RESUMEN
The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis emphasize the necessity to find new antitubercular targets and drugs. The antigen 85 (Ag85) complex from Mycobacterium tuberculosis plays important roles in the biosynthesis of major components of the mycobacterial cell envelope. For this reason, Ag85 has emerged as an attractive drug target. Recently, ebselen was identified as an effective inhibitor of the Ag85 complex through covalent modification of a cysteine residue proximal to the Ag85 active site and is therefore a covalent, allosteric inhibitor. To expand the understanding of this process, we have solved the x-ray crystal structures of Ag85C covalently modified with ebselen and other thiol-reactive compounds, p-chloromercuribenzoic acid and iodoacetamide, as well as the structure of a cysteine to glycine mutant. All four structures confirm that chemical modification or mutation at this particular cysteine residue leads to the disruption of the active site hydrogen-bonded network essential for Ag85 catalysis. We also describe x-ray crystal structures of Ag85C single mutants within the catalytic triad and show that a mutation of any one of these three residues promotes the same conformational change observed in the cysteine-modified forms. These results provide evidence for active site dynamics that may afford new strategies for the development of selective and potent Ag85 inhibitors.
Asunto(s)
Aciltransferasas/química , Antígenos Bacterianos/química , Cisteína/química , Inhibidores Enzimáticos/química , Aciltransferasas/genética , Aciltransferasas/metabolismo , Regulación Alostérica , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Azoles/química , Azoles/farmacología , Biocatálisis/efectos de los fármacos , Dominio Catalítico , Cloromercuribenzoatos/química , Cloromercuribenzoatos/farmacología , Cristalografía por Rayos X , Cisteína/genética , Cisteína/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Enlace de Hidrógeno/efectos de los fármacos , Yodoacetamida/química , Yodoacetamida/farmacología , Isoindoles , Modelos Moleculares , Estructura Molecular , Mutación , Compuestos de Organoselenio/química , Compuestos de Organoselenio/farmacología , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
The activities of specific transport systems were determined before and after large portions of the surface membrane had been interiorized by phagocytosis of inert particles. In five separate transport systems in rabbit polymorphonuclear leukocytes (adenosine and two adenine transport systems) and alveolar macrophages (adenosine and lysine transport systems), the rate of transport was unaffected even after an estimated 35-50% of the membrane had been internalized. Studies of the kinetics of lysine and adenosine transport, exchange diffusion of lysine transport in alveolar macrophages, and the specificities of adenine transport in polymorphonuclear leukocytes indicate that the nature of the membrane transport systems is not altered by phagocytosis. Therefore the constancy of transport indicates that the number of carriers remains the same before and after phagocytosis. It was also shown that this constancy of transport did not depend on the introduction into the surface of new transport sites during phagocytosis. Therefore transport sites are preserved on the surface during the internalization of membrane which accompanies phagocytosis. The results are best explained by the concept that the membrane is mosaic in character with geographically separate transport and phagocytic sites.
Asunto(s)
Membrana Celular/metabolismo , Fagocitosis , Adenina/metabolismo , Adenosina/metabolismo , Animales , Transporte Biológico , Isótopos de Carbono , Cloromercuribenzoatos/metabolismo , Difusión , Femenino , Cinética , Lisina/metabolismo , Macrófagos , Masculino , Isótopos de Mercurio , Métodos , Neutrófilos/metabolismo , Peritoneo , Conejos , TritioRESUMEN
The ultrastructural localization of D-amino acid oxidase (DAO) was studied cytochemically by detecting sites of hydrogen peroxide production in human polymorphonuclear leukocytes (PMNs). Reaction product, which forms when cerous ions react with H2O2 to form an electron-dense precipitate, was demonstrated on the cell surface and within the phagosomes of phagocytically stimulated cells when D-amino acids were provided as substrate. Resting cells showed only slight activity. The competitive inhibitor D,L-2-hydroxybutyrate greatly reduced the D-amino acid-stimulated reaction while KCN did not. The cell surface reaction was abolished by nonpenetrating inhibitors of enzyme activity while that within the phagosome was not eliminated. Dense accumulations of reaction product were formed in cells which phagocytosed Staphylococcus aureus in the absence of exogenous substrate. No reaction product formed with Proteus vulgaris while an intermediate amount formed when Escherichia coli were phagocytosed. Variation in the amount of reaction product with the different bacteria correlated with the levels of D-amino acids in the bacterial cell walls which are available for the DAO of PMNs. An alternative approach utilizing ferricyanide as an electron acceptor was also used. This technique verified the results obtained with the cerium reaction, i.e., the DAO is located in the cell surface and is internalized during phagocytosis and is capable of H2O2 production within the phagosome. The present finding that DAO is localized on the cell surface further supports the concept that the plasma membrane is involved in peroxide formation in PMNs.
