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Thermal stability of ribosomal ribonucleic acid from baby hamster kidney cells

Martin, S. J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1966 EN
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1. RNA has been prepared from baby hamster kidney cells by extraction with a phenol–EDTA mixture and further purified by passing through a column of Sephadex G-25 that had been equilibrated with water. 2. Aging of the total RNA extracts at 4° or heating at 95° followed by rapid cooling caused a conversion of 28s RNA into material sedimenting in sucrose gradients at approx. 18s. 3. When heated RNA was re-extracted with phenol the sedimentation profile was not returned to that of the unheated RNA. 4. The 28s and 18s RNA fractions were collected separately from sucrose gradients by precipitation with 2vol. of ethanol and passed through a Sephadex G-25 column equilibrated with water. 5. Heat treatment of purified 28s RNA at 95° caused the sedimentation coefficient to increase to approx. 40s, whereas similar treatment of 18s RNA caused no significant increase. If the RNA was heated before the Sephadex G-25 treatment the sedimentation coefficient of the 28s and 18s RNA decreased to approx. 12s and 8s. 6. Heating mixtures of purified 28s and 18s RNA at 95° caused some aggregation of 18s material with the 28s fraction.

Preparation and properties of sialomucopolysaccharides obtained from rat brain

Brunngraber, Eric G.; Brown, Barbara D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1967 EN
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Sialomucopolysaccharides were released from the defatted protein residue by the proteolytic action of papain after extraction of rat whole brains with chloroform–methanol (2:1, v/v). Further purification is achieved by dialysis to remove low-molecular-weight fragments and by precipitation of nucleic acids and glucuronic acid-containing mucopolysaccharides by treatment with cetylpyridinium chloride. Gel filtration of the sialomucopolysaccharides through Sephadex G-200 removes the major portion of the impurities that absorb light in the ultraviolet region. The sialomucopolysaccharides were fractionated on DEAE-Sephadex to yield a population of sialomucopolysaccharides that show an increase in N-acetylneuraminic acid content and a decrease in fucose content as the concentration of chloride required to elute the individual components is increased. On gel filtration on Sephadex G-200, those sialomucopolysaccharide molecules rich in N-acetylneuraminic acid and poor in fucose appear to be larger molecules than those rich in fucose and poor in N-acetylneuraminic acid. A structure is proposed in which all sialomucopolysaccharide molecules are assumed to possess the same repeating unit consisting of hexosamine and hexose. The molecules differ from each other in the number of fucose and N-acetylneuraminic acid residues attached to the basic structure. Most of the hexosamine is present as glucosamine...

The immunological and physicochemical properties of mitogenic proteins derived from Phaseolus vulgaris

Yachnin, S.; Svenson, R. H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1972 EN
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A commercially available phytohaemagglutinin derived from Phaseolus vulgaris (PHAP) was subjected to CM Sephadex and Sephadex G-150 chromatography. Two major mitogenic fractions were isolated; one (L-PHAP), appeared homogeneous by polyacrylamide gel electrophoresis, and was virtually lacking in haemagglutinating activity, while the other (H-PHAP) was a potent haemagglutinin. H-PHAP was a complex material as shown by its behaviour on CM Sephadex chromatography. Electrophoretic analysis revealed three distinct bands of protein, all migrating cathodally to L-PHAP. With increasing cathodal mobility H-PHAP subfractions showed diminishing mitogenicity, increasing haemagglutinating potency, and the appearance of the ability to precipitate serum proteins non-specifically. The latter property, present in crude PHAP, was not displayed by L-PHAP. The mitogenic activity of all H-PHAP subfractions was potentiated by the presence of autologous red blood cells.

The mediation of tissue eosinophilia in hypersensitivity reactions. II. Separation of a delayed eosinophil chemotactic factor from macrophage chemotactic factors.

Hirashima, M; Honda, M; Hayashi, H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1976 EN
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In anaphylactic cutaneous lesions induced by DNP-ascaris extract in the guinea-pig, the time-course of delayed tissue eosinophilia was found to parallel that of the macrophage reaction, reaching its peak in 24 h. Macrophages could be differentiated from lymphocytes by the numerous lysosomal granules which stained for acid phosphatase. Extracts from such skin lesions contained a delayed eosinophil chemotactic factor and two different macrophage chemotactic factors. Most of the delayed eosinophil chemotactic factor was separated from the two macrophage chemotactic factors by gel filtration on Sephadex G-100 and Sephadex G-200 in that order. The eosinophil chemotactic factor after re-chromatography on Sephadex G-I99 showed no or little chemotactic activity for macrophages.

