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Sea urchin (Anthocidaris crassispina) egg zinc-binding protein. Cellular localization, purification and characterization.

Ohtake, H; Suyemitsu, T; Koga, M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/04/1983 EN
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Gel-filtration analysis of cytosol fraction obtained from unfertilized sea-urchin (Anthocidaris crassispina) eggs on Sephadex G-75 revealed the presence of two Zn-binding-protein fractions. The major Zn-binding protein fraction had a low molecular weight and a low absorbance at 280 nm, properties similar to those of the metallothionein found in the regenerating rat liver. These fractions were further purified by DEAE-cellulose and Sephadex G-50 chromatography. Homogeneity of the Zn-binding protein was judged by polyacrylamide-disc-gel electrophoresis and gel-permeation chromatography in the presence of 6 M-guanidinium chloride. The molecular weight determined by gel-permeation chromatography was 3900. This value is in good agreement with the minimum molecular weight calculated from the amino acid composition, which was 3655. Zn-binding protein is composed of 36 amino acid residues and the distinctive features include an extremely high content of cysteine, which accounted for one-third of the total amino acid residues, and a complete absence of aromatic amino acids, as well as of methionine, histidine and arginine. Zn-binding protein contained 4.1 g-atoms of zinc per mol and a trace of cadmium, but no copper, iron or calcium. The molar ratio of reactive thiol groups to metal ion was calculated to be 2.73:1. Possible roles of this Zn-binding protein in the homoeostasis of zinc in unfertilized sea-urchin eggs are discussed.

Isolation, characterization and the role of rabbit testicular arysulphatase A in fertilization.

Farooqui, A A; Srivastava, P N
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/08/1979 EN
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Arysulphatase A was purified from rabbit testis. The purification was accomplished by a four-step procedure involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, SP(sulphopropyl)-Sephadex and affinity chromatography on concanavalin A-Sepharose. The specific activity of purified preparation was 135 mumol/min per mg of protein, which represented an increase of 900-fold above that of the crude homogenate. The purified enzyme (20-50 micrograms) was found to move electrophoretically as a single band on polyacrylamide gel at pH 7.2 and 8.4. The homogeneous enzyme was shown to be a glycoprotein with 0.8% (w/w) of N-acetylneuraminic acid and 20% neutral sugar. The treatment of purified enzyme with bacterial neuraminidase had no effect on enzyme activity or kinetic properties, but it changed the elution prolife of rabbit testis arylsulphatase A through DEAE-Sephadex. The purified enzyme was strongly inhibited by Cu2+, Fe3+ and Ag+. It hydrolysed several sulphate esters including cerebroside 3-sulphate, ascorbic acid 2-sulphate and steroid sulphates. Pure arysulphatase was effective in dispersing the cumulus cells of rabbit ova.

Granulocyte/macrophage-, megakaryocyte-, eosinophil- and erythroid-colony-stimulating factors produced by mouse spleen cells

Burgess, Antony W.; Metcalf, Donald; Russell, Sue H. M.; Nicola, Nicos A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/02/1980 EN
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The formation of mature haemopoietic cells is controlled by hormones that specifically stimulate the progenitor cells of the granulocyte/macrophage, eosinophil, megakaryocyte and erythroid pathways. PWMSC medium (pokeweed-mitogen-stimulated spleen-cell-conditioned medium) is known to contain the biological activities that control the clonal proliferation of these four progenitor cells in vitro in semi-solid agar cultures. In this study the molecular properties of these biological activities were characterized, and all four colony-stimulating factors appear to be associated with glycoproteins. These factors were precipitated between 50 and 80%-satd. (NH4)2SO4 and could be concentrated by ultrafiltration over a 10000-mol.wt.-cut-off hollow-fibre membrane. Megakaryocyte- and erythroid-colony-stimulating factors were lost when the conditioned medium was dialysed at low ionic strength (<0.03m). Neither asialo- nor sialo-erythropoietin was detectable in concentrated PWMSC medium or in the fractions purified from it by gel filtration on Sephadex G-150. The factors bound to concanavalin A–Sepharose were eluted with α-methyl-d-glucopyranoside (0.10m). Analysis by gel filtration on Sephadex G-150 indicated that the apparent molecular-weight distributions of all colony-stimulating factors were identical (37000). Treatment with neuraminidase did not alter the biological activities of any of these factors...

