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Deacylation of acetyl-coenzyme A and acetylcarnitine by liver preparations.

Snoswell, A M; Tubbs, P K
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/05/1978 EN
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16.55107%
The breakdown of acetylcarnitine catalysed by extracts of rat and sheep liver was completely abolished by Sephadex G-25 gel filtration, whereas the hydrolysis of acetyl-CoA was unaffected. Acetyl-CoA and CoA acted catalytically in restoring the ability of Sephadex-treated extracts to break down acetylcarnitine, which was therefore not due to an acetylcarnitine hydrolase but to the sequential action of carnitine acetyltransferase and acetyl-CoA hydrolase. Some 75% of the acetyl-CoA hydrolase activity of sheep liver was localized in the mitochondrial fraction. Two distinct acetyl-CoA hydrolases were partially purified from extracts of sheep liver mitochondria. Both enzymes hydrolysed other short-chain acyl-CoA compounds and succinyl-CoA (3-carboxypropionyl-CoA), but with one acetyl-CoA was the preferred substrate.

Cytosol oestrogen receptor of lactating mammary gland. Effect of heparin on the aggregation of the receptor and interaction of the receptor with heparin-Sepharose.

Auricchio, F; Rotondi, A; Sampaolo, P; Schiavone, E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/04/1978 EN
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1. An oestrogen receptor is present in low-salt cytosol of the mammary gland of lactating mice as a large aggregate; it is excluded from gel matrix when filtered on a Sephadex G-200 column and sediments at 7S in sucrose gradients. After incubation of cytosol with heparin, the receptor is dissociated. On a Sephadex G-200 column, it is included in the gel matrix and eluted as a protein with mol.wt. 260000 and a Stokes radius of 6.8nm; it sediments at 6S in sucrose gradients. 2. Dissociation of the mammary-gland cytosol oestrogen receptor seems to be the result of interaction of the oestrogen-receptor complex with heparin. This receptor interacts with heparin covalently bound to Sepharose, thereafter sedimenting at 6S. By using this interaction, the cytosol receptor was purified 200-fold compared with the homogenate, with a yield of 70%. 3. The cytosol receptor that was not incubated or was incubated with heparin was much smaller during sucrose-gradient centrifugation than during gel filtration. This discrepancy can be explained by pressure-induced dissociation during high-speed centrifugation. This possibility is supported by the decrease in the sedimentation coefficient of the receptor with increased duration of centrifugation.

Characterization of two acid proteinases found in rabbit skin homografts.

Jasani, B; Jasani, M K; Talbot, M D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/02/1978 EN
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Two types of acid proteinase activity found in rabbit skin homografts were characterized by studying the effect of temperature, pH and polyacrylamide-gel electrophoresis. Their chromatographic behaviour was characterized on DEAE-cellulose, Sephadex G-75, G-100 and G-200, and their molecular weights were estimated by gel filtration. One of the acid proteinases in the homograft resembled cathepsin D (EC 3.4.23.5) of normal skin. The other acid proteinase differed from cathepsin D with respect to heat inactivation, pH optimum and molecular weight; it was not inactivated on heating at 60 degrees C for 60 min, its pH optimum was 2.5 and its molecular weight measured by Sephadex G-100 chromatography was 100 000. In all these respects, the heat-stable proteinase resembles cathepsin E (EC 3.4.23.5) of rabbit polymorphonuclear leucocytes.

Studies on a nucleoprotein prepared from rat liver polysomes by digestion with T1 ribonuclease

Hawtrey, A. O.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1969 EN
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16.55107%
1. Treatment of rat liver polysomes in a buffer containing 2·5mm-magnesium chloride with T1 ribonuclease at a concentration of 330units/ml. of reaction medium at 37° for 2hr. leads to the production of an insoluble nucleoprotein. 2. On the bases of analysis for protein and RNA and of u.v.-absorption spectra the nucleoprotein appears to have lost approx. 60% of the structural RNA originally present in the ribosome. Degradation of 3H-labelled polysomes (structural RNA labelled with orotic acid) with T1 ribonuclease leads to nucleoprotein preparations retaining approx. 30% of the radioactivity originally present in the polysomes. By means of sucrose-density-gradient centrifugation it is shown that the nucleoprotein preparations are free of single 73s ribosomes and ribosomal subunits. No evidence for the presence of 28s and 18s structural RNA was obtained on examination of extracted nucleoprotein-particle RNA by means of sucrose-density-gradient centrifugation. 3. Digestion of washed polysomes carrying 14C-labelled nascent peptide chains with T1 ribonuclease gives a nucleoprotein particle that retains approx. 70% of the original labelled chains. Treatment of labelled nucleoprotein particles with 1mm-puromycin in the absence of transfer factors releases 20% of the labelled chains. Addition of GTP (0·48μmole) increases this release to 37%. 4. Treatment of nucleoprotein particles carrying 14C-labelled peptide chains with either EDTA (50mm) or ammonium chloride (0·5m) brings about a small release of labelled material (approx. 15%). 5. Disruption of nucleoprotein particles carrying 14C-labelled peptide chains with either sodium dodecyl sulphate or 2m-lithium chloride...

