by DEAE-Sephadex chromatography as previously described [1]. Further fractionation methods utilized in the present paper included ammonium sulfate precipitation. SE-Sephadex chromatography, isoelectric focusing, and chromatography on Bio-Gel P-60. 3. A yield of 53 mg enzyme was obtained per 100 g commercial cellulase. The specific activity of the purified enzyme represented an approximately 60-fold increase from that of the commercial product. 4. The isolated enzyme was homogeneous in polyacrylamide gel electrophoresis at pH 8.0 by isoelectric focusing in a polyacrylamide gel and also in free zone electrophoresis. 5. No enzyme activity towards Avicel (mieroerystalline cellulose) or towards carboxymethylcellulose could be detected in the purified material under the assay conditions used. 6. A molecular weight of 47 000 was determined for the enzyme by dodecylsulfate-polyacrylamide gel electrophoresis. The same molecular weight value was obtained by chromatography of the native enzyme on Bio-Gel P-100 and also by chromatography of the reduced and alkylated enzyme on Sepharose 6B in 6 M guanidine-HCl. 7. The amino-acid analysis showed that the enzyme was rich in aromatic (9.5%) and acidic (19.0%) amino acids. The half-cystine content was 6 residues per molecule indicating 3 disulfide bridges, since no free sulfhydryl group was detectable. The purified enzyme contained no carbohydrate (as hexose). 8. The enzyme was isoelectric at pH 5.74 (10 °C) and the electrophoretic mobility was determined to be 1.49 × 10-5 cm² s-1 V-1 at pH 8.0. 9. The Km-values for the enzyme towards the substrates p-nitrophenyl-β-D-glucoside, cellobiose, and cellotetraose were found to be 0.28, 1.5, and 0.35 mM, respectively. [ABSTRACT FROM AUTHOR]

A two-dimensional fractionation procedure has been developed fro separating radioactively-labelled oligonucleotides of up to 50 residues long, using uniformly 32P-labelled 5S RNA of Escherichia coli as a model compound. The method uses ionophoresis on cellulose acetate of pH 3.5 in the frist dimension; and ascending chromatography with a concentrated mixture of oligonucleotides on thin layers of mixed DEAE-cellulose and cellulose in the second dimension. [ABSTRACT FROM AUTHOR]

32P-labelled N-formyl methionyl transfer RNA from Escherichia coli strain CA265 was purified by chromatography on a column DEAE Sephadex followed by chromatography on benzoylated DEAE cellulose. Nucleotides from ribonuclease T1 and pancreatic ribonuclease digests of tRNAF were separated and their sequences determined. tRNAF consists of 77 nucleotides of which pCG is 5′-terminal and CAACCOH is 3′-terminal. [ABSTRACT FROM AUTHOR]

There is extensive, published research on the use of high energy irradiation for the modification of textiles, hut no reports pertaining to the use of this technique for the direct fixation of dyes could be found. This study demonstrates that dyes containing activated vinyl groups can be fixed with good yields on cellulose by means of high energy irradiation. The majority of the experiments were performed using high energy electrons, but gamma rays from a cobalt-60 source were shown to give similar results. The mechanism is believed to be grafting by vinyl addition to polymer radicals formed on irradiation. The fixed dyes exhibit good fastness and cannot be extracted from the fiber with an azeotrope of pyridine/water, or boiling N,N-dimethylformamide. To estimate the potential for fixation of a particular dye by irradiation, a method was developed which is based on redox initiated copolymerization of acrylamide and dye and subsequent chromatography. Simultaneous fixation of dye and N-methylolacrylamide has also been achieved, and the coapplication of dye and finishing agent to attain dyeing and durable press in one operation has been successfully demonstrated by a plant scale trial using a 500 kv electron accelerator system. [ABSTRACT FROM AUTHOR]

A purification procedure to prepare highly purified DNA-dependent RNA polymerase from Bacillus subtilis vegetative cells is described. The enzyme consists of four different subunits, β′, β, α (molecular weights 154000 for β′ and β, 56000 for α and 43000 for α) in a molar ratio 1:1:1:2. By phosphocellulose chromatography RNA polymerase has been disseociated in α subunit and core enzyme, containing β′, β and α subunits in a molar ratio 1:1:2 the α subunit stimulates the activity of core polyrnerase with ϕ 29 DNA but not with poly[d(A--T)]. The general properties of the enzyme are also described. [ABSTRACT FROM AUTHOR]

Yeast DNA polymerases A and B were purified 5000- 10000-fold by chromatography on DEAEcellulose, phosphocellulose, DNA-agarose and DEAE-Sephadex. In acrylamide gel electrophoresis in the presence of sodium dodecylsulfate, both enzymes give rise to three main bands. The enzymes are equally susceptible to inhibition by the - SH reagents N-ethylmaleimide and p-chloromercuribenzoate but differ in their sensitivity to cytosine-arabinoside triphosphate, polymerase A being considerably more sensitive to this nucleotide analog. Whereas DNA polymerase A prefers nicked DNA and poly[d(A-T)] as template-primer, polymerase B is most active with poly(dA) · (dT)10 · Km values for deoxyribonucleoside triphosphates were determined as 3.7-3.9 μM for enzyme A and 1.8-2.4 μM for enzyme B. During active growth of cells polymerase activity increases about 1.4-fold over the amount found in resting cells, which is mainly if not exclusively due to an increased level of DNA polymerase A. This together with other properties suggests a role of this enzyme m DNA replication. [ABSTRACT FROM AUTHOR]