Asunto(s)
D-Aminoácido Oxidasa/análisis , Neutrófilos/enzimología , Membrana Celular/enzimología , Cloromercuribenzoatos/farmacología , Cianuros/farmacología , D-Aminoácido Oxidasa/metabolismo , Ferricianuros/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Hidroxibutiratos/farmacología , FagocitosisRESUMEN
A biochemical and cytochemical study has been made of the distribution of ATPase in mature and differentiating phloem cells of Nicotiana tabacum and of the substrate specificity and effects of fixation on enzyme activity. Homogenates of unfixed leaf midveins and midveins fixed in formaldehyde-glutaraldehyde were assayed for enzyme activity by determining the amount of P(i), liberated per milligram of protein from various substrates in a 30 min period at pH 7.2. In fresh homogenates, hydrolysis of ATP was not significantly different from that of ITP, CTP, and UTP. Hydrolysis of GTP was slightly higher than that of ATP. ATP hydrolysis by fresh homogenates was 17% more extensive than that of ADP, 76% more extensive than that of 5'-AMP, and was inhibited by fluoride and p-chloromercuribenzoate (PCMB). There was little or no hydrolysis of the competitive inhibitors 2'- and 3'-AMP nor with the alternate substrates p-nitrophenylphosphate (PNP) or beta-glycerophosphate (beta-GP). In homogenates of material fixed in formaldehyde-glutaraldehyde for 1(1/4) h, ATPase activity was 13% preserved. Hydrolysis of ATP by fixed homogenates was not significantly different from that of ADP, 5'-AMP, ITP, CTP, and GTP. Hydrolysis of UTP was lower. Fluoride and PCMB inhibited fixed ATPase activity. The results of cytochemical localization experiments using a lead phosphate precipitation technique were in agreement with the biochemical results. Similar localization patterns were obtained with the nucleoside triphosphates ATP, CTP, GTP, ITP, and UTP. Activity was also localized with ADP and 5'-AMP but not with the competitive inhibitors 2'- and 3'-AMP, nor with PNP or beta-GP. Little or no reaction product was deposited in other controls incubated without substrate or with substrate plus fluoride, PCMB, or N-ethylmaleimide. ATPase activity was demonstrated chiefly at the plasma membrane of mature and differentiating phloem cells and was associated with the P-protein of mature sieve elements. It is suggested that the phloem transport system derives its energy from the demonstrated nucleoside triphosphatase activity.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Plantas/enzimología , Adenosina Monofosfato/farmacología , Adenosina Trifosfatasas/antagonistas & inhibidores , Unión Competitiva , Diferenciación Celular , Membrana Celular/enzimología , Cloromercuribenzoatos/farmacología , Etilmaleimida/farmacología , Fluoruros/farmacología , Formaldehído , Glutaral , Histocitoquímica , Técnicas Histológicas , Cinética , Microscopía Electrónica , Células Vegetales , Plantas/efectos de los fármacos , Plantas Tóxicas , Nicotiana/enzimologíaRESUMEN
The initial rate of thymidine-(3)H incorporation into the acid-soluble pool by cultured Novikoff rat hepatoma cells was investigated as a function of the thymidine concentration in the medium. Below, but not above 2 microM, thymidine incorporation followed normal Michaelis-Menten kinetics at 22 degrees , 27 degrees , 32 degrees , and 37 degrees C with an apparent K(m) of 0.5 microM, and the V(max) values increased with an average Q(10) of 1.8 with an increase in temperature. The intracellular acid-soluble (3)H was associated solely with thymine nucleotides (mainly deoxythymidine triphosphate [dTTP]). Between 2 and 200 microM, on the other hand, the initial rate of thymidine incorporation increased