Partial purification and characterization of a novel neutral proteinase from human uterine cervix

Ito, Akira; Ihara, Hiroko; Mori, Yo
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/02/1980 EN
Relevância na Pesquisa
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1. Human uterine cervical stroma was found to contain a Ca2+-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×105 by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein...

The identification of the folate conjugates found in rat liver 48 h after the administration of radioactively labelled folate tracers.

Connor, M J; Blair, J A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/01/1980 EN
Relevância na Pesquisa
16.55107%
About 70% of the radioactivity retained in the livers of rats dosed 48 h earlier with radioactively labelled folate was incorporated into two folate conjugates. The major derivative was purified and isolated by Sephadex G-15, DEAE-cellulose and DEAE-Sephadex ion-exchange column chromatography and paper chromatography. It was identified as 10-formylpteroylpentaglutamate by a combination of spectral, microbiological, chemical and chromatographic techniques. The minor conjugate, though less well characterized, exhibited similar properties and was assigned the structure 10-formylpteroyltetraglutamate. 10-Formylpteroylpentaglutamate (2.0nmol/g) and 10-formylpteroyltetraglutamate (0.25nmol/g) comprised about 20% of the total endogenous hepatic folate as determined by microbiological assay (Lactobacillus casei after conjugase treatment.

Control of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in isolated hepatocytes by glucose and glucagon. Role of a low-molecular-weight stimulator of phosphofructokinase

Van Schaftingen, Emile; Hue, Louis; Hers, Henri-Géry
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/12/1980 EN
Relevância na Pesquisa
16.55107%
1. Recycling of metabolites between fructose 6-phosphate and triose phosphates has been investigated in isolated hepatocytes by the randomization of carbon between C(1) and C(6) of glucose formed from [1-14C]galactose. 2. Randomization of carbon atoms was regularly observed with hepatocytes isolated from fed rats and was then little influenced by the concentration of glucose in the incubation medium. It was decreased by about 50% in the presence of glucagon. 3. Randomization of carbon atoms by hepatocytes isolated from starved rats was barely detectable at physiological concentrations of glucose in the incubation medium, but was greatly increased with increasing glucose concentrations. It was nearly completely suppressed by glucagon. These large changes can be attributed to parallel variations in the activity of phosphofructokinase. 4. The main factors that appear to control the activity of phosphofructokinase under these experimental conditions are the concentration of fructose 6-phosphate, the concentration of fructose 1,6-bisphosphate and also the affinity of the enzyme for fructose 6-phosphate. 5. The affinity of phosphofructokinase for fructose 6-phosphate was diminished by incubation of the cells in the presence of glucagon and also by filtration of an extract of hepatocytes through Sephadex G-25 and by purification of the enzyme. When assayed at 0.25 or 0.5mm-fructose 6-phosphate...

Isolation and characterization of dihydropteridine reductase from human liver.

Firgaira, F A; Cotton, R G; Danks, D M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/07/1981 EN
Relevância na Pesquisa
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Dihydropteridine reductase (EC 1.6.99.7) was purified from human liver obtained at autopsy by a three-step chromatographic procedure with the use of (1) a naphthoquinone affinity adsorbent, (2) DEAE-Sephadex and (3) CM-Sephadex. The enzyme was typically purified 1000-fold with a yield of 25%. It gave a single band on non-denaturing and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and showed one spot on two-dimensional gel electrophoresis. The molecular weight of the enzyme was determined to be 50000 by sedimentation-equilibrium analysis and 47500 by gel filtration. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, a single subunit with mol.wt. 26000 was observed. A complex of dihydropteridine reductase with NADH was observed on gel electrophoresis. The isoelectric point of the enzyme was estimated to be pH 7.0. Amino acid analysis showed a residue composition similar to that seen for the sheep and bovine liver enzymes. The enzyme showed anomalous migration in polyacrylamide-gel electrophoresis. A Ferguson plot indicated that this behaviour is due to a low net charge/size ratio of the enzyme under the electrophoretic conditions used. The kinetic properties of the enzyme with tetrahydrobiopterin, 2-amino-4-hydroxy-6...