Vitamin A and carotenoids. The enzymic conversion of β-carotene into retinal in hog intestinal mucosa

Fidge, Noel H.; Smith, Frank Rees; Goodman, DeWitt S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1969 EN
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16.55107%
The conversion of β-carotene into retinal was studied in vitro with enzyme preparations from homogenates of hog intestinal mucosa. The hog mucosal enzyme was purified about 27-fold by precipitation with ammonium sulphate, chromatography on DEAE-Sephadex and gel filtration on Sephadex G-200. The reaction displayed a narrow optimum pH range (approx. 7·8–8·2). The enzyme was stimulated strongly by the addition of thiols, and was inhibited by thiol inhibitors and by the chelating agents αα′-bipyridyl and o-phenanthroline. The reaction required the addition of an appropriate detergent (or bile salt); maximal activity was obtained by addition of an appropriate combination of detergents and lipid (specifically Tween 40, sodium glycocholate and sphingomyelin). The reaction displayed Michaelis kinetics with Km1·3×10−6m and Vmax.1·1nmole of retinal formed/hr. (for 0·7mg. of enzyme protein). The properties of the hog enzyme are similar to those previously reported for a less purified rat enzyme preparation.

The formation of short fibres from native cellulose by components of Trichoderma koningii cellulase

Halliwell, G.; Riaz, M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1970 EN
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16.55107%
Cellulolytic enzyme components of culture filtrates of Trichoderma koningii were fractionated on ionic and non-ionic forms of Sephadex and on cellulose powder (Whatman) and examined for their ability to hydrolyse soluble carboxymethyl-cellulose, and to saccharify, solubilize and form short fibres from native undegraded cellulose of the type found in cotton. DEAE-Sephadex provided two CM-cellulase components and a C1 component; the C1 component acted weakly and solely on cotton, forming soluble products but not short fibres. The ability to form short fibres was confined almost wholly to one of the CM-cellulase components which completely degraded cotton, minimally to soluble products and extensively to short fibres. The latter action was unaffected by the presence of the other two components. The two CM-cellulase components solubilized cellulose synergistically whereas the short-fibre-forming component and C1 component were inhibitory.

Active accumulation of tetracycline by Escherichia coli

Franklin, T. J.; Higginson, B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1970 EN
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16.55107%
1. At low concentrations of tetracycline (10μg/ml) net accumulation of the drug by Escherichia coli cells ceased after 7–10min. 2. At higher concentrations of tetracycline (>30μg/ml) the period of net accumulation of the drug was significantly extended. 3. The efflux of tetracycline from E. coli cells transferred from medium containing 10μg of tetracycline/ml to drug-free medium was a rapid temperature-dependent process and was accelerated by 2,4-dinitrophenol. 4. As the concentration of tetracycline in the preloading phase was increased, the rate of subsequent efflux of the drug progressively declined. The efflux of drug from cells preloaded in medium containing 200μg of tetracycline/ml was negligible, although efflux was readily provoked by 2,4-dinitrophenol, by N-ethylmaleimide or by omission of glucose from the medium. 5. The initial rate of uptake of tetracycline by E. coli cells was linearly proportional to the concentration of tetracycline in the medium up to the maximum concentration of drug obtainable under the experimental conditions used (400μg/ml, 0.83mm). 6. Although N-ethylmaleimide strongly inhibited the accumulation of tetracycline by E. coli, no evidence was obtained for the direct involvement of thiol groups in the transport process. It was concluded that N-ethylmaleimide inhibited accumulation by interruption of the energy supply of the cells. 7. Osmotic shock of E. coli cells did not significantly affect the influx of tetracycline...