Histamine release from human leucocytes. A serum factor necessary for the induction of histamine release and desensitization by protein A.

Petersson, B A; Stålenheim, G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1977 EN
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The ability of human leucocytes to release histamine on protein A treatment is lost when the cells are washed repeatedly. It is, however, possible to restore the sensitivity to protein A treatment by incubating the leucocytes in serum. Treatment of the cells with purified IgG does not restore the activity. The material responsible for the resensitization is eluted both in the second and the third protein peak when serum is chromatographed on Sephadex G-200, indicating the possible existence of several active factors. Material with low immunoglobulin content, but with retained capacity to resensitize leucocytes to release histamine on protein A treatment, was obtained by repeated chromatography of peak III material on the Sephadex G-200 column. Furthermore, material from the second and third peaks from Sephadex G-200 deprived of their IgG by passage through a protein A Sepharose or a DEAE-cellulose column had the same capacity to resensitize the leucocytes as unseparated material. When serum was separated by Pevikon block electrophoresis, most of the activity was detected in the alpha and beta regions but only little in the gamma region. The serum fractionations indicate that neither IgG nor the other immunoglobulins are the factor(s) responsible for resensitizing the leucocytes to release histamine on protein A treatment. Beside being necessary for protein A-induced histamine release...

In vitro macrophage chemotactic generation from serum immunoglobulin G by neutrophil neutral seryl protease.

Ishida, M; Honda, M; Hayashi, H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1978 EN
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A neutral protease with a molecular weight of about 14,00 was separated at acid pH from rabbit neutrophils and then partially purified by elution on DEAE-Sephadex, CM-Sephadex and Sephadex G-75 in that order. This enzyme was inactivated by diisopropyl fluorophosphate (DFP), phenylmethyl sulphonylfluoride (PMSF), soybean trypsin inhibitor (SBTI), or elastatinal, suggesting a seryl protease resembling elastase, but it failed to digest elastin-orcein. The enzyme seemed different from histonase of rabbit neutrophils because of its haemoglobin (3HHb)-degrading ability and of inactivation by heparin. The protease generated in vitro macrophage chemotactic activity from guineapig serum IgG. This chemotactic factor had a molecular weight similar to that of IgG and its chemotacic generation was accompanied by release of dialysable peptide(s). No generation of macrophage chemotactic activity from IgG was induced in vitro by elastase from pig pancreas or by neutral thiol protease from rabbit neutrophils.

Cadmium-binding proteins of three marine molluscs and characterization of two cadmium-binding glycoproteins from the hepatopancreas of a whelk, Buccinum tenuissimum.

Dohi, Y; Kosaka, K; Ohba, K; Yoneyama, Y
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1986 EN
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The cadmium-binding proteins were shown to exist in the hepatopancreas of three molluscs, a whelk, Buccinum tenuissimum, a turbo, Batillus cornutus, and a squid, Todarodes pacificus. Cadmium was efficiently accumulated in nature to a mean concentration of 119, 33, and 50 micrograms/g wet tissue in the hepatopancreas of three species of molluscs, and 30%, 11%, and 43% of the element in each tissue of whelk, turbo, and squid was extracted to the soluble fraction, respectively. Separation of the soluble fraction by Sephadex G-75 in the presence of 2-mercaptoethanol revealed that cadmium was mainly bound to the protein fraction FII of molecular weight 10,000. Two cytoplasmic cadmium-binding glycoproteins from the hepatopancreas of Buccinum tenuissimum were purified to homogeneity by Sephadex G-75 gel filtration and double DEAE-Sephadex A-25 chromatographies in the presence of 2-mercaptoethanol. These two cadmium-binding glycoproteins, termed FIIA and FIIB, had molecular weights of 8000 and 13,000 and consisted of 52 and 94 amino acid residues, respectively. Three and two cysteine residues in FIIA and FIIB, respectively, were found and two more half-cystine were also detected in FIIB. The sugar contents of FIIA and FIIB were about 20.5% and 8.7% by weight...

