RESUMEN
1. The short-circuit current response of toad bladders to high concentrations of aldosterone (2 x 10(-7) M) has been investigated following saline exposure of toads. 2. Limited saline exposure appears to enhance the aldosterone-stimulated response. 3. Additional saline exposure results in a reduced aldosterone response. 4. Overnight pre-incubation of isolated bladders proved a better pretreatment condition for study of an aldosterone short-circuit current response. 5. Plasma aldosterone concentrations and studies of [3H]aldosterone binding in the bladders have failed to demonstrate significant effects of saline exposure on endogenous aldosterone concentrations.
Asunto(s)
Aldosterona/farmacología , Potenciales de la Membrana/efectos de los fármacos , Cloruro de Sodio/farmacología , Vejiga Urinaria/fisiología , Aldosterona/metabolismo , Animales , Bufo marinus/fisiología , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Técnicas In Vitro , Receptores de Glucocorticoides/metabolismo , Factores de Tiempo , Vejiga Urinaria/metabolismoRESUMEN
Kangaroo IGF-II has been purified from western grey kangaroo (Macropus fuliginosus) serum and characterised in a number of in vitro assays. In addition, the complete cDNA sequence of mature IGF-II has been obtained by reverse-transcription polymerase chain reaction. Comparison of the kangaroo IGF-II cDNA sequence with known IGF-II sequences from other species revealed that it is very similar to the human variant, [Ser29]-hIGF-II. Both the variant and kangaroo IGF-II contain an insert of nine nucleotides that encode the amino acids Leu-Pro-Gly at the junction of the B and C domains of the mature protein. The deduced kangaroo IGF-II protein sequence also contains three other amino acid changes that are not observed in human IGF-II. These amino acid differences share similarities with the changes described in many of the IGF-IIs reported for non-mammalian species. Characterisation of human IGF-II, kangaroo IGF-II, chicken IGF-II and [Ser29]-hIGF-II in a number of in vitro assays revealed that all four proteins are functionally very similar. No significant differences were observed in the ability of the IGF-IIs to bind to the bovine IGF-II/cation-independent mannose 6-phosphate receptor or to stimulate protein synthesis in rat L6 myoblasts. However, differences were observed in their abilities to bind to IGF-binding proteins (IGFBPs) present in human serum. Kangaroo, chicken and [Ser29]-hIGF-II had lower apparent affinities for human IGFBPs than did human IGF-II. Thus, it appears that the major circulating form of IGF-II in the kangaroo and a minor form of IGF-II found in human serum are structurally and functionally very similar. This suggests that the splice site that generates both the variant and major form of human IGF-II must have evolved after the divergence of marsupials from placental mammals.
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/aislamiento & purificación , Macropodidae/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Pollos/metabolismo , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Datos de Secuencia Molecular , Radioinmunoensayo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Relación Estructura-ActividadRESUMEN
Insulin-like growth factor-I (IGF-I) and IGF-II have been purified to homogeneity from kangaroo (Macropus fuliginosus) serum, thus this represents the first report of the purification, sequencing and characterisation of marsupial IGFs. N-Terminal protein sequencing reveals that there are six amino acid differences between kangaroo and human IGF-I. Kangaroo IGF-II has been partially sequenced and no differences were found between human and kangaroo IGF-II in the 53 residues identified. Thus the IGFs appear to be remarkably structurally conserved during mammalian radiation. In addition, in vitro characterisation of kangaroo IGF-I demonstrated that the functional properties of human, kangaroo and chicken IGF-I are very similar. In an assay measuring the ability of the proteins to stimulate protein synthesis in rat L6 myoblasts, all IGF-I proteins were found to be equally potent. The ability of all three proteins to compete for binding with radiolabelled human IGF-I to type-1 IGF receptors in L6 myoblasts and in Sminthopsis crassicaudata transformed lung fibroblasts, a marsupial cell line, was comparable. Furthermore, kangaroo and human IGF-I react equally in a human IGF-I RIA using a human reference standard, radiolabelled human IGF-I and a polyclonal antibody raised against recombinant human IGF-I. This study indicates that not only is the primary structure of eutherian and metatherian IGF-I conserved, but also the proteins appear to be functionally similar.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/aislamiento & purificación , Macropodidae/metabolismo , Secuencia de Aminoácidos , Análisis de Varianza , Animales , Bioensayo , Pollos , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/aislamiento & purificación , Factor II del Crecimiento Similar a la Insulina/metabolismo , Datos de Secuencia Molecular , Radioinmunoensayo , Ensayo de Unión Radioligante , Salmón , Homología de Secuencia de AminoácidoRESUMEN