Enzymes of 2-oxo acid degradation and biosynthesis in cell-free extracts of mixed rumen micro-organisms.

Bush, R S; Sauer, F D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/08/1976 EN
Relevância na Pesquisa
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The enzymes of 2-oxo acid decarboxylation and 2-oxo acid synthesis (EC 1.2.7.1 and EC 1.2.7.2) were isolated and partially purified from cell-free extracts of rumen micro-organisms. The lyase was active with pyruvate, 3-hydroxypyruvate and 2-oxobutyrate. The synthase was active with acetate, 2-oxoglutarate or succinate. Pyruvate synthase was separated from pyruvate lyase by Sephadex G-200 gel filtration. With Sephadex filtration, approximate mol.wts. of 310000 and 210000 were determined for pyruvate lyase and pyruvate synthase respectively.

Purification of an exo-beta-D-glucanase from cell-free extracts of Candida utilis.

Notario, V; Villa, T G; Villanueva, J R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/12/1976 EN
Relevância na Pesquisa
16.55107%
beta-Glucanase present in cell-free extracts from Candida utilis was isolated and purified 562-fold by procedures that include adsorption on DEAE-Sephadex A-50 and filtration through columns of Sephadex G-50, G-100 and G-200, Bio-Gel P-10, and Concanavalin A-Sepharose 4B. The purified enzyme appeared homogeneous on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (S20,w = 1.74S). The enzyme behaved as an acidic glycoprotein (pI4.1) with 68% carbohydrate and a high content of acidic amino acids. The mol.wt. was estimated to be 20000 from gel filtration and polyacrylamide-gel electrophoresis and 36000 from sedimentation experiments. Studies on the hydrolysis of different substrates showed that the enzyme is an unspecific beta-glucanase able to break down both (1 leads to 3)-eta- and (1 leads to 6)-beta-linkages by an exo-splitting mechanism. Glucono-delta-lactone, Zn2+ and Hg2+ inhibited the enzyme activity.

Purification and properties of bovine pituitary follitropin.

Cheng, K W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/12/1976 EN
Relevância na Pesquisa
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A reproducible procedure was developed for the purification of follitropin from frozen bovine pituitary glands. The method involved precipitation with (NH4)2SO4 and acetone, followed by ion-exchange column chromatography on CM-cellulose and DEAE-cellulose and gel filtration on Sephadex G-100. A specific radioligand-receptor assay for follitropin was used to locate the activity in eluates after column chromatography and gel filtration. The potency of the highly purified bovine follitropin as measured by Steelman-Pohley bioassay was 164 times that of NIH-FSH-S1 standard preparation. They yield of bovine follitropin was 2.9 mg/kg of frozen pituitary glands. Electrophoretically, bovine follitropin was more acidic in nature and migrated further towards the anode than lutropin and thyrotropin. The elution volume of bovine follitropin by gel filtration on Sephadex G-100 was very similar to that of bovine lutropin. The amino acid composition of bovine follitropin was similar to that of sheep and human follitropin, being rich in lysine, aspartic acid, threonine, serine, glutamic acid and half-cystine.

Properties and N-terminal sequence of allophycocyanin from the unicellular rhodophyte Cyanidium caldarium.

Brown, A S; Troxler, R F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/06/1977 EN
Relevância na Pesquisa
16.55107%
Allophycocyanin from the unicellular rhodophyte Cyanidium caldarium was purified by (NH4)2SO4 fractionation and ion-exchange chromatography on brushite (calcium phosphate) columns and on DEAE-Sephadex A-25 columns. The specific absorption coefficient (A0.1%1cm) at 650nm of purified allophycocyanin was 6.35 in 0.05M-potassium phosphate buffer, pH7.0. Absorption maxima of allophycocyanin occurred at 650, 618 (shoulder), 350 and 275 nm. Circular-dichroic spectra displayed positive-ellipticity bands at 658 and 630 nm and a major negative-ellipticity band at 340nm. Computer analysis of the circular-dichroic spectrum of allophycocyanin from 207 to 243 nm indicated 42% alpha-helix and 58% beta-form. The estimated molecular weight of purified allophycocyanin on calibrated Sephadex G-200 columns at pH7.0. was 196000. Electrophoretic examination of allophycocyanin on sodium dodecyl sulphate/polyacrylamide gels revealed a single band with apparent mol.wt. 16000. The presence of two polypeptide subunits, with nearly the same molecular weight, was revealed on polyacrylamide gels by using a modified electrophoresis buffer. Spectral analysis of the allophycocyanin subunits resolved by ion-exchange chromatography on Bio-Rex 70 columns indicated that a single phycocyanobilin chromophore was present on each polypeptide chain. Treatment of allophycocyanin with 8M-urea (pH3.0) and subsequent removal of urea by dialysis against water yielded a derivative phycobiliprotein with spectroscopic characteristics similar to those of phycocyanin. The original allophycocyanin spectrum was regenerated after incubation in phosphate buffer...