Liver specific antigens: purification and characterization

Meyer Zum Büschenfelde, K. H.; Miescher, P. A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1972 EN
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Human liver has been homogenated and submitted to 150,000 g in order to investigate the supernatant for the presence of liver specific antigens. Gel-filtration on Sephadex G-100 revealed liver specific antigenicity in the first peak. Gel-filtration of the first peak on Sephadex G-200 revealed two peaks each exhibiting liver specific antigenicity. Immunochemical and physicochemical analysis made it possible to define the liver-specific antigen present in the first peak as a lipoprotein with the characteristics of a high molecular weight, low density protein. It is partly located in the membrane of liver cells. The liver specific protein of the second Sephadex G-200 peak has a molecular weight of about 190,000. It is located within the cytoplasm of hepatocytes.

THE SYNTHESIS AND TURNOVER OF RAT LIVER PEROXISOMES : I. Fractionation of Peroxisome Proteins

Leighton, Federico; Poole, Brian; Lazarow, Paul B.; De Duve, Christian
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/05/1969 EN
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Rat liver peroxisomes isolated by density gradient centrifugation were disrupted at pH 9, and subdivided into a soluble fraction containing 90% of their total proteins and virtually all of their catalase, D-amino acid oxidase, L-α-hydroxy acid oxidase and isocitrate dehydrogenase activities, and a core fraction containing urate oxidase and 10% of the total proteins. The soluble proteins were chromatographed on Sephadex G-200, diethylaminoethyl (DEAE)-cellulose, hydroxylapatite, and sulfoethyl (SE)-Sephadex. None of these methods provided complete separation of the protein components, but these could be distributed into peaks in which the specific activities of different enzymes were substantially increased. Catalase, D-amino acid oxidase, and L-α-hydroxy acid oxidase contribute a maximum of 16, 2, and 4%, respectively, of the protein of the peroxisome. The contribution of isocitrate dehydrogenase could be as much as 25%, but is probably much less. After dissolution of the cores at pH 11 , no separation between their urate oxidase activity and their protein was achieved by Sephadex G-200 chromatography.

RABBIT MACROPHAGE INTERFERONS : II. SOME PHYSICOCHEMICAL PROPERTIES AND ESTIMATIONS OF MOLECULAR WEIGHTS

Smith, Thomas J.; Wagner, Robert R.
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 31/03/1967 EN
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Antiviral factors present in cultures of rabbit peritoneal macrophages or rabbit kidney (RK) cells infected with Newcastle disease virus (NDV) and those in cultures of uninfected macrophages all fulfilled the biological and physicochemical criteria for classification as interferons. Virus-induced macrophage and RK interferons were slightly more stable to heat or acid than "spontaneously produced" or endotoxin-induced macrophage interferon. Interferon activity in serum of NDV-infected rabbits was decidedly more labile than NDV-induced macrophage interferon. However, these differences in lability were too slight to serve as a useful basis for distinguishing one rabbit interferon from another. Rabbit interferons from various sources could be differentiated by filtration through Sephadex G-100 and their molecular weights estimated by comparison with elution profiles of a series of marker proteins of known molecular weight. Each of four different preparations of rabbit interferons was found to contain more than one molecular component. Elution peaks for three NDV-induced interferons were equivalent to the following molecular weights: RK ≃44,000–45,000 and > 134,000 (variable and < 1% when present); macrophage ≃37,000, 44,000–45...

PATHOGENESIS OF EXPERIMENTAL CHOLERA : PREPARATION AND ISOLATION OF CHOLERAGEN AND CHOLERAGENOID

Finkelstein, Richard A.; LoSpalluto, Joseph J.
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/07/1969 EN
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Choleragen, a diarrheagenic protein enterotoxin elaborated by Vibrio cholerae, has been isolated from the supernate of fermenter cultures by steps involving ammonium sulfate precipitation, DEAE cellulose, Sephadex G-75, and Agarose A-5m chromatography. The resulting product appears to be pure according to immunoelectrophoretic, disc electrophoretic, ultracentrifugal, and immunologic criteria. Sephadex gel filtration and membrane filtration studies suggest a molecular size of 61,000. The isolated product is highly active in inducing experimental cholera in infant and adult rabbit models. It also elicits, in small dosage, an increased vascular permeability in skin. These observations indicate that choleragenicity and increased vascular permeability are intimately associated phenomena and may be manifestations of the same basic mechanism. An additional, antigenically identical, protein has also been isolated by the same procedures. The latter substance, termed "choleragenoid", lacks the permeability effect and choleragenicity of the choleragen moiety. Its size (estimated from Sephadex gel filtration at 42,000) is smaller than that of choleragen and it also differs in charge. Choleragenoid may prove useful as a nontoxic immunogen to protect against pathologic effects of V. cholerae infection.

