RESUMEN
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
Dictyostelium cells express a G-protein-coupled adenylyl cyclase, ACA, during aggregation and an atypical adenylyl cyclase, ACG, in mature spores. The ACG gene was disrupted by homologous recombination. acg- cells developed into normal fruiting bodies with viable spores, but spore germination was no longer inhibited by high osmolarity, a fairly universal constraint for spore and seed germination. ACG activity, measured in aca-/ACG cells, was strongly stimulated by high osmolarity with optimal stimulation occurring at 200 milliosmolar. RdeC mutants, which display unrestrained protein kinase A (PKA) activity and a cell line, which overexpresses PKA under a prespore specific promoter, germinate very poorly, both at high and low osmolarity. These data indicate that ACG is an osmosensor controlling spore germination through activation of protein kinase A.
Asunto(s)
Adenilil Ciclasas/fisiología , Dictyostelium/enzimología , Proteínas Fúngicas/fisiología , Proteínas Protozoarias , Esporas Fúngicas/fisiología , Equilibrio Hidroelectrolítico , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Dictyostelium/fisiología , Mutagénesis Insercional , Transducción de SeñalRESUMEN
Studies of the cysteine proteinases of the cellular slime mold Dictyostelium discoideum have been aided by a simple acid treatment step that was incorporated into the standard one-dimensional gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay procedure. The step involved immersing the separating gel in 10% (v/v) glacial acetic acid for 30-60 s immediately after electrophoresis. This modified approach revealed the presence of acid-activatable forms of some enzymes with noticeable increases in their ability to hydrolyze gelatin, a substrate present in the sodium dodecyl sulfate-polyacrylamide gels, and peptidyl amidomethylcoumarins. The activation has been analyzed using extracts of dormant spores from which cysteine proteinase activity had previously appeared low or virtually absent. The major acid-activatable proteinase had an apparent molecular mass of 48 kDa. Its activation was not due to autocatalysis as it was not prevented by mercuric chloride, an inhibitor of the enzyme, and was not accompanied by a significant change in electrophoretic mobility. It was most likely due to a conformational change and/or the removal of a low molecular weight inhibitor. The acid treatment has also revealed the presence of acid-activatable cysteine proteinases in vegetative cells, in which cysteine proteinase activity is present at high levels, as well as among enzymes from the developmental cells which have much lower cysteine proteinase activity. Indeed novel developmental forms were detected at some stages. These results provide additional insight concerning cysteine proteinase expression at various stages during development in the slime molds. A developmental model is presented which suggests that the crypticity of the cysteine proteinases in dormant spores may be governed by proton pumps and endogenous lysosomotropic agents.
Asunto(s)
Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Dictyostelium/enzimología , Acetatos , Ácido Acético , Secuencia de Aminoácidos , Animales , Dictyostelium/crecimiento & desarrollo , Electroforesis en Gel de Poliacrilamida/métodos , Activación Enzimática , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Conformación Proteica , Especificidad por SustratoRESUMEN
RasG protein levels in dormant and germinating spores of Dictyostelium discoideum strains JC1 and SG1 were estimated by Western blotting. RasG levels were very low in dormant spores and remained low during the lag period, regardless of whether spores were heat activated or treated with autoactivator during the early stages of spore germination. RasG levels increased late during spore swelling just prior to the emergence stage of germination. These data are consistent with a requirement for RasG during vegetative growth.
Asunto(s)
Dictyostelium/fisiología , Proteínas Fúngicas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas ras , Animales , Esporas Fúngicas/fisiologíaRESUMEN
Cellular communication dictates all stages of growth and development in the cellular slime molds. Dictyostelium discoideum utilizes a number of signal molecules for cell-to-cell communication, including growth and density factors, cAMP, ammonia, differentiation-inducing factor, discadenine, and spore autoactivator. A source and sink model is presented in which the assimilation of ammonia plays a major role in determining cell fate and pattern formation. This model emphasizes a recycling of ammonia by prespore cells, the accumulation of free hydrophilic and neutral amino acids, and their incorporation into proteins associated with sporulation and (or) germination. If spore cAMP signalling is regulated by the relative concentrations of discadenine and autoactivator, and its disruption triggers the initiation of the spore germination cascade, then the accumulation of intracellular cAMP may be necessary for both sporulation and dormancy maintenance.
Asunto(s)
Dictyostelium/crecimiento & desarrollo , Adenina/análogos & derivados , Adenina/fisiología , Secuencia de Aminoácidos , Amoníaco/metabolismo , Animales , Secuencia de Bases , AMP Cíclico/fisiología , Dictyostelium/genética , Dictyostelium/fisiología , Proteínas Fúngicas/metabolismo , Sustancias de Crecimiento/metabolismo , Hexanonas/metabolismo , Datos de Secuencia Molecular , Morfogénesis/fisiología , Esporas Fúngicas/fisiologíaRESUMEN
Comparisons of the sensitivities of one-dimensional (1D) and two-dimensional (2D) electrophoreses to detect genetic variability have generally shown that the 2D approach appears to be two- to five-fold less sensitive than conventional 1D approaches. Concerns about the validity of this conclusion have arisen because such comparisons have involved mainly enzymic proteins in 1D approaches versus a complex mixture of soluble proteins in most 2D analyses. Comparisons involving the absolute number of variants detected, using 1D and 2D sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), denatured mouse blood proteins isolated from C3HeB/FeJ and C57B1/6J inbred strains of mice, and highly sensitive silver staining, indicate that the latter uncovers at least as much variability as the former. Although the relative percentage of variable bands (1D SDS-PAGE) was greater than the relative percentage of variable spots (2D SDS-PAGE) when proteins of intact erythrocytes were surveyed, both techniques uncovered approximately equal percentages of variable proteins when the mouse erythrocyte proteins were partitioned into membrane and lysate components. Therefore, the simpler 1D SDS-PAGE was found to be as effective as 2D SDS-PAGE in detecting protein variability. Since 1D SDS-PAGE separates proteins primarily on the basis of molecular weight and to a lesser degree on other primary protein sequence alterations, much of the variability observed by 2D SDS-PAGE may be due to these same features and unit charge differences may not play a significant role in detecting variability in the proteins studied. This differs from enzymic proteins, where such charge differences appear to be responsible for much of the variability. This study also indicated that decreasing the number of proteins in samples (membranes and lysates vs whole erythrocytes) increased the ability of both of these techniques to resolve differences. Mating studies indicated that most of the differences detected with both techniques were inherited and were not artifacts.
Asunto(s)
Proteínas Sanguíneas/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida/métodos , Animales , Animales Salvajes , Proteínas Sanguíneas/genética , Electroforesis en Gel Bidimensional , Femenino , Variación Genética , Masculino , Ratones , Ratones Endogámicos , Especificidad de la EspecieRESUMEN
Type II restriction endonucleases cleave double stranