RESUMEN
We screened for pollen-specific kinase genes, which are potential signal transduction components of pollen-pistil interactions, and isolated two structurally related receptor-like kinases (RLKs) from tomato, LePRK1 and LePRK2. These kinases are similar to a pollen-expressed RLK from petunia, but they are expressed later during pollen development than is the petunia RLK. The abundance of LePRK2 increases when pollen germinates, but LePRK1 remains constant. Both LePRK1 and LePRK2 are localized to the plasma membrane/cell wall of growing pollen tubes. Both kinase domains have kinase activity when expressed in Escherichia coli. In phosphorylation assays with pollen membrane preparations, LePRK2, but not LePRK1, is phosphorylated, and the addition of tomato style, but not leaf, extracts to these membrane preparations results at least partially in specific dephosphorylation of LePRK2. Taken together, these results suggest that LePRK1 and LePRK2 play different roles in postpollination events and that at least LePRK2 may mediate some pistil response.
Asunto(s)
Estructuras de las Plantas/metabolismo , Polen/enzimología , Proteínas Quinasas/metabolismo , Solanum lycopersicum/enzimología , Secuencia de Aminoácidos , Pared Celular/enzimología , Escherichia coli , Expresión Génica , Genes de Plantas , Germinación , Técnicas In Vitro , Solanum lycopersicum/genética , Solanum lycopersicum/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Fosforilación , Polen/metabolismo , Proteínas Quinasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The Km values of the enzyme for ATP, GDP and dTDP were 0.2 +/- 0.008 mM, 0.125 +/- 0.012 mM and 0.4 +/- 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65 degrees C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [gamma-32P]ATP or thiophosphorylated with [35S]GTP gamma S. The incubation of the 32P-labelled phosphoenzyme with unlabelled nucleoside 5'-diphosphates resulted in the formation of 32P-labelled nucleoside 5'-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5'-diphosphates, GTP was preferentially formed.
Asunto(s)
Nucleósido-Difosfato Quinasa/aislamiento & purificación , Proteínas Protozoarias/aislamiento & purificación , Trypanosoma cruzi/enzimología , Adenosina Trifosfato/metabolismo , Animales , Reacciones Cruzadas , Guanosina Difosfato/metabolismo , Cinética , Nucleósido-Difosfato Quinasa/inmunología , Nucleósido-Difosfato Quinasa/metabolismo , Fosforilación , Conformación Proteica , Desnaturalización Proteica , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Solubilidad , Especificidad de la Especie , Especificidad por Sustrato , Temperatura , Nucleótidos de Timina/metabolismo , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/inmunologíaRESUMEN
The LAT52 gene of tomato is expressed in a pollen-specific manner. It is shown that LAT52 encodes a heat-stable, glycosylated protein that traverses the secretory pathway when expressed in a baculovirus expression system. The LAT52 protein shows some similarity with Kunitz trypsin inhibitors and with pollen proteins from maize, rice and olive, but the biological function of these pollen proteins is unknown. To test whether the LAT52 protein plays an important role during pollen development, tomato plants were transformed with an antisense LAT52 gene driven by the LAT52 promoter. Because the LAT52 gene is expressed gametophytically, only 50% of the pollen of the primary transformants would be expected to express the antisense construct. Selfprogeny of 19 of the primary transformants showed the predicted 3:1 segregation for a single locus insertion of the linked kanamycin-resistance gene. However, the self-progeny of the other 32 primary transformants showed a 1:1 segregation pattern and could not transmit the linked kanamycin-resistance gene through the male. A subset of these 1:1 segregation class plants was examined in detail. The pollen showed lower levels of LAT52 mRNA and LAT52 protein when compared with wild-type. In vitro, approximately 50% of the pollen grains appear to hydrate abnormally; this anomaly is not present when the same pollen grains are incubated in a medium with higher water potential. In vivo pollination experiments showed that the growth of around 50% of the pollen tubes is arrested in the style. The 3:1 segregation class plants showed no significant differences from untransformed control plants. Taken together, the results show a direct correlation between the reduced expression of LAT52 protein and abnormal pollen function, and suggest that the LAT52 protein plays a role in pollen hydration and/or pollen germination.
Asunto(s)
ADN sin Sentido/genética , Germinación , Proteínas de Plantas/genética , Polen/crecimiento & desarrollo , Solanum lycopersicum/genética , Secuencia de Aminoácidos , Fertilización , Solanum lycopersicum/crecimiento & desarrollo , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente/genética , Alineación de SecuenciaRESUMEN
G-protein subunits were characterized from Medicago sativa (alfalfa) seedlings. Crude membranes and GTP-Sepharose-purified fractions were electrophoresed on SDS/polyacrylamide gels and analysed by Western blotting with 9193 (anti-alpha common) and AS/7 (anti-alpha t, anti-alpha i1 and anti-alpha i2) polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 (anti-beta) antibody, of about 37 kDa, was also detected. The 43 kDa polypeptide bound specifically [alpha-32P]GTP by a photoaffinity reaction and was ADP-ribosylated by activated cholera toxin, but not by pertussis toxin. Irradiation of etiolated Medicago sativa protoplast preparations at 660 nm for 1 min produced a maximal increase in the guanosine 5'-[gamma-thio]triphosphate (GTP[35S])-binding rate. After this period of irradiation, the binding rate tended to decrease. The effect of a red-light (660 nm) pulse on the binding rate was reversed when it was immediately followed by a period of far-red (> 730 nm) illumination. These results may suggest that activation of GTP[S]-binding rate was a consequence of conversion of phytochrome Pr into the Ptr form.