linearly with an increase in thymidine concentration in the medium and was about the same at all four temperatures. Pretreatment of the cells with 40 or 100 microMp-chloromercuribenzoate for 15 min or heat-shock (49.5 degrees C, 5 min) markedly reduced the saturable component of uptake without affecting the unsaturable component or the phosphorylation of thymidine. The effect of p-chloromercuribenzoate was readily reversed by incubating the cells in the presence of dithiothreitol. Persantin and uridine competitively inhibited thymidine incorporation into the acid-soluble pool without inhibiting thymidine phosphorylation. At concentrations below 2 microM, thymidine incorporation into DNA also followed normal Michaelis-Menten kinetics and was inhibited in an apparently competitive manner by Persantin and uridine. The apparent K(m) and K(i) values were about the same as those for thymidine incorporation into the nucleotide pool. The over-all results indicate that uptake is the rate-limiting step in the incorporation of thymidine into the nucleotide pool as well as into DNA. The cells possess an excess of thymidine kinase, and thymidine is phosphorylated as rapidly as it enters the cells and is thereby trapped. At low concentrations, thymidine is taken up mainly by a transport reaction, whereas at concentrations above 2 microM simple diffusion becomes the principal mode of uptake. Evidence is presented that indicates that uridine and thymidine are transported by different systems. Upon inhibition of DNA synthesis, net thymidine incorporation into the acid-soluble pool ceased rapidly. Results from pulse-chase experiments indicate that a rapid turnover of dTTP to thymidine may be involved in limiting the level of thymine nucleotides in the cell.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , ADN de Neoplasias/biosíntesis , Neoplasias Hepáticas/metabolismo , Timidina/metabolismo , Animales , Transporte Biológico , Línea Celular , Sistema Libre de Células , Cloromercuribenzoatos/farmacología , Cromatografía en Papel , Difusión , Dipiridamol/farmacología , Ditiotreitol/farmacología , Cinética , Neoplasias Experimentales/metabolismo , Ratas , Temperatura , Timidina/administración & dosificación , Timidina Quinasa/metabolismo , Nucleótidos de Timina/metabolismo , Tritio , Uridina/metabolismo , Uridina/farmacologíaRESUMEN
Postnuclear supernates from homogenates of purified neutrophil polymorphonuclear leukocytes (PMNs) from human blood were fractionated by zonal sedimentation and isopycnic equilibration in sucrose gradients. The fractions were characterized biochemically by measuring protein content and the activities of eight enzymes. Selected fractions were further analyzed by electron microscopy. In both centrifugation systems, azurophil and specific granules could be resolved almost completely. Azurophil granules sediment three to four times faster than the specifics and have an average density of 1.23. They contain all the peroxidase of the cells, large portions of four lysosomal hydrolases, and about half of the total lysozyme, and therefore appear to be, in biochemical terms, very similar to the azurophil granules of rabbit PMNs. The specific granules, which have an average density of 1.19, contain the remaining half of the lysozyme but appear to be free of the other components of the azurophil granules, and of alkaline phosphatase. Isopycnic equilibration disclosed a minor lysosomal population, which strongly overlaps the specific granules, and made possible the identification of a membrane-fraction which is characterized by the presence of the thiol-sensitive acid 4-nitrophenyl phosphatase and of alkaline phosphatase.