SUBSTRATES OF HAGEMAN FACTOR : I. Isolation and Characterization of Human Factor XI (PTA) and Inhibition of the Activated Enzyme by α1-Antitrypsin

Heck, Louis W.; Kaplan, Allen P.
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 30/11/1974 EN
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Unactivated partial thromboplastin antecedent (PTA) has been purified by sequential chromatography of plasma on quaternary aminoethyl Sephadex, sulphoprophyl Sephadex, Sephadex G-150, and passage over an anti-IgG immunoadsorbant. The preparation gave a single band after alkaline disc gel electrophoresis, sodium dodecyl sulfate (SDS) gel electrophoresis and isoelectric focusing in acrylamide gels and was found to have a mol wt of 175,000 by gel filtration, 163,000 by SDS gel electrophoresis, and an isoelectric point of 8.8–9.4 (peak 9.0–9.1). Pre-PTA was activated directly by activated Hageman factor or by Hageman factor prealbumin fragments. Its coagulant activity was inhibited by DFP, soybean trypsin inhibitor and trasylol but not by lima bean trypsin inhibitor or ovomucoid trypsin inhibitor indicating that activated PTA possesses the same inhibition profile utilizing these reagents as does plasma kallikrein. A major plasma inhibitor of activated PTA was found to be a 65,000 mol wt α-globulin which was isolated free of α1-chymotrypsin inhibitor, inter α-trypsin inhibitor, α2-macroglobulin, and the other known inhibitors of activated PTA, the activated first component of complement (C1 INH), and antithrombin III. Its physicochemical properties were identical to α1-antitrypsin...

Neuroprotective Activities of Enzymatically Hydrolyzed Peptides from Porcine Hide Gelatin

Wang, Shaoyun; Wang, Deng-Shun; Wang, Rui
Fonte: e-Century Publishing Corporation Publicador: e-Century Publishing Corporation
Tipo: Artigo de Revista Científica
Publicado em 10/08/2008 EN
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Central nervous system disorders, including cerebrovascular disease, neurodegenerative diseases and head trauma are the most common cause of severe disability in adults and share a number of pathophysiological features. The therapeutic strategy of neuroprotection has been well accepted as one of the promising approaches in treating such brain disorders, and searching for the effective neuroprotective agents is still an open-ended task for neurologists and neuro-pharmacologists. In this study, we report for the first time that the enzymatic hydrolysates from type-B porcine hide gelatin has potent neuroprotective activity against H2O2- or serum deprivation-induced injuries of cultured SH-SY5Y cells. The peptides used in this study were prepared from type-B porcine hide gelatin digested with pepsin and papain. The neuroprotective activity of the porcine hide gelatin hydrolysate (PHH) was evaluated using MTT reduction assay. From the pre-screening of PHH, we found that the whole porcine hide gelatin hydrolysate obtained from papain digestion (PHH-I) showed significant neuroprotective activities (P<0.05). After further separation of PPH-I through SP-Sephadex C-50 and Sephadex G-25, only the fraction with smaller molecular weight from Sephadex G-25 (PHH-Ic) demonstrated potent neuroprotective activities (P<0.01). The active fraction showed a molecular mass between 1...

4-Thiouridine induces dose-dependent reduction of oedema, leucocyte influx and tumour necrosis factor in lung inflammation

Evaldsson, C; Rydén, I; Rosén, A; Uppugunduri, S
Fonte: Blackwell Science Inc Publicador: Blackwell Science Inc
Tipo: Artigo de Revista Científica
Publicado em /02/2009 EN
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Recent reports demonstrate a role for nucleotides as inflammatory modulators. Uridine, for example, reduces oedema formation and leucocyte infiltration in a Sephadex-induced lung inflammation model. Tumour necrosis factor (TNF) concentration was also reduced. Previous in vivo observations indicated that 4-thiouridine might have similar effects on leucocyte infiltration and TNF release. The aim of this study was thus to investigate the effects of 4-thiouridine in greater detail. We used a Sephadex-induced acute lung inflammation model in Sprague–Dawley rats. The dextran beads were instilled intratracheally into the lungs, which were excised and examined after 24 h. Sephadex alone led to massive oedema formation and infiltration of macrophages, neutrophils and eosinophils. Microgranulomas with giant cell formations were clearly visible around the partially degraded beads. A significant increase in bronchoalveolar lavage fluid (BALF) content of TNF and leukotrienes was also seen. 4-Thiouridine co-administration affected all variables investigated in this model, i.e. oedema, microscopic and macroscopic appearance of lung tissue, total leucocyte and differential leucocyte counts in BALF, TNF and leukotrienes C4 (LTC4), LTD4and LTE4 in BALF...