Purification of membrane-bound galactosyltransferase from rat liver microsomal fractions

Fraser, Ian H.; Mookerjea, Sailen
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/06/1977 EN
Relevância na Pesquisa
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1. Rat liver microsomal preparations incubated in 1% Triton X-100 at 37°C for 1h released about 60% of the membrane-bound UDP-galactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. The supernatant galactosyltransferase which was solubilized but not purified by this treatment had a higher molecular weight than the serum enzyme as shown by Sephadex G-100 column chromatography. 2. The galactosyltransferase present in the high-speed supernatant was purified 680-fold by an affinity-column-chromatographic technique by using a column of activated Sepharose 4B coupled with α-lactalbumin. The galactosyltransferase ran as a single band on polyacrylamide gels and contained no sialyltransferase, N-acetylglucosaminyltransferase or UDP-galactose pyrophosphatase activities. 3. The purified membrane enzyme had properties similar to serum galactosyltransferase. It had an absolute requirement for Mn2+ that could not be replaced by Ca2+, Mg2+, Zn2+ or Co2+, and was active over a wide pH range (6–8) with a pH optimum of 6.5. The apparent Km for UDP-galactose was 10.8μm. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose. 4. The molecular weight of the membrane enzyme was 65000–70000...

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats.

Ragnotti, G; Aletti, M G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1975 EN
Relevância na Pesquisa
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1. The effect of phenobarbitone on the rate of protein synthesis and on the sedimentation patterns of various liver subcellular fractions containing ribosomes was studied in rats. 2. Phenobarbitone treatment increased the incorporation of [114C]leucine into protein by all preparations, provided they had not been subjected to preliminary treatment with Sephadex G-25. The phenobarbitone-induced effect on incorporation was associated with a gain in liver weight and a higher degree of polyribosomal aggregation. 3. Preparations that were treated with Sephadex G-25 incorporated more radioactivity into protein, but did not show the response to phenobarbitone treatment. 4. When the influence of starvation and phenobarbitone was studied separately on membrane-bound and membrane-free polyribosomes, it was shown that whereas both classes of polyribosomes were affected by starvation, apparently only the former class was susceptible to phenobarbitone stimulation of protein synthesis. 5. The decreased capacity for protein synthesis of polyribosomes from starved rats was independent of their association with the membranes of the endoplasmic reticulum, but resulted from polyribosomal disaggregation, from an intrinsic defect of the polyribosomes themselves and from changes in composition of the cell cap. 6. The results are discussed in relation to the problem of the control of protein biosynthesis and of the functional separation of membrane-bound and membrane-free polyribosomes.

Quantitative interpretation of concentration-dependent migration in gel chromatography of reversibly polymerizing solutes.

Baghurst, P A; Nichol, L W; Ogston, A G; Winzor, D J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1975 EN
Relevância na Pesquisa
16.55107%
A theoretical expression is derived for concentration dependence of elution volume in the gel chromatography of a non-interacting solute. Experimental results for bovine serum albumin on Sephadex G-100 are shown to be in good agreement with the predicted gel-chromatographic behaviour. The theoretical treatment of concentration dependence is extended to include a solute undergoing rapid reversible polymerization (nA in equilibrium C). Computer simulation of gel-chromatographic data for monomer-dimer systems on Sephadex G-100 is used to illustrate the deficiencies of earlier empirical approaches, and also the potential of the present treatment, of allowing for non-chemical concentration dependence in gel chromatography of polymerizing solutes.