Asunto(s)
Gránulos Citoplasmáticos , Neutrófilos/citología , Fosfatasa Ácida/metabolismo , Fosfatasa Alcalina/metabolismo , Proteínas Sanguíneas/análisis , Fraccionamiento Celular , Membrana Celular/enzimología , Centrifugación por Gradiente de Densidad , Centrifugación Zonal , Cloromercuribenzoatos/farmacología , Gránulos Citoplasmáticos/análisis , Gránulos Citoplasmáticos/enzimología , Etilmaleimida/farmacología , Fluoruros/farmacología , Glucuronidasa/metabolismo , Glicósido Hidrolasas/metabolismo , Hexosaminidasas/metabolismo , Humanos , Lisosomas/enzimología , Manosa , Microscopía Electrónica , Muramidasa/metabolismo , Neutrófilos/análisis , Neutrófilos/enzimología , Peroxidasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Tensoactivos/farmacología , Tartratos/farmacologíaRESUMEN
An enzyme having esterase activity accumulates in the culture media of mouse peritoneal mononuclear leukocytes during differentiation of the cells into macrophages. It has a pH optimum of 6.5 and shows aryl esterase characteristics. The esterase differs from another macrophage hydrolase, acid phosphatase, in its mainly extracellular distribution.
Asunto(s)
Diferenciación Celular , Esterasas/metabolismo , Macrófagos , Monocitos/enzimología , Fosfatasa Ácida/metabolismo , Animales , Cloromercuribenzoatos/farmacología , Medios de Cultivo , Técnicas de Cultivo , Esterasas/antagonistas & inhibidores , Concentración de Iones de Hidrógeno , Ratones , Monocitos/citología , NaftalenosRESUMEN
The cellular site of binding of dexamethasone by specific glucocorticoid receptors in cultured hepatoma cells was investigated with the use of certain mercurials. p-Chloromercuribenzene sulfonate and p-chloromercuribenzoate inhibit the binding of steroid by receptors in cell-free extracts, but they allow the steroid-receptor complex to form in whole cells. In contrast, HgCl(2) inhibits binding both in extracts and cells. Since both organic mercury compounds, unlike HgCl(2), do not readily enter intact cells, it appears that the specific steroid binding occurs inside the cell rather than at the cell membrane.
Asunto(s)
Sitios de Unión , Dexametasona/metabolismo , Receptores de Droga , Animales , Autorradiografía , Sitios de Unión/efectos de los fármacos , Carcinoma Hepatocelular , Sistema Libre de Células , Células Cultivadas , Cloruros/farmacología , Cloromercuribenzoatos/farmacología , Neoplasias Hepáticas , Mercurio/farmacología , Ratas , Ácidos Sulfónicos/farmacología , TritioRESUMEN
A protein with characteristics similar to actomyosin has been isolated from whole brain of rat and cat. It is soluble in 0.6 molar potassium chloride and insoluble in 0.1 molar potassium chloride. It superprecipitates with magnesium ions and adenosine triphosphate. It has adenosine triphosphatase activity stimulated by either magnesium or calcium ions. Both superprecipitation and adenosine triphosphatase activity are inhibited by p-chloromercuribenzoate and Mersalyl but not by ouabain.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Química Encefálica , Proteínas Musculares , Proteínas del Tejido Nervioso/análisis , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Calcio/farmacología , Gatos , Precipitación Química , Cloromercuribenzoatos/farmacología , Depresión Química , Magnesio/farmacología , Compuestos Organomercuriales/farmacología , Cloruro de Potasio/farmacología , Ratas , Estimulación QuímicaRESUMEN
An unstable hemoglobin variant termed Hb Louisville, was found in four members of a Caucasian family, who were suffering from a mild hemolytic anemia. The variant showed a decreased stability upon warming at 65 degrees C and an increased tendency to dissociate in the presence of sulfhydryl group-blocking agents. The structural abnormality was identified as a replacement of phenylalanyl residue in position 42 (CD1) by a leucyl residue. Substitution of this phenylalanyl residue, which participates in the contact with heme, by a nonpolar leucyl residue has apparently less severe consequences than a replacement of the same residue by a polar seryl residue as in Hb Hammersmith. Oxygen equilibrium studies of total hemolysate from one Hb Louisville heterozygote indicated a decreased oxygen affinity, a marked decrease in heme-heme interaction, and a normal Bohr effect. Studies with isolated Hb Louisville were not made because it was not possible to separate the variant from normal Hb A.