A protein kinase from Dictyostelium discoideum which phosphorylates the synthetic peptide, calmodulin-dependent protein kinase substrate (CDPKS, amino acid sequence: PLRRTLSVAA) and is stimulated by Ca2+/calmodulin is described. This is the first report of a protein kinase with these characteristics in D. discoideum. The enzyme was partially purified by Q-Sepharose chromatography. The protein kinase is very labile, and rapidly loses Ca2+/calmodulin-dependence upon standing at 4 degrees C, even in the presence of protease inhibitors, making further purification and characterisation difficult. In the active fractions, a 55 kDa polypeptide is labelled with [gamma-32P]ATP in vitro under conditions in which intramolecular rather than intermolecular reactions are favoured. The phosphorylation of this peptide is stimulated in the presence of Ca2+ and calmodulin but not Ca2+ alone. Ca2+/calmodulin-dependent stimulation is inhibited in the presence of the calmodulin antagonist, trifluoperazine (TFP). It is proposed that the 55 kDa polypeptide may represent the autophosphorylated form of the enzyme.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Dictyostelium/enzimología , Proteínas Quinasas/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Calmodulina/antagonistas & inhibidores , Cromatografía en Agarosa , Dictyostelium/crecimiento & desarrollo , Electroforesis en Gel de Poliacrilamida , Peso Molecular , Fosforilación , Proteínas Quinasas/química , Trifluoperazina/farmacologíaRESUMEN
To examine further the role of the enzyme glutamine synthetase in Dictyostelium discoideum we report here the effects of a specific glutamine synthetase inhibitor, methionine sulfoximine, on the growth and differentiation of this organism. Vegetative AX3 cells grown in the presence of methionine sulfoximine did not complete culmination in the normal time but were blocked at the finger stage. In these cells glutamine synthetase activity was almost completely abolished. However, methionine sulfoximine did not affect the level of glutamine synthetase mRNA, suggesting that there is no link between glutamine synthetase activity and mRNA transcription. Eventually glutamine synthetase activity reappeared and at the time culmination occurred. These results suggest that glutamine synthetase plays an important role in the assimilation of ammonia during the later stages of development in D. discoideum and that this assimilation is necessary for the completion of culmination.
Asunto(s)
Dictyostelium/efectos de los fármacos , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Metionina Sulfoximina/farmacología , Amoníaco/metabolismo , Animales , Dictyostelium/crecimiento & desarrollo , Dictyostelium/metabolismoAsunto(s)
Glándulas Suprarrenales/fisiología , Aldosterona/farmacología , Riñón/metabolismo , Fosfolípidos/biosíntesis , Adrenalectomía , Animales , Isótopos de Carbono , Núcleo Celular/metabolismo , Colina/metabolismo , Cromatografía en Capa Delgada , Corticosterona/farmacología , Desoxicorticosterona/farmacología , Femenino , Hidrocortisona/farmacología , Riñón/citología , Riñón/efectos de los fármacos , Cinética , Membranas/metabolismo , Microsomas/metabolismo , Mitocondrias/metabolismo , Fosfatos/metabolismo , Fosfatidilcolinas/biosíntesis , Isótopos de Fósforo , RatasAsunto(s)
Inositol/biosíntesis , Plantas/metabolismo , ARN de Transferencia/biosíntesis , ARN/biosíntesis , Ribosomas/metabolismo , Adenosina Trifosfato , Centrifugación por Gradiente de Densidad , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Peso Molecular , Fosfatos , Ácidos Fosfóricos/biosíntesis , Isótopos de Fósforo , Desarrollo de la Planta , Semillas/metabolismo , Solubilidad , Ácido TricloroacéticoAsunto(s)
Glándulas Suprarrenales/fisiología , Glucocorticoides/metabolismo , Riñón/metabolismo , ARN/metabolismo , Fracciones Subcelulares/metabolismo , Adrenalectomía , Animales , Isótopos de Carbono , Núcleo Celular/metabolismo , Centrifugación por Gradiente de Densidad , Grupo Citocromo c/metabolismo , Retículo Endoplásmico/metabolismo , Femenino , Semivida , Riñón/citología , Riñón/enzimología , Cinética , Mitocondrias/metabolismo , Ácido Orótico/metabolismo , Polirribosomas/metabolismo , ARN/biosíntesis , ARN Nucleotidiltransferasas/metabolismo , Ratas , Ribosomas/metabolismo , Succinato Deshidrogenasa/metabolismoAsunto(s)
Retículo Endoplásmico/metabolismo , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Animales , Isótopos de Carbono , Centrifugación por Gradiente de Densidad , Semivida , Riñón/citología , Riñón/metabolismo , Hígado/citología , Hígado/metabolismo , Masculino , Ácido Orótico/metabolismo , Polirribosomas/metabolismo , RatasAsunto(s)
Aldosterona/farmacología , ARN Polimerasas Dirigidas por ADN/análisis , Riñón/enzimología , Glándulas Suprarrenales/fisiología , Adrenalectomía , Animales , Núcleo Celular/enzimología , Femenino , Riñón/citología , Riñón/efectos de los fármacos , Cinética , Magnesio , Manganeso , Ratas , TritioRESUMEN
1. The intracellular cysteine and glutathione concentrations were measured in Escherichia coli under a variety of growth conditions. 2. An inverse relation between intracellular cysteine concentration and the specific activity of the sulphate-activating enzymes was found. 3. This is compatible with the view that the intracellular cysteine concentration controls the rate of synthesis of these enzymes.