Amino acid activation in mammalian brain. Purification and characterization of tryptophan-activating enzyme from buffalo brain

Liu, C.-C.; Chung, C.-H.; Lee, M.-L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1973 EN
Relevância na Pesquisa
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l-Tryptophan-activating enzyme [l-tryptophan–tRNA ligase (AMP), EC 6.1.1.2] of water-buffalo brain was purified to near homogeneity by heat and pH treatments, ammonium sulphate fractionation, column chromatography on DEAE-cellulose, hydroxyapatite and Amberlite CG-50, and gel filtration on Sephadex G-200. The purified enzyme catalyses tryptophanyl-tRNA formation with yeast tRNA, but not with Escherichia coli tRNA. The enzyme exhibits multiple peaks of activity in Sephadex gel filtration with molecular weights corresponding to 155000, 105000 and 50000. However, only one peak of activity with molecular weight of 155000 can be detected when the enzyme is subjected to gel filtration at high concentration. Disc gel electrophoresis in the presence of sodium dodecyl sulphate reveals a single band with molecular weight of 55000. The activity of the enzyme is concentration dependent. Different Km and Vmax. values are obtained at different enzyme concentrations. These data suggest that this enzyme may exist in different quaternary structures, each with its own kinetic constants. The enzyme activity is inhibited by p-chloromercuribenzoate, and is not protected by the presence of the substrates, l-tryptophan, Mg2+, ATP, in any combination.

The degradation of l-histidine in the rat. The formation of imidazolylpyruvate, imidazolyl-lactate and imidazolylpropionate

Emes, Alan V.; Hassall, Harold
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1973 EN
Relevância na Pesquisa
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1. Soluble and mitochondrial forms of histidine–pyruvate aminotransferase were separated from rat liver preparations by chromatography on DEAE-cellulose. 2. These enzymes were characterized with respect to substrate specificity, substrate affinity, pH optimum, stability and molecular weight by chromatography on Sephadex G-200. 3. Each enzyme has a relatively broad specificity showing significant activity towards l-phenylalanine and l-tyrosine and catalysing transamination with a number of monocarboxylic 2-oxo acids. 2-Oxoglutarate is not a substrate for either enzyme. 4. The molecular weights of the two enzymes, by chromatography on Sephadex G-200, are in the range 130000–150000. 5. The formation in vitro of imidazolyl-lactate from imidazolylpyruvate and NADH was demonstrated by using liver preparations. 6. From a study of imidazolyl-lactate–NAD+ oxidoreductase activity after electrophoresis of liver preparations on polyacrylamide gel, and from an examination of the activity of l-lactate–NAD+ oxidoreductase (EC 1.1.1.27) towards imidazolylpyruvate, it is concluded that this latter enzyme is responsible for the formation of imidazolyl-lactate in the liver. 7. Preparations of bacteria obtained from rat faeces form imidazolylpropionate from l-histidine and urocanate without further subculture. The amount of imidazolylpropionate formed is increased under anaerobic conditions and more so in an atmosphere of H2. It is suggested that the gut flora of the rat contribute largely...

The response of the small intestine to vitamin D. Isolation and properties of chick intestinal polyribosomes

Emtage, J. Spencer; Lawson, D. Eric M.; Kodicek, Egon
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1974 EN
Relevância na Pesquisa
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Undegraded polyribosome preparations may be obtained from chick intestinal mucosa if ribonuclease activity is strictly controlled. This is best achieved by homogenization of the mucosa directly in rat liver cell-sap. 2. The extent of amino acid incorporation by chick intestinal polyribosomes is greatly influenced by the source of the cell-sap. Sephadex-treated intestinal cell-sap caused impaired incorporation and release of completed polypeptide chains, whereas Sephadex-treated rat liver cell-sap promoted the polymerization of up to 90 amino acids per ribosome. Under optimum conditions 30–35% of the nascent polypeptide chains are completed and released. 3. The preparation of an antiserum against the calcium-binding protein formed in response to vitamin D is described. It is shown that the antiserum is highly specific for calcium-binding protein. 4. This antiserum was used to investigate the ability of chick intestinal polyribosomes to synthesize calciumbinding protein. Only polyribosomes from chicks receiving vitamin D have the ability to synthesize calcium-binding protein. Moreover, the product formed in vitro has the same electrophoretic mobility as calcium-binding protein synthesized in vivo. 5. It is concluded that one of the main functions of vitamin D in the small intestine is to induce the synthesis de novo of calcium-binding protein.