Asunto(s)
Anemia Hemolítica/sangre , Hemoglobinas Anormales/análisis , Secuencia de Aminoácidos , Aminoácidos/análisis , Cloromercuribenzoatos , Cromatografía por Intercambio Iónico , Recuento de Eritrocitos , Cuerpos de Heinz , Hematócrito , Humanos , Hidantoínas , Hidroximercuribenzoatos , Oxígeno/sangre , Linaje , Péptidos/análisis , EspectrofotometríaRESUMEN
Transport of D-glucose across human erythrocyte membranes occurs via a facilitated diffusion process which demonstrates influx-efflux asymmetry. The mechanism of the asymmetry has been studied by estimating unidirectional fluxes in the presence or absence of trans equilibrium hexose. In the absence of transhexose, the half-saturation constant for efflux at 15 degrees C was approximately 10 mM as compared with 27 mM for influx; the corresponding values for maximal transfer rates (mumol/min per ml cell H(2)O) were approximately 51 vs. 18. The estimation of kinetic parameters, including the constant F(s), which is the ratio of maximal transfer rate/half-saturation constant, indicates a unique effect of intracellular hexose on the transfer system. Further evidence to support this conclusion was obtained by studying the effects of noncompetitive inhibitors on efflux vs. influx. N-ethylmaleimide, p-chloromercuribenzenesulfonate, and dichloroallyldiethylstilbestrol all inhibited efflux much more than influx. Glucose rendered the transport system more reactive to N-ethylmaleimide as assayed by efflux, whereas influx was much less affected. The results support the hypothesis that the transport system exists in two states. Transition from one state to the other is dependent on the presence of intracellular hexose.
Asunto(s)
Eritrocitos/metabolismo , Glucosa/metabolismo , Transporte Biológico/efectos de los fármacos , Isótopos de Carbono , Membrana Celular/metabolismo , Cloromercuribenzoatos/farmacología , Dietilestilbestrol/farmacología , Difusión , Etilmaleimida/farmacología , Humanos , Técnicas In Vitro , Cinética , MatemáticaRESUMEN
Skin fibroblasts from two siblings with hydroxylysine-deficient collagen collagen (Ehlers-Danlos syndrome, type VI) contained normal levels of collagen prolyl hydroxylase activity but were markedly deficient in collagen lysyl hydroxylase activity. The deficiency was evident in all fractions of cell lysates, in low and high ionic strength buffers, and in detergent. Assays of mixtures of wild-type and mutant cell lysates indicated no activation of mutant enzyme by factors in wild-type cells or inhibition of normal enzyme by material in mutant cells. Wild type or mutant cells cultured with ascorbic acid (50 mug/ml of culture medium, added daily) contained approximately the same level of lysyl hydroxylase activity as cells cultured without ascorbate, but prolyl hydroxylase activity without ascorbate was depressed in both an average of 41%. The mutant lysyl hydroxylase was less stable at 37 degrees C than the wild type and did not form high molecular weight aggregates in low ionic strength buffers, as did the control enzyme. The activity of the mutant enzyme was maximally stimulated after dialysis against buffer solutions containing 10 mM dithiothreitol. When assayed in 100 muM dithiothreitol, the mutant enzyme exhibited a higher apparent Km for ascorbate (20 muM) than the wild type (4 muM). In 1.0 mM dithiothreitol the mutant enzyme's apparent Km for ascorbate was reduced to 5 muM. Wild type and mutant enzymes had the same apparent Km for alpha-keto-glutarate (20 muM). The properties of prolyl hydroxylase in wild type and mutant cells were identical: apparent Km's for ascorbate and alpha-ketoglutarate were 100 muM and 20 muM, respectively. If mutant enzyme protein with altered kinetic properties is the only enzyme functioning to hydroxylate lysyl residues in collagen, the variations in hydroxylysine content observed in collagen from different tissues in the subjects reported here could be in part due to differences in cofactor concentrations and in rate and sequence of events in collagen synthesis in different tissues.