Partial purification and properties of an enzyme from rat liver that catalyses the sulphation of l-tyrosyl derivatives

Mattock, P.; Jones, J. G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1970 EN
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1. An enzyme that catalyses the transfer of sulphate from adenosine 3′-phosphate 5′[35S]-sulphatophosphate to l-tyrosine methyl ester and tyramine was purified approx. 70-fold from female rat livers. 2. The partially purified preparation is still contaminated with adenosine 3′-phosphate 5′-sulphatophosphate–phenol sulphotransferase (EC 2.8.2.1), but a partial separation of the two enzymes can be achieved by chromatography on columns of Sephadex G-200 and DEAE-Sephadex. 3. The enzyme responsible for the sulphation of l-tyrosine methyl ester and tyramine is activated by dithiothreitol, 2-mercaptoethanol and GSH, the degree of activation being more marked with preparations previously stored at 0 or −10°C. In contrast, the enzymic sulphation of p-nitrophenol is inhibited by all three thiols. Again, there is a quantitative difference in the degree of inhibition of the two enzymes by o-iodosobenzoate, p-chloromercuribenzoate, N-ethylmaleimide and iodoacetate. 4. Mixed-substrate experiments support the hypothesis that the enzyme responsible for the sulphation of l-tyrosine methyl ester and tyramine is separate from that responsible for the sulphation of p-nitrophenol. However, p-nitrophenol is a potent inhibitor of the sulphation of both tyrosyl derivatives whereas these latter compounds have no effect on the sulphation of p-nitrophenol.

The enzymes of proline biosynthesis in Escherichia coli. Their molecular weights and the problem of enzyme aggregation.

Hayzer, D J; Moses, V
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/07/1978 EN
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16.55107%
1. By using Bio-Gel A1.5M and Sephadex G-150 columns, crude cell-free extracts of Escherichia coli were fractionated to demonstrate the existence of a proline-biosynthetic aggregate. 2. Sephadex G-150 resolves two glutamyl kinases that are inhibited by proline, with mol.wts. of 125000 and 38000, the reactions of which are Mg2+-dependent. The heavier species is more sensitive to inhibition by proline. 3. Gamma-Glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase (EC 1.5.1.2) have mol.wts. of approx. 125000 and 190000 respectively, the specific activity of the latter being 5 X 10(3)-fold greater than either of the other two biosynthetic enzymes or of the total pathway in vivo. 4. Bio-Gel A1.5M chromatography gave a single glutamyl kinase of mol.wt. 250000, and the possibility of this being a constituent of an enzyme complex is discussed.

Purification and properties of gamma-glutamylcyclotransferase from human erythrocytes.

Board, P G; Moore, K A; Smith, J E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/08/1978 EN
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16.55107%
1. GAMMA-Glutamylcyclotransferase was purified 10000-fold from human erythrocytes. 2. The purification steps involved fractionation with (NH4)(2)SO(4) and chromatography on Sephadex G-75, DEAE-cellulose and hydroxyapatite. The purified enzyme was found to be homogeneous on density-gradient polyacrylamide-gel electrophoresis. 3. The maximum reaction rate was observed at pH9.0 and the apparent Km value for gamma-glutamyl-L-alanine was 2.2mM. 4. The molecular weight (25250) of the purified enzyme agreed well with the value (25500) in fresh haemolysates, indicating no apparent structural modification of the enzyme during purification. However, rapid processing of the blood through the initial (NH4)(2)SO(4) and Sephadex-chromatography steps was required to prevent formation of a high-molecular-weight aggregate with substantially lower specific activity. 5. gamma-Glutamylcyclotransferase catalyses the formation of 5-oxoproline from gamma-glutamyl dipeptides. The role of this enzyme in erythrocytes is of particular interest, because gamma-glutamyl-L-cysteine serves as a substrate for both gamma-glutamylcyclotransferase and glutathione synthetase. Thus the cyclotransferase could modulate glutathione synthesis.

Isolation and characterization of the subunits of bovine follitropin.