RNA affinity tags for purification of RNAs and ribonucleoprotein complexes

Srisawat, Chatchawan; Engelke, David R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/2002 EN
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Intrinsic affinity tags are useful tools for the study of macromolecular targets. Although polypeptide affinity tags are routinely used in purification and detection of protein complexes, there has been a relative lack of powerful RNA affinity tags that can be embedded within RNA sequences. Here, the preparation and use of two RNA affinity tags against Sephadex or streptavidin are described. The two tags have different strengths that make them appropriate for slightly different uses. One is a high-affinity ligand for streptavidin that can be specifically eluted by competition with biotin under otherwise native binding conditions. The other tag binds selectively to Sephadex beads, and can be eluted by competition with the soluble dextran that composes Sephadex. When properly placed within another RNA molecule, the tags can be used to effect dramatic purification of RNA or ribonucleoprotein complexes from complex mixtures of cellular RNA.

Aislamiento e identificación de productos naturales bioactivos

Pérez Quintiana, María Alejandra.
Fonte: Universidade da Corunha Publicador: Universidade da Corunha
Tipo: Trabalho de Conclusão de Curso
SPA
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Traballo fin de grao (UDC.CIE). Química. Curso 2014/2015; [Resumen] En este trabajo de fin de grado se ha procurado adquirir los conocimientos necesarios para llevar a cabo el aislamiento, purificación y determinación estructural de compuestos naturales bioactivos. Para ello, se ha dividido en tres partes fundamentales que conlleva el trabajo de investigación en este campo de la química de productos naturales de origen marino. Parte I. En la primera parte de la memoria se estudia una fracción de la esponja Ircina strobilina empleando métodos cromatográficos. Para el aislamiento de los metabolitos presentes en dicha fracción se utiliza una columna de exclusión molecular Sephadex LH-20 y la posterior purificación de las fracciones obtenidas mediante HPLC. Parte II. En la segunda parte se realiza la caracterización completa de un macrociclo de origen marino PM120021 empleando espectroscopia de RMN mono y bidimensional para la elucidación de su estructura plana. Parte III. La tercera parte del trabajo se centra en el empleo de cálculos computacionales que permitieron determinar la estereoquímica relativa de dos centros quirales adyacentes los cuales poseían átomos de O y N. Este estudio se hace a partir de las constantes teóricas homo y heteronucleares realizando una comparación de las mismas con las constantes experimentales obtenidas previamente por otros miembros del grupo de investigación. Se realizan los cálculos computacionales empleando dos tipos de funcionales de densidad (DFT) lo que permite decidir cuál es más adecuado asumiendo un buen compromiso entre coste computacional y exactitud.; [Resumo] Neste traballo de fin de grao procurouse adquirir os coñecementos necesarios para levar a cabo o illamento...

Widespread IgE-mediated hypersensitivity in the Sudan to the 'green nimitti' midge, Cladotanytarsus lewisi (Diptera: Chironomidae) II. Identification of a major allergen.

Gad El Rab, M O; Thatcher, D R; Kay, A B
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1980 EN
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A major allergen has been identified in an aqueous extract of the 'green nimitti' midge, Cladotanytarsus lewisi (Diptera: Chironomidae). Following chromatography on Sephadex G-100 allergenic activity, as assessed by skin ('prick') testing, eluted as two closely related peaks (pools I and II) at about 50% bed volume. When these pools were applied separately to columns of CM-cellulose, activity in each eluted with 0 . 05 M NaCl. Isoelectric focusing of the unfractionated allergen gave a single peak of activity at pI 4 . 3. By SDS--PAGE, biological activity in the whole 'green nimitti' extract and the material eluting from both pools I and II of the Sephadex G-100 column migrated to the same positions and were associated with a molecular size of 15,000--20,000 daltons. Skin test reactivity of the unfractionated material and the Sephadex G-100 pool I and II eluates were all destroyed following incubation with trypsin, chymotrypsin, thermolysin and neuraminidase. These experiments indicate that a major allergen derived from the 'green nimitti' midge, a cause of widespread and severe immediate-type allergy in the Sudan, is an acidic glycoprotein of 15,000--20,000 molecular weight.