Asunto(s)
Colágeno/metabolismo , Síndrome de Ehlers-Danlos/enzimología , Oxigenasas de Función Mixta/deficiencia , Ácido Ascórbico/farmacología , Cloromercuribenzoatos/farmacología , Ditiotreitol/farmacología , Síndrome de Ehlers-Danlos/genética , Fibroblastos/enzimología , Humanos , Hidroxilisina/deficiencia , Técnicas In Vitro , Cinética , Mercaptoetanol/farmacología , Mutación , Procolágeno-Prolina Dioxigenasa/metabolismoRESUMEN
Lysates prepared from the amebocytes of Limulus polyphemus, the horseshoe crab, are gelled by endotoxin. Studies were carried out to characterize the components of amebocyte lysate and to examine the kinetics of their reaction with endotoxin. Analysis of amebocyte lysate using sucrose density gradients showed two peaks at 46% and 86% gradient volumes. G50 and G75 Sephadex column chromatography resulted in three protein peaks. One fraction contained a clottable protein, which had a molecular weight of approximately 27,000, and was heat stable. Another fraction contained a high molecular weight, heat labile material, which was activated by endotoxin and reacted with the clottable protein to form a gel. The rate of the reaction between endotoxin and amebocyte lysate was dependent upon the concentration of endotoxin and the concentration of the fraction containing the high molecular weight material. The activity of this fraction was inhibited by diisopropyl fluorophosphate, parachloromercuribenzoate, and para-chloromercuriphenyl sulfonate, suggesting that enzymatic activity depended upon serine hydroxyl and sulfhydryl groups. The reaction between endotoxin and the fractions of lysate was temperature and pH dependent. The data suggest that endotoxin activates an enzyme which then gels the clottable protein contained in amebocyte lysate.
Asunto(s)
Células Sanguíneas/análisis , Coagulación Sanguínea/efectos de los fármacos , Braquiuros/fisiología , Endotoxinas/farmacología , Animales , Centrifugación por Gradiente de Densidad , Cloromercuribenzoatos , Cromatografía en Gel , Escherichia coli , Concentración de Iones de Hidrógeno , Inmunodifusión , Inmunoelectroforesis , Isoflurofato , Cinética , Estimulación Química , Ácidos Sulfónicos , TemperaturaRESUMEN
The 490 quinone, a natural sulfhydryl-arylating reagent from the mushroom, Agaricus bisporus, markedly inhibited L1210 murine leukemia DNA polymerase alpha while resulting in little inhibition of DNA polymerase beta from this source. This quinone was more strongly inhibitory than p-chloromercuri-benzoate or N-ethylmaleimide and was less readily neutralized by sulfhydryl-containing molecules such as dithioerythritol. Preliminary experiments indicate that DNA protects DNA polymerase alpha from inhibition by the 490 quinone. The inhibition of DNA synthesis by quinone 490 may contribute significantly to the cytotoxicity of this compound and to the potential of gamma-L-glutaminyl-4-hydroxybenzene as an antitumor agent.