Asunto(s)
Proteínas de Unión al GTP/análisis , Medicago sativa/química , Células Fotorreceptoras/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Secuencia de Aminoácidos , Western Blotting , Membrana Celular/química , Toxina del Cólera/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteínas de Unión al GTP/metabolismo , Guanosina Trifosfato/metabolismo , Luz , Medicago sativa/metabolismo , Datos de Secuencia Molecular , Radioisótopos de AzufreRESUMEN
A guanosine 5'-[gamma-[35S]thio]triphosphate-binding activity was detergent-extracted from Trypanosoma cruzi membranes. This binding activity was co-eluted from gel-filtration columns with a factor which, in a heterologous reconstitution system, blocks glucagon stimulation of adenylate cyclase activity in liver membranes. ADP-ribosylation of these membranes by pertussis toxin eliminated this blocking capacity. Incubation of T. cruzi membranes with activated pertussis toxin and [adenylate-32P]NAD+ led to the incorporation of radioactivity into a labelled product with an apparent M(r) of approx. 43,000. Crude membranes were electrophoresed on SDS/polyacrylamide gels and analysed, by Western blotting, with GA/1 anti-alpha common, AS/7 anti-alpha t, anti-alpha i1 and anti-alpha i2 polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 anti-beta antibody, of about 30 kDa, was also detected in the membrane fraction.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Proteínas Protozoarias/análisis , Trypanosoma cruzi/química , Adenosina Difosfato/metabolismo , Toxina de Adenilato Ciclasa , Secuencia de Aminoácidos , Animales , Proteínas de Unión al GTP/análisis , Proteínas de Unión al GTP/inmunología , Guanosina Trifosfato/metabolismo , Sustancias Macromoleculares , Proteínas de la Membrana/análisis , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Toxina del Pertussis , Proteínas Protozoarias/inmunología , Radioisótopos de Azufre , Trypanosoma cruzi/fisiología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
An adenylate cyclase activity was partially characterized in the cyanobacterium Anabaena sp. The enzyme activity is found in soluble cell fractions and shows an apparent molecular weight of about 183,400. This adenylate cyclase is activated by Ca2+ and bovine brain or spinach calmodulin and it is inhibited by EGTA and some phenothiazine derivatives. Furthermore, Anabaena sp. extracts contain a calmodulin-like activity which stimulates bovine brain cyclic AMP phosphodiesterase and the Anabaena adenylate cyclase. EGTA and phenothiazine derivatives block the cyanobacterial modulator effect.
Asunto(s)
Adenilil Ciclasas/metabolismo , Calmodulina/fisiología , Cianobacterias/enzimología , Calcio/fisiología , Activación EnzimáticaRESUMEN
Phorbol esters stimulate cyclic adenosine 3',5'-monophosphate (cAMP) accumulation in hamster spermatozoa under conditions for in vitro capacitation. The 20-50-fold elevation of cAMP levels induced by 1 microM phorbol 12-myristate 13-acetate (PMA) in spermatozoa depends on the presence of sodium bicarbonate in the medium (ED50: 15 mM) and it is independent of extracellular pH. Sodium bicarbonate stimulates adenylate cyclase activity in membrane preparations by 4-fold (ED50: 40 mM). After solubilization, the bicarbonate-sensitive moiety elutes as a single peak of 55 kDa in a gel filtration column. Blockers of bicarbonate chloride antiporters diisothiocyanate stilbene 2,2'-disulfonic acid (DIDS) or acetamido 4'-isothiocyanate stilbene 2,2'-disulfonic acid (SITS) inhibit the bicarbonate dependent PMA effect on cAMP in living spermatozoa (ED50: 100 microM). Maximal (85%) inhibition in cAMP accumulation is observed at 1 mM. Motility is inhibited only at high concentrations of the blockers. Pretreatment of living cells with 1 mM DIDS does not affect membrane adenylate cyclase activity which remains responsive to bicarbonate. These results suggest that controlled transport of bicarbonate through the sperm plasma membrane could be associated to the regulation of cAMP synthesis.