Cheng, K W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/10/1978 EN
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16.55107%
Highly purified bovine follitropin was dissociated into its alpha- and beta-subunits after treatment with 1 M-propionic acid. The dissociated subunits were fractionated by chromatography on DEAE-cellulose and further purified by gel filtration on Sephadex G-100. The isolated alpha- and beta-subunits were biologically inactive, but their recombinants regenerated 80% of the follitropin activity. The alpha-subunit of bovine follitropin recombined with the beta-subunits of bovine lutropin and thyrotropin to regenerate 70% of lutropin and 50% of thyrotropin activities respectively. The beta-subunit of bovine follitropin recombined with the alpha-subunit of either bovine lutropin or thyrotropin to regenerate about 75% of follitropin activity. Recombinations were monitored by specific radioligand-receptor assays and polyacrylamide-gel electrophoresis. The elution volumes of the alpha- and beta-subunits of bovine follitropin after gel filtration on Sephadex G-100 were almost identical. The amino acid composition of bovine follitropin-alpha was low in histidine, arginine, isoleucine and leucine, but relatively high in lysine, threonine and glutamic acid. The bovine follitropin-beta contained one methionine residue and low amounts of histidine and phenylalanine...

beta-Bungarotoxin. Separation of two discrete proteins with different synaptic actions.

MacDermot, J; Westgaard, R H; Thompson, E J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/10/1978 EN
Relevância na Pesquisa
16.55107%
beta-Bungarotoxin, a specific presynaptic blocking agent, was prepared in two stages from the crude venom of Bungarus multicinctus by ion-exchange chromatography on the weakly acidic ion exchanger, CM-Sephadex, and on the strongly acidic ion exchanger, sulphopropyl-Sephadex. By these procedures it was purified to a single protein, which was shown by reduction to contain two polypeptide chains with mol.wts. of less than 15000. During purification of beta-bungarotoxin three other proteins were isolated. Two of these proteins have similar molecular weights, subunit structure and physiological properties to the major protein component. This latter is referred to as beta-bungarotoxin, since it has the same physiological properties as those described for unpurified beta-bungarotoxin by other workers. The first protein has very different physiological effects and biochemical properties from beta-bungarotoxin. This protein has a single class of polypeptide chains with an apparent molecular weight that is lower than the main beta-bungarotoxin protein, and appears to block synaptic transmission by a predominantly postsynaptic effect. It has been suggested [Oberg & Kelly (1976) J. Neurobiol. 7, 129-141] that the action of beta-bungarotoxin depends on its phospholipase A activity; however...

Purification and characterization of the ferredoxin component of 25-hydroxycholecalciferol 1 alpha-hydroxylase.

Kulkoski, J A; Ghazarian, J G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/02/1979 EN
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16.55107%
The chick kidney mitochondrial iron--sulphur protein (ferredoxin), a component of the NADPH--cytochrome P-450 reductase functional in the 1 alpha-hydroxylation of 25-hydroxycholecalciferol, was purified to homogeneity by chromatography on DEAE-cellulose, gel filtration on Sephadex G-100 and preparative electrophoresis on polyacrylamide gel. A novel NADPH--cytochrome c reductase assay utilizing crude renal NADPH--ferredoxin reductase was used for the detection of the ferredoxin. A mol. wt. of 53 000 was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by Sephadex G-100 gel filtration of the 125I-labelled ferredoxin. The ferredoxin has a sedimentation constant (S 20, w) of 2.66S, an A411/A280 of 0.4, and a molar absorptivity of 7300 cm-1 . M-1. The electron-paramagnetic-resonance spectrum after reduction with Methyl Viologen and dithionite was characteristic of ferredoxins with signals at g = 1.956 and 2.025. Two iron and two labile sulphur atoms per molecule of ferredoxin were released by acid. Ouchterlony immunodiffusion tests by using goat anti-(bovine adrenal ferredoxin) antiserum showed precipitin reactions with the bovine adrenal ferredoxin and the chick renal ferredoxin as antigens, suggesting that the renal ferredoxin shares antigenic determinants(s) with the natural adrenal antigen. Amino acid analysis showed that of the total number of residues per molecule of ferredoxin...

Brain arylamidase. Purification and characterization of the soluble bovine enzyme

Brecher, A. S.; Suszkiw, J. B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1969 EN
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16.55107%
1. An enzyme acting on aminoacyl-β-naphthylamides has been isolated from the soluble fraction of bovine brain and purified 205-fold by means of ammonium sulphate fractionation, hydroxyapatite adsorption and DEAE-Sephadex column chromatography. 2. Arylamidase requires thiol groups for retention of its activity, is heat-labile and is susceptible to freezing. p-Chloromercuribenzoate and N-ethylmaleimide inactivate the enzyme rapidly. 3. Metal ions are not required for its activity, but stimulation by Mn2+ and Mg2+ and inactivation by Co2+ and Zn2+ are observed. 4. Optimum pH7·5 in phosphate buffer was exhibited for all substrates tested except l-leucyl-β-naphthylamide, for which optimum pH is 6·5. 5. Km values for a number of substrates have been obtained and substrate inhibition at high concentrations was demonstrated. 6. The molecular weight is approx. 70000 as determined by Sephadex-gel filtration.

