RESUMEN
In the presence of germination signals, dormant spores of Dictyostelium discoideum rapidly germinate to start a new life cycle. Previously we have shown that half of the actin molecules in spores are maintained in a tyrosine-phosphorylated state, and a decline of the actin phosphorylation levels is a prerequisite for spore swelling. In this study, we have established d-glucose as a trigger molecule for the actin dephosphorylation. Present in a nutrient germination medium, d-glucose both may act as a trigger molecule and/or may serve as a substrate within a pathway for actin dephosphorylation depending upon spore age. However, the glucose-induced actin dephosphorylation was insufficient for spores to swell. Other factors in the nutrient medium were required for complete germination of young spores aged 1 to 5 days. In contrast, dispersion in nonnutrient buffer was necessary and sufficient for a decline of actin phosphorylation levels and even the emergence of amoebae in older spores (6 days and beyond). Moreover, the dephosphorylation pathway in the older spores was independent of energy production. We propose that the diversification of the actin dephosphorylation pathway may enable spores to increase their probability of germination upon spore aging.
Asunto(s)
Actinas/metabolismo , Dictyostelium/fisiología , Glucosa/metabolismo , Anaerobiosis , Animales , Medios de Cultivo , Dictyostelium/efectos de los fármacos , Glucosa/farmacología , Cinética , Estadios del Ciclo de Vida , Fosforilación , Fosfotirosina/metabolismo , Esporas/fisiología , Factores de TiempoAsunto(s)
Dictyostelium/fisiología , Transducción de Señal/fisiología , Adenilil Ciclasas/metabolismo , Amoníaco/metabolismo , Animales , Bacterias/metabolismo , AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dictyostelium/genética , Dictyostelium/metabolismo , Ambiente , Modelos Biológicos , Mutagénesis , Compuestos de Amonio Cuaternario/metabolismoRESUMEN
Acid-activatable cysteine proteinases of Dictyostelium discoideum were first identified in spore extracts of strain SG1 using gelatin/SDS/PAGE, followed by acid treatments. Here we utilized the technique of acid activation to identify cryptic cysteine proteinases throughout auto-induced and heat-induced spore germination of D. discoideum strain SG2 and SG1. The major acid-activatable cysteine proteinase identified in SG2 and SG1 spore extracts was ddCP38 (D. discoideum cysteine proteinase with a molecular mass of 38 kDa) and ddCP48, respectively. Further investigation of these enzymes revealed that they were also base deactivatable with a treatment of ammonium chloride directly following acid activation. However, the most intriguing observation was the reversibility of the effects of base deactivation on the enzymes following a second treatment with acetic acid. Thus, we hypothesize that, unlike most mammalian cysteine proteinases which generally require the cleavage of a pro-peptide region for activation, these cysteine proteinases of D. discoideum likely undergo reversible conformational changes between latent and active forms. Moreover, we were able to detect these cryptic cysteine proteinases in the vegetative cells and early aggregates of both strains SG1 and SG2. Studies using 4-[(2S, 3S)-3-carboxyoxiran-2-ylcarbonyl-L-leucylamido]buty lguanidine, a cysteine proteinase inhibitor, revealed that acid activation of a portion of these proteinases was still achievable even after incubation with the inhibitor, further supporting the concept of two stable and reversible conformational arrangements of the enzymes. Thus, we speculate that the pH shuffles that modulate proteinase conformation and activity in vitro may be a reflection of the in vivo regulation of these enzymes via H+-ATPases and ammonia.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Dictyostelium/enzimología , Proteínas Protozoarias/metabolismo , ATPasas de Translocación de Protón Vacuolares , Amoníaco/metabolismo , Animales , Cicloheximida/farmacología , Cisteína Endopeptidasas/aislamiento & purificación , Dictyostelium/fisiología , Activación Enzimática , Concentración de Iones de Hidrógeno , ATPasas de Translocación de Protón/metabolismo , Proteínas Protozoarias/aislamiento & purificación , EsporasRESUMEN
Experimental data show that opiates interfere with calcium influx in the cell and that some calcium-channel blockers are analgesic. We therefore studied the effect of the calcium-receptor blocker nifedipine on the analgesic effect of morphine in the rat, using tail-flick responses, and in humans, using measurements of the intensity of postoperative pain. In both the experimental animals and humans nifedipine significantly (P less than 0.001) increased the analgesic effect of morphine independently of any effect on the metabolism of morphine. Respiratory and cardiovascular functions were not significantly changed by nifedipine. The data indicate that Ca2+ is important in mediating the analgesic effects of opiates and suggest that calcium-receptor blockers might find a place in the treatment of pain.