Asunto(s)
Adenilil Ciclasas/metabolismo , Bicarbonatos/farmacología , AMP Cíclico/metabolismo , Epidídimo/enzimología , Forbol 12,13-Dibutirato/farmacología , Sodio/farmacología , Capacitación Espermática/efectos de los fármacos , Espermatozoides/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Transporte Biológico , Cricetinae , Activación Enzimática/efectos de los fármacos , Epidídimo/efectos de los fármacos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Masculino , Forbol 12,13-Dibutirato/farmacocinética , Bicarbonato de Sodio , Espermatozoides/efectos de los fármacos , Espermatozoides/enzimologíaRESUMEN
Mice receiving reserpine (1 mg/kg/day) during 5 days develop behavioral supersensitivity. To study the possible molecular correlates of these adaptive changes we compared binding parameters of D1 and D2 receptors and adenylate cyclase activity in striata from normal and reserpinized mice. Saturation curves using [3H]SCH 23390 showed no changes in maximum binding capacity (Bmax) or Kd of striatal D1 receptors taken from control or 5 days reserpine-treated mice. However, [3H]spiperone saturation curves showed a 31% increase in D2 receptors Bmax with no changes in Kd. Dopamine competition of [3H]SCH 23390 and [3H]spiperone binding in mouse striatum was also performed. Analysis of data by LIGAND showed that dopamine recognizes two subpopulations for D1 and for D2 receptors. The proportion of receptors in the high affinity state (D1high and D2high) were increased in reserpine-treated animals. The addition of 100 microM GTP produced a complete conversion of D1high and D2high receptors into their low-affinity states in striata from control and reserpinized mice. Five days of reserpine treatment increased basal adenylate cyclase activity of mouse striatum in the presence of Mn++ or Mg++ ions. Concentration curves with dopamine, NaF or forskolin revealed shifts to the left and higher maximum responses without changes in EC50 values in striata from reserpinized mice. Thus, a prolonged reserpine treatment produces marked changes in D1 and D2 receptors increasing the proportion of high affinity state subpopulations and the total Bmax of D2 receptors. Also, dopamine function may be enhanced through an increment of the catalytic component of striatal adenylate cyclase.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Receptores Dopaminérgicos/efectos de los fármacos , Reserpina/farmacología , Adenilil Ciclasas/análisis , Animales , Benzazepinas/metabolismo , Colforsina/farmacología , Magnesio/farmacología , Masculino , Manganeso/farmacología , Ratones , Receptores de Dopamina D1 , Receptores de Dopamina D2 , Espiperona/metabolismoRESUMEN
Adenylate cyclase catalytic subunits from Neurospora crassa membranes may interact with regulatory factors from membranes of bovine retinal rod outer segments (pretreated with N-ethylmaleimide), reconstituting a heterologous system which, in the presence of light, is catalytically active in assay mixtures containing MgATP. Maximal activation was observed at 550 nm. Transducin-depleted retinal membranes were not capable of reconstituting the heterologous light-stimulated adenylate cyclase system. Addition of a transducin preparation to depleted membranes restored the reconstitution capacity of these membranes. A similar heterologous adenylate cyclase system was reconstituted with Neurospora and mouse retinal whole membranes (pretreated with N-ethylmaleimide). Membranes from mice suffering photoreceptor degeneration (rd homozygotes) did not reconstitute an heterologous adenylate cyclase system.
Asunto(s)
Adenilil Ciclasas/análisis , Luz , Neurospora crassa/enzimología , Neurospora/enzimología , Retina/enzimología , Adenosina Difosfato Ribosa/metabolismo , Animales , Bovinos , GMP Cíclico/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
An adenylate cyclase activity in Medicago sativa L. (alfalfa) roots was partially characterized. The enzyme activity remains in the supernatant fluid after centrifugation at 105,000 g and shows in crude extracts an apparent Mr of about 84,000. The enzyme is active with Mg2+ and Ca2+ as bivalent cations, and is inhibited by EGTA and by chlorpromazine. Calmodulin from bovine brain or spinach leaves activates this adenylate cyclase.
Asunto(s)
Adenilil Ciclasas/metabolismo , Medicago sativa/enzimología , Inhibidores de Adenilato Ciclasa , Calcio/farmacología , Calmodulina/farmacología , Centrifugación por Gradiente de Densidad , Clorpromazina/farmacología , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Ácido Egtácico/farmacología , Flufenazina/farmacologíaRESUMEN
The effects of temperature, pH, divalent cations, 2-mercaptoethanol (Et-SH), N-ethylmaleimide (NEM), and phenylmethylsulfonyl fluoride (PMSF) on the dihydrotestosterone (DHT) binding to sex steroid binding protein from Bufo arenarum (baSBP) were examined. The temperature curve indicated that the binding remained stable up to 50 degrees C and the pH curve showed maximum binding between pH 7 and 9. The incubations of baSBP with divalent cations, NEM and Et-SH demonstrated that baSBP require disulfides and sulfhydryl groups for steroid binding or to maintain an adequate protein conformation. On the other hand, PMSF had no effect on the binding, consequently, serine residues appear not to be involved in DHT binding to baSBP. These results indicate that baSBP has a behavior resembling that of human SBP.