Asunto(s)
ADN Polimerasa II/antagonistas & inhibidores , ADN Polimerasa I/antagonistas & inhibidores , Leucemia L1210/tratamiento farmacológico , Inhibidores de la Síntesis del Ácido Nucleico , Reactivos de Sulfhidrilo/farmacología , Animales , Antineoplásicos , Sitios de Unión , Cloromercuribenzoatos/farmacología , ADN Polimerasa I/metabolismo , ADN Polimerasa II/metabolismo , ADN de Neoplasias/biosíntesis , Ditiotreitol/farmacología , Evaluación Preclínica de Medicamentos , Etilmaleimida/farmacología , Leucemia L1210/enzimología , Leucemia L1210/metabolismo , Ratones , Ratones Endogámicos DBA , Reactivos de Sulfhidrilo/metabolismo , Reactivos de Sulfhidrilo/uso terapéuticoRESUMEN
Glucocorticoid-binding macromolecules were examined in Morris hepatomas 7787, 5123tc, 3683F, 7800, and 3683 and the Reuber hepatoma H-35 with the use of the synthetic glucocorticoid, triamcinolone acetonide. The physical properties of the triamcinolone acetonide-binding macromolecules of the hepatomas indicate that they are specific glucocorticoid receptors. The equilibrium association constants (Ka), sedimentation coefficients, and sensitivity to sulfhydryl-blocking reagents were found to be similar when hepatoma receptors were compared with the known properties of the liver receptor. Probably the most convincing criterion that the triamcinolone acetonide-binding macromolecules from the hepatomas are specific receptors is that 50 to 90% of the receptor can be depleted from hepatoma cytosol by treating rats with cortisol. In adrenalectomized tumor-bearing rats, the receptor levels in hepatomas 7787, 7800, 5123tc, and H-35 are comparable to or greater than receptor levels of host liver. However, tryptophan oxygenase was not responsive to glucocorticoids in hepatoma 7800 although receptor levels were quite high, and there were no indications that the receptor molecules were altered. Hepatomas 3683 and 3686F have low levels of receptor which may be related to resistance of these tumors to glucocorticoid treatment.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Hígado/metabolismo , Receptores de Glucocorticoides , Receptores de Esteroides , Animales , Cloromercuribenzoatos/farmacología , Citosol/metabolismo , Inducción Enzimática/efectos de los fármacos , Hidrocortisona/farmacología , Neoplasias Experimentales/metabolismo , Ratas , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides/metabolismo , Triamcinolona Acetonida/metabolismo , Triamcinolona Acetonida/farmacología , Triptófano Oxigenasa/biosíntesis , Tirosina Transaminasa/biosíntesisRESUMEN
A number of affinity materials for the purification of dipeptidyl amino-peptidase (dipeptidylpeptide hydrolase, EC 3.4.14.1) have been prepared and tested. These material include peptide and amino acid inhibitors bound to agarose and reversible sulfhydryl adsorbents. Several of these materials are effective affinity adsorbents. The most useful material to be employed in combination with earlier purification methods is acetoxy-anilinomercuri-Sepharose. This removes proteins which are contaminants of some preparations and yields consistently high specific activities. Results with affinity and hydrophobic columns indicate that the primary interactions of the enzyme with amino acid and peptide derivative inhibitors are ionic in nature. This results is in agreement with the conclusions reached in studies of the interactions of these inhibitors with dipeptidyl aminopeptidase in solution.
Asunto(s)
Catepsinas/aislamiento & purificación , Cloromercuribenzoatos , Cromatografía de Afinidad , Cromatografía en Agarosa , Inhibidores de ProteasasRESUMEN
Hemoglobin (Hb II) of the blood clam Anadara broughtonii has a alpha 2 beta 2 sub-unit structure in athe oxy form with a sedimentation constant of 4.8 S. When deoxygenated, Hb II polymerizes with a major component, S20,w = 11.5 (above 150 microM in heme). Deoxy polymerization was not observed in a highly diluted protein below 20 microM (in heme). Gel filtration of Hb II in the deoxygenated state indicated that the major component has an apparent molecular weight of 195 000, which corresponds to a dodecamer. However, the sedimentation pattern and the elution profile of gel filtration showed the polymerization to be somewhat asymmetric. These results suggest that deoxy Hb II may polymerize with different polymerization states. We examined oxygen equilibria of Hb II in a range of 3--180 microM (in heme). Influences of the polymerization on its oxygen affinity and cooperativity were found to be very small. We have also found that the deoxy polymerization was completely prevented when all the sulfhydryl groups of the hemoglobin molecule were modified with p-chloromercuribenzoate.