The cell wall of Bacillus licheniformis N.C.T.C. 6346. Isolation of low-molecular-weight fragments from the soluble mucopeptide

Hughes, R. C.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1968 EN
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1. Soluble mucopeptide was prepared by lysozyme treatment of acid-extracted walls of Bacillus licheniformis N.C.T.C. 6346 and separated into fractions differing in molecular size by chromatography on Sephadex G-25 and G-50. 2. About 16% of the weight of soluble mucopeptide has a weight-average molecular weight in excess of 20000. About one half has a weight-average molecular weight of less than 2000 and the balance of soluble mucopeptide is of intermediate size. 3. In the mucopeptide fractions isolated from Sephadex there is a correlation between the weight-average molecular weight, the number of non-reducing muramic acid residues and the proportion of diaminopimelic acid residues recovered after treatment with 1-fluoro-2,4-dinitrobenzene. 4. The extent of cross-linking between peptide side chains is relatively low, even in mucopeptide material of the large molecular size. 5. The small amount of residual phosphorus present in preparations of B. licheniformis soluble mucopeptide remains associated mainly with mucopeptide material of large molecular size. 6. The mucopeptide components of lowest molecular weight are not produced as artifacts during the preparation of soluble mucopeptide, but are apparently incorporated in the insoluble mucopeptide present in walls of exponentially growing cells. 7. Soluble mucopeptide isolated in a complex with acidic polymers after lysozyme treatment of walls of B. licheniformis N.C.T.C. 6346 and Bacillus subtilis W23 retains a high molecular weight when the covalent bonds between mucopeptide and the acidic polymers are broken. 8. Pure fragments were isolated from B. licheniformis soluble mucopeptide. A major component...

An electrophoretic investigation of groundnut proteins: the structure of arachins A and B

Tombs, M. P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1965 EN
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16.55107%
1. The proteins of the groundnut cotyledon have been fractionated and analysed by DEAE-Sephadex chromatography and acrylamide-gel electrophoresis. Seventeen components were detected. 2. A new method is described for the preparation of arachin, using calcium precipitation. The product contains at least 99% of arachin. 3. The theory of acrylamide-gel electrophoresis is developed and applied to the arachin system to predict the molecular weight of one sub-unit of arachin. 4. A variant form of arachin, arachin B, has been discovered. Of 81 nuts, 27 contained only arachin B, 53 contained both arachin A and B, and one contained arachin A only. This is almost certainly a polymorphism of arachin; this is the first example of polymorphism to be reported in plant proteins. 5. A combination of controlled denaturation, electrophoretic analysis, ultracentrifuge and Sephadex filtration data has shown that arachin A contains four different kinds of peptide chains (α, β, γ and δ). Arachin B contains only β, γ and δ chains. 6. The most probable structure for arachin B, mol.wt. 330000 form, is 8 β, 2 γ and 2 δ chains, and for arachin A, 4 α, 4 β, 2 γ and 2 δ chains. Arachin without β chains was not found. 7. The α and β chains have mol.wts. of about 35000 and the γ and δ chains of about 10000. 8. Three N-terminal groups were found: the α and β chains both terminate in glycine; the γ and δ chains terminate in isoleucine and glutamic acid. 9. Arachin contains no carbohydrate. 10. Disulphide bonds are not important in arachin: there are none between the α...

The composition and physicochemical properties of hyaluronic acids prepared from ox synovial fluid and from a case of mesothelioma

Preston, B. N.; Davies, M.; Ogston, A. G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1965 EN
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16.55107%
1. Materials containing hyaluronic acid have been prepared by filtration (Ogston & Stanier, 1950) from ox synovial fluid and from a protein-rich human mesothelioma fluid. The ox material has been deproteinized by treatment with chloroform and pentanol and by gradient elution on DEAE-Sephadex; several fractions were obtained by the latter method. These materials can be stored in solution at −20° without change of properties. The ox material contained 21% of protein; all other preparations contained less than 6% of protein. 2. The two materials have been compared by sedimentation and viscosity and shown to be closely similar. Treatment of the ox material with neuraminidase caused no change in its viscosity behaviour. 3. Information about the molecular configuration of the ox material has been obtained from measurements of light-scattering and viscosity. The results, though consistent with a highly extended configuration, are not consistent with a linear random-coil configuration. It is tentatively suggested that the structure may have some degree of branching and of cross-linking, which give it a rigidity with respect to expansion of the molecular domain that would not be possessed by a random coil. 4. The deproteinized material recovered from DEAE-Sephadex...