Alkaline phosphatase from pig kidney. Method of purification and molecular properties

Wachsmuth, Ernst D.; Hiwada, Kunio
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1974 EN
Relevância na Pesquisa
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Alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes was solubilized from membrane precipitates by butan-1-ol at a critical pH of 7.0. The 12000-fold purification procedure included (NH4)2SO4 precipitation, DEAE-and TEAE-cellulose chromatography, Sephadex G-200 gel filtration and neuraminidase digestion followed by DEAE-cellulose chromatography. The purified protein contained 20% (w/w) carbohydrate and had mol.wt. 150000–156000 as estimated by Sephadex filtration and ultracentrifuge analysis. It was a tetrameric glycoprotein consisting of identical subunits, and it had a molecular activity at 25°C of 2600s−1 per tetramer. Its concentration in kidney was estimated to be 8.5–8.8mg/kg.

Isolation of oligomers of 5,6-dihydroxyindole-2-carboxylic acid from the eye of the catfish

Ito, Shosuke; Nicol, J. A. Colin
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1974 EN
Relevância na Pesquisa
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The reflecting material of the tapetum lucidum of the sea catfish (Arius felis) was chromatographed on Sephadex LH-20 in methanol–dimethyl sulphoxide–formic acid. Two components were present: one, showing an absorption maximum at 330nm, was tapetal pigment; the other, at 257nm, was an associated nucleoside. The tapetal pigment was extracted in methanol–HCl and isolated by adsorption chromatography on Sephadex LH-20. It yielded a methoxy methyl ester on treatment with diazomethane, and permanganate oxidation gave pyrrole-2,3,5-tricarboxylic acid. From the information provided by u.v. and i.r. spectra of the pigment and its methoxy methyl ester, from elemental analyses and from the oxidation products, we suggest that the tapetal pigment is derived from oxidative coupling of 5,6-dihydroxyindole-2-carboxylic acid. A molecular-weight determination and chromatography of the methoxy methyl ester indicate that the pigment is a mixture of oligomers, among which the tetramers probably predominate. We consider that the monomers are joined mainly by C-C linkages at positions 4 and 7. A synthetic pigment having spectral properties nearly identical with those of the natural pigment was prepared by enzymic oxidation of 5,6-dihydroxyindole-2-carboxylic acid with mushroom tyrosinase. The identity of the tapetal pigment with the synthetic pigment was further confirmed by comparing u.v. and i.r. spectra of their methoxy methyl esters. Formation of the tapetal pigment from tyrosine and relationships of the tapetal pigment to melanin are discussed.

Evaluation of equilibrium constants by affinity chromatography

Nichol, Lawrence W.; Ogston, Alexander G.; Winzor, Donald J.; Sawyer, William H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1974 EN
Relevância na Pesquisa
16.55107%
Theoretical expressions are derived for affinity chromatography of systems comprising an acceptor A with one binding site for attachment to a functional group X on the column matrix and one site for interaction with a small ligand B that specifically affects its elution. From a general relationship covering all possible interactions between A, B and X simpler expressions are derived for affinity systems in which only two equilibria operate. Methods are suggested whereby these simpler systems may be characterized in terms of the two pertinent equilibrium constants and the concentration of matrix-bound constituent. The means by which the theory may be adapted to affinity chromatography of acceptors with multiple binding sites for ligand is also illustrated. Results of partition experiments on the Sephadex G-100–lysozyme–d-glucose system in acetate–chloride buffer (I=0.17m), pH5.4, are used to demonstrate the feasibility of evaluating quantitatively affinity-chromatography interactions. Values of 30m−1 and 1.2×106m−1 are obtained for the equilibrium constants for the reactions of lysozyme with glucose and Sephadex respectively, there being only an occasional binding site in the polysaccharide matrix (approximately 1 in 105 glucose residues). In a second experimental study the phytohaemagglutinin from Ricinus communis is subjected to frontal chromatography on Sepharose 4B in the presence of different concentrations of d-galactose...