From spinach leaves (Spinacea oleracea L.) two isoenzymes of glucosephosphate isomerase can be separated by DEAE-cellulose ion-exchange chromatography. Isoenzyme 1 eluting at high ionic strength shows a faster mobility during disc gel eleotrophoresis than isoenzyme 2 eluting at low ionic strength. For both isoenzymes the pH-optima and Km-values were very similar for either direction. However, the Km-values with glucose-6-phosphate as substrate (Km = 8.0 mM and 5.9 mM for isoenzyme 1 and 2, respectively) were 20-fold higher than with fructose-6-phosphate as substrate (Km = 300 μM for both isoenzyxnes). The molecular weight of both isoenzymes was estimated by sedimentation velocity centrifugation analysis to be 120000. The subcellular distribution reveals that isoenzyme 1 is located within the chloroplasts and isoenzyme 2 in the non-particulate cell fraction (cytosol). The results give further support to the view that in plants there exist two sets of isoenzymes for the oxidative pentose phosphate cycle, one set located in the chloroplasts, the other one in the non-particulate cell fraction (cytosol).

Rat liver thioredoxin, which could be reduced nonenzymatically by dithiothreitol, was identified by its ability to function as hydrogen donor in the conversion of ribonucleotides to deoxyribonucleotides catalyzed by ribonucleotide reductase both from the same tissue and from Escherichia coli. The latter enzyme was used to detect the reduced form of the thioredoxin during its purification which involved treatment at pH 5, chromatography on Sephadex G-50, heat treatment, and DEAE-cellulose chromatography. The isolated material was approximately 50% pure as determined by polyacrylamide electrophoresis. Liver thioredoxin is a heat-stable protein of low molecular weight. [ABSTRACT FROM AUTHOR]

Human lactoferrin, the red iron-binding protein from milk, was prepared by chromatography on DEAE-cellulose of the milk proteins soluble in 2 M ammonium sulfate. Several preparations differing in iron content were obtained. Apolactoferrin was prepared by dialyzing the protein against 0.1 M citric acid. Saturation experiments with iron ascorbate indicated the iron : protein ratio at saturation to be 0.025 micromoles of metal per mg of protein. Assuming 80,000 to be the molecular weight of lactoferrin, this would correspond to a ratio of two iron atoms per molecule. With copper, a similar molar proportion at saturation was obtained. Gasometrie measurements were used to calculate the amount of carbon dioxide liberated upon dissociation of the metal-protein complexes by acidification. One mole of CO2 was freed per atom of iron or copper. [ABSTRACT FROM AUTHOR]

Collagenase was found to attack the collagen molecule at both ends at 10°. Six molecular fragments of different lengths were observed and could be correlated with the intact molecule as long-spacing segment fragments. It was possible to isolate the 2,600, 1,250 and 780 Å fragments by fractional ammonium sulphate precipitation. The sharp helix-coil transition on denaturation shows that the fragments have an intact triple helix. It was possible to separate the two shorter fragments into three components (the α1, α2 and α3 chain fragments) by chromatography on CM cellulose. The α1 and α3 chains have very similar amino acid compositions and peptide maps for both these fragments. This suggests that the differences between these two components are probably due to telopeptides and not to the amino acid sequence of the main chain. [ABSTRACT FROM AUTHOR]

Antibodies against Esch. Coli WF 96 and WF 61 present in human colostrums and serum were fractionated by DEAE-cellulose chromatography. Using the haemagglutination test it was found that the antibodies present in colostrums were recovered in the fraction containing the bulk of γA-globulin, whereas the antibodies present in serum were recovered in the fraction containing the bulk of γM-globulin. In the presence of human or guinea-pig complement the antibodies present in colostrums did not lyse red cells coated with bacterial polysaccharides whereas the antibodies present in serum were lytic. When the properties of γA and γM antibodies were studied using a bacteriolytic system, it was observed the γA-globulin lysed bacteria only in the presence of both complement and lysozyme; in this respect γAbacterial antibodies behaved differently from γM antibodies which were bacteriolytic in the presence of complement alone, without lysozyme. The effect of treating γA and γM antibodies with 2-mercaptoethanol at neutral pH and of heating at 56° was investigated. [ABSTRACT FROM AUTHOR]

An account is given of an investigation in which ampholytes of suint from degreased Australian merino wool were isolated by ion-exchange chromatography and the free amino acids were separated from the peptides by chromatography on Sephadex impregnated with cupric ions. Suint contains 21 free amino acids, of which nineteen have been identified. Possible sources of free amino acids are considered; they are probably ovine metabolic products, but there is some evidence to suggest bacterial contamination. [ABSTRACT FROM AUTHOR]

A soluble protein extract, obtained after [SUP35]S labeling exponential cultures of Cryptothecodinium cohnii for 4-5 generations, was fractionated on DNA-cellulose. The protein eluted at high salt concentrations from this homologous DNA-containing matrix was then separated by chromatography on BioGel P150 and P200 sieve gels. Two prominent chromatographic species of 22,000 and 35,000 daltons plus a substantial amount of material in the molecular weight range of 80,000-140,000 were resolved. The application of this analytical procedures to the study of transcriptional control in the cell cycle is discussed. [ABSTRACT FROM AUTHOR]