Expolygalacturonase from Suspension Cultures of Marchantia polymorpha1: Its Presence and Involvement in Pectic Polysaccharide Degradation

Konno, Haruyoshi; Yamasaki, Yoshiki; Katoh, Kenji
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1983 EN
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Polygalacturonase was isolated from cell suspension cultures of a thalloid liverwort, Marchantia polymorpha. The enzyme in the `buffer-soluble' protein fraction was dialyzed at pH 5.2 and further purified 91-fold by a combination of chromatographic techniques including CM-Sephadex, Sephacryl S-200, DEAE-Sephadex, and Sephadex G-200. The purified enzyme had an optimum activity in the pH range at 3.6 to 3.8 and molecular weight of 76,000 daltons, and its activity was not stimulated by cations. The enzyme was identified as an exohydrolase from viscometric data and chromatographic analysis of the reaction products.

Bacteriolytic enzymes from Staphylococcus aureus. Purification of an endo-β-N-acetylglucosaminidase

Wadström, T.; Hisatsune, K.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1970 EN
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On cultivation of Staphylococcus aureus in a complex liquid medium, bacteriolytic activity is found extracellularly. The maximal amount was found at the end of the exponential growth phase in batch culture, but in continuous culture run under similar conditions the yield was doubled. Isoelectric focusing of dialysed crude culture supernatants showed that the bacteriolytic activity of all four strains studied (M18, 524, Wood 46 and Duncan) was heterogeneous. The most alkaline peak of activity (isoelectric point 9.5±0.1) was assayed against Micrococcus lysodeikticus turbidimetrically. This bacteriolytic activity was purified more than 70-fold after continuous dialysis by adsorption on CM-Sephadex, precipitation with ethanol, heat purification, isoelectric focusing and Sephadex G-100 chromatography. The purified enzyme (isoelectric point 9.6±0.1) was found to give a single band on polyacrylamide-gel and cellulose acetate electrophoresis and was devoid of all 14 staphylococcal enzymes and toxins assayed for. The molecular weight is 70000±5000 as estimated by Sephadex G-100 and G-200 chromatography. The marked instability of the partially and highly purified enzyme was investigated. The mode of action and some properties of this enzyme are given in the following papers (Wadström & Hisatsune...

The relaxing protein system of striated muscle. Resolution of the troponin complex into inhibitory and calcium ion-sensitizing factors and their relationship to tropomyosin

Schaub, M. C.; Perry, S. V.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1969 EN
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1. A method involving isoelectric precipitation and chromatography on SE-Sephadex (sulphoethyl-Sephadex) is described for the preparation of the troponin complex free of tropomyosin from low-ionic-strength extracts of natural actomyosin and myofibrils. 2. Purified troponin complex required tropomyosin to inhibit the Mg2+-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin in the presence of ethanedioxybis(ethylamine)tetra-acetate. An upper limit of 35000 for the `molecular weight' of the troponin complex was derived from the amounts required to bring about 50% of the maximum inhibition of the Mg2+-stimulated adenosine triphosphatase activity of desensitized actomyosin of known concentration. 3. In the presence of dissociating reagents the troponin complex could be dissociated into inhibitory and Ca2+-sensitizing factors, which could be isolated separately on SE-Sephadex. The inhibitory factor inhibited the Mg2+-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin independently of the concentration of free Ca2+ in the medium. 4. The Ca2+-sensitizing factor changed its electrophoretic mobility on polyacrylamide gel in the presence of ethanedioxybis(ethylamine)tetra-acetate. It formed a complex with the inhibitory factor at low ionic strength and the original biological activity of the troponin complex could be restored on mixing the inhibitory factor with the Ca2+-sensitizing factor in the ratio of about 3:2. 5. Evidence is presented indicating that the ability of tropomyosin preparations to restore relaxing-protein-system activity to the troponin complex and their inhibitory effect on the Ca2+-stimulated adenosine triphosphatase activity of desensitized actomyosin are two properties of different stability to preparative procedures and tryptic digestion. This suggests that the relaxing protein system of muscle may contain another as yet uncharacterized component.