Asunto(s)
Nucleótidos de Adenina/biosíntesis , Cisteína/metabolismo , Represión Enzimática , Escherichia coli/metabolismo , Sulfatos/metabolismo , Isótopos de Carbono , Cromatografía en Papel , Etilmaleimida/farmacología , Glutatión/metabolismo , Isótopos de Azufre , Factores de TiempoRESUMEN
NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent Kms for alpha-ketoglutarate, NADPH and NH4+ are 1.2 mM, 9.7 microM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 microM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn2+ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 microM) yet N-ethylmaleimide does not. In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.
Asunto(s)
Dictyostelium/enzimología , Glutamato Deshidrogenasa/aislamiento & purificación , Adenosina Difosfato/farmacología , Adenosina Monofosfato/farmacología , Animales , AMP Cíclico/farmacología , Etilmaleimida/farmacología , Glutamato Deshidrogenasa (NADP+) , Guanosina Trifosfato/farmacología , Concentración de Iones de Hidrógeno , Hidroximercuribenzoatos/farmacología , Peso Molecular , MorfogénesisRESUMEN
The cellular slime mould Polysphondylium violaceum contains two vegetative stage specific acidic (casein) kinases. These two enzymes have been partially purified and their properties investigated. Both utilise casein as their preferred substrate but they can be distinguished in a number of ways, including their responses to spermine, heparin and salt. In addition, they have different affinities for their substrates and different pH activity profiles. It is suggested that they may play a role in a vegetative specific function such as cell division.
Asunto(s)
Mixomicetos/enzimología , Proteínas Quinasas/análisis , Caseína Quinasas , Heparina/farmacología , Concentración de Iones de Hidrógeno , Magnesio/farmacología , Mixomicetos/crecimiento & desarrollo , Nucleótidos/farmacología , Cloruro de Potasio/farmacología , Proteínas Quinasas/aislamiento & purificación , Proteínas Quinasas/fisiología , Espermina/farmacología , Especificidad por SustratoRESUMEN
Incubation of Dictyostelium discoideum cells with selenate is known to inhibit vegetative growth. In this paper we show that in the presence of selenate macromolecules accumulate which can be converted to sulphated products once the selenate is removed. The presence of cycloheximide, an inhibitor of protein synthesis, during the subsequent incubation does not prevent this conversion but tunicamycin, an inhibitor of glycosylation does. It is concluded that, in the presence of selenate, precursors accumulate as unglycosylated proteins, suggesting that feedback inhibition of glycosylation may be operated.
Asunto(s)
Dictyostelium/efectos de los fármacos , Dictyostelium/metabolismo , Compuestos de Selenio/farmacología , Sulfatos/metabolismo , Animales , Cicloheximida/farmacología , Dictyostelium/crecimiento & desarrollo , Glicosilación/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Ácido Selénico , Tunicamicina/farmacologíaRESUMEN
Pyruvate kinase (EC 2.7.1.40) from aggregating Dictyostelium discoideum cells has been purified to homogeneity. It has a monomeric molecular weight of 66kD and is tetrameric in low ionic strength buffers. The enzyme is not regulated by fructose 1,6-bisphosphate or by alanine and appears to resemble the M1 isoenzyme from rat liver most closely, although its activity is not inhibited by ATP.