Asunto(s)
Bivalvos/metabolismo , Hemoglobinas , Animales , Cloromercuribenzoatos , Ligandos , Peso Molecular , Oxígeno , Conformación ProteicaRESUMEN
1. Oligomeric Hb I and II of Anadara broughtonii, which are unusual with respect to having no Bohr effect, were shown to have a R-T transformation on ligand-binding on the basis of the following experimental results. (a) Iodoacetamide reacted preferentially with the oxyiforms of the hemoglobins. (b) CD spectra at the far-ultraviolet regions significantly changed on ligand-binding. (c) 1-Anilinonaphthalene-8-sulfonate bound to the hemoglobins with a preference for deoxyforms. From these results and previous findings [1], it is concluded that the absence of the Bohr effect in these hemoglobins is due to the lack of the Bohr proton ionizing groups in the molecules. 2. Hb I and II treated with p-chloromercuribenzoate, designated as PMB-I and PMB-II, showed greatly increased oxygen affinity and decreased cooperativity. CD spectra at the far-ultraviolet of the PMB-Hb in the oxygen liganded state gave similar patterns to those of native oxygenated Hb. However no changes in the spectra were observed on deoxygenation. These findings suggest that the PMB-I and PMB-II retain their native oxy conformation even in the deoxy states. The PMB-modification might prevent the initial ligand-induced conformational change within the protomers.
Asunto(s)
Bivalvos/metabolismo , Hemoglobinas , Regulación Alostérica , Animales , Cloromercuribenzoatos , Cisteína , Yodoacetamida , Ligandos , Oxígeno , Conformación ProteicaRESUMEN
Glycolic acid oxidase has been isolated from human liver and purified over 3000-fold to a specific activity of 123 U/mg protein by a 5-step procedure. The preparation gave a single protein band on polyacrylamide gel electrophoresis, required flavin mononucleotide for catalytic activity, had a pH optimum between 8.2-8.8 depending on the substrate, and had a molecular weight of 105 000. The enzyme has a broad specificity towards alpha-hydroxy acids. Glycolate (Km = 3.3 . 10(-4) M) was the most effective substrate. The enzyme was stable for several months when stored as an (NH4)2SO4 precipitate or in 15% glycerol. Since glycolate inhibits the oxidation of glyoxylate to oxalate by glycolic acid oxidase, it is suggested that glycolic acid oxidase contributes to the synthesis of oxalate in vivo when the glyoxylate concentration is high and the glycolate concentration is low.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Hígado/enzimología , Oxidorreductasas de Alcohol/aislamiento & purificación , Cloromercuribenzoatos/farmacología , Glicolatos , Humanos , Cinética , Peso Molecular , Especificidad por SustratoRESUMEN
Myosin from rabbit white skeletal muscle was treated with 10 mM EDTA in 150 mM phosphate buffer. After precipitation of myosin by dialysis against a 14-fold volume of water, EDTA-treated myosin, myosin before treatment and the supernatant from the treatment of myosin with EDTA were examined on sodium dodecyl sulphate-polyacrylamide gels by electrophoresis. It has been found that the quantity of LC2 light chains diminished after treatment with EDTA, and the supernatant contained the LC2 light chains. Treatment of myosin with EDTA in the presence of Mg2+ does not change the stoichiometry of the LC2 light chain and the supernatant is free from LC2 light chains. The treatment of myosin with p-chloromercuri-benzoate leads to dissociation of the same amount of LC2 light chains. It is suggested that divalent cations and thiol groups are engaged in the attachment of LC2 light chain to the myosin molecule.