Ribonucleic acids from barley leaves

Srivastava, B. I. Sahai
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1965 EN
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16.55107%
1. The total RNA and the RNA present in 27000g pellet (probably composed of chloroplasts, nuclei and mitochondria) and in 27000g supernatant (probably composed of microsomes and soluble proteins) fractions (separated by centrifugation at 27000g of a leaf homogenate prepared in 0·5m-sucrose–0·02m-tris–HCl, pH7·6) of barley leaves were extracted by phenol–sodium lauryl sulphate and their elution profiles on Sephadex G-200 and on ECTEOLA-cellulose anion-exchanger were examined and their nucleotide compositions and the melting curves were determined. 2. The pellet and the supernatant fractions contained respectively about 55% and 20% of the total RNA, whereas 25% of the total RNA was lost during homogenization of the leaf tissue with sucrose–buffer. 3. The total RNA or the RNA from pellet or supernatant fractions, which by its behaviour on Sephadex G-200 columns was found to be predominantly of high molecular weight (i.e. of ribosomal origin), produced about 13 peaks on ECTEOLA-cellulose columns. The RNA species in the pellet and supernatant fractions probably resembled each other in molecular size or secondary structure or both. However, they were present in relatively different amounts in these fractions. 4. The Tm (i.e. the temperature at which 50% of the maximal increase in extinction had occurred) of total RNA and of RNA from pellet fraction was 64·5° whereas Tm of RNA from the supernatant fraction was 73°. The total RNA and the RNA from pellet fraction also resembled each other in nucleotide composition...

Demonstration of a glycoprotein derived from the 24p3 gene in mouse uterine luminal fluid.

Chu, S T; Huang, H L; Chen, J M; Chen, Y H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/06/1996 EN
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A glycoprotein in mouse uterine luminal fluid was purified to homogeneity via a series of purification steps involving Sephadex G-100 chromatography, Sephadex G-50 chromatography and HPLC on a reverse-phase C18 column, in that order. Automated Edman degradation was unable to determine the N-terminal residue of the glycoprotein and the partial sequences determined from its trypsin digests were found to be identical with the protein sequence deduced from 24p3 cDNA. The core protein and the total amount of carbohydrate together gave a molecular mass of 25.8 kDa. Results from the characterization of the glycopeptide bond indicated the presence of N-linked carbohydrate but no O-linked carbohydrate in the protein, which has two potential sites for N-linked carbohydrate at Asn81 and Asn85, as deduced from analysis of the primary structure. The core protein was shown to have a molecular mass equal to that of the putative protein deduced from cDNA, suggesting that this protein may contain no signal peptide. Results of Northern-blot analysis for various tissues of adult mice revealed that the 24p3 gene was expressed in lung, spleen, uterus, vagina and epididymis.

Characterization and purification of a lipoxygenase inhibitor in human epidermoid carcinoma A431 cells.

Chen, C J; Huang, H S; Lee, Y T; Yang, C Y; Chang, W C
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/10/1997 EN
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A lipoxygenase inhibitor in the cytosolic fraction of human epidermoid carcinoma A431 cells was characterized and purified. The cytosolic inhibitor lost the inhibitory activity upon heating at 75 degrees C for 15 min or pretreating with 1 mg/ml trypsin at 37 degrees C for 60 min. Cytosol, after dialysis, lost the inhibitory activity but its inhibitory activity recovered when 1 mM GSH was added to the dialysate. The inhibitory activity of cytosol was also abolished by treatment either with 1 mM iodoacetate at 4 degrees C for 1 h or with 0.5 mM H2O2. The pI of the inhibitor was approx. 7.0. In addition to 12-lipoxygenase, the inhibitor inhibited the activities of 5-lipoxygenase and fatty acid cyclo-oxygenase in a cell-free system. The inhibitor was purified by a series of column chromatographies using CM Sephadex C-50, Sephadex G-100 SF and Mono P columns. A major 22 kDa protein was obtained that was distinct from selenium-dependent glutathione peroxidase.