Asunto(s)
Dictyostelium/enzimología , Piruvato Quinasa/aislamiento & purificación , Dictyostelium/crecimiento & desarrollo , Fructosadifosfatos/farmacología , Cinética , Peso Molecular , Conformación Proteica , Piruvato Quinasa/química , Piruvato Quinasa/metabolismoRESUMEN
The utilization of [35S]sulphate by bacterially grown amoebae of Dictyostelium discoideum strain NP73 was examined in this study. During vegetative growth the sulphation of at least ten macromolecules was observed. These macromolecules had molecular masses less than 66 kDa and isoelectric points below 5. Simple tests indicated that the sulphate linkage was periodate-sensitive but not acid-labile which implied that carbohydrate moieties, rather than tyrosine residues, were sulphated. Pulse-chase experiments indicated that the sulphated macromolecules were extremely stable during vegetative growth, but that secretion occurred on starvation, resulting in the loss of the sulphated macromolecules to the extracellular medium. Incorporation of [35S]sulphate into these macromolecules by amoebae declined rapidly within 2 h of starvation on membrane filters. In contrast, incorporation by amoebae starving in suspension culture continued for 6-8 h. Similar patterns of [35S]sulphate incorporation were observed for two other strains of D. discoideum (strains AX2 and NC4) and for Polysphondylium violaceum. Since in a previous study it was shown that the sulphation inhibitor, sodium selenate, arrests the growth of D. discoideum [Davis, S.J. & Wheldrake, J.F. (1985) FEMS Micro Lett. 30, 353-358], it is suggested that the sulphation of these macromolecules is necessary for the vegetative growth of D. discoideum.
Asunto(s)
Dictyostelium/metabolismo , Sulfatos/metabolismo , Dictyostelium/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Sustancias Macromoleculares , Radioisótopos de Azufre , Tirosina/metabolismoRESUMEN
The properties and developmental regulation of the protein phosphatases of Dictyostelium discoideum were examined. When crude extracts from vegetative cells were separated on a Mono Q column (FPLC) three protein phosphatase peaks, designated P1, P2 and P3 were found. When aggregation and culmination cells were examined only one protein phosphatase peak was observed. This corresponded to phosphatase P1 of vegetative cells. All three of the vegetative cell phosphatase were inhibited by heparin and mammalian phosphatase inhibitor-2, both of which are specific for type-1 protein phosphatases. Trifluoperazine, which inhibits type-2 protein phosphatases, had little effect on any peaks while levamisole, an alkaline phosphatase inhibitor, stimulated P2, slightly inhibited P3 and had no effect on P1. These results demonstrate the existence of two vegetative phase specific protein phosphatases in D. discoideum and one which occurs during all phases of the life cycle. The protein phosphatases isolated from vegetative cells all appear to be type-1 enzymes.
Asunto(s)
Dictyostelium/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Fosfatasa Ácida/metabolismo , Dictyostelium/crecimiento & desarrollo , Heparina/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidoresRESUMEN
The cellular slime mould Polysphondylium violaceum contains a cAMP-dependent protein kinase resembling the mammalian type I enzyme. The appearance of this enzyme is developmentally regulated. The level of kinase activity is very low in vegetative cell and increases more than tenfold during differentiation. The catalytic subunit of this cAMP-dependent protein kinase has a native molecular weight of 60-80 kDa, an isoelectric point of 5.7 and an apparent Km for ATP and Kemptide of 50 and 13.4 microM respectively. It is characterised by its sensitivity to a synthetic inhibitor specific for cAMP-dependent protein kinase. The regulatory subunit has a molecular weight of 50 kDa.
Asunto(s)
Mixomicetos/enzimología , Proteínas Quinasas/genética , Diferenciación Celular , AMP Cíclico/fisiología , Citosol/enzimología , Inducción Enzimática , Proteínas Quinasas/aislamiento & purificaciónRESUMEN
The NAD-dependent glutamate dehydrogenase (GDH) from Dictyostelium discoideum was purified 1101-fold with a yield of 23.4%. The enzyme has an apparent Mr of 356 kDa, determined using Sephacryl S400, and a subunit molecular weight of 54 kDa on SDS-polyacrylamide gel electrophoresis. The Kms for alpha-ketoglutarate, NADH, and NH4+ are 0.36 +/- 0.03 mM, 16.0 +/- 0.1 microM, and 34.5 +/- 2.7 mM, respectively. The purified enzyme has a pH optimum of pH 7.25-7.5. At 0.1 mM, ADP and AMP stimulate GDH activity 25 and 102%, respectively. Half-maximal activity in the presence of 0.1 mM AMP for alpha-ketoglutarate, NADH, and NH4+ is reached at 2.3 +/- 0.1 mM, 71.4 +/- 5.5 microM, and 27.9 +/- 3.6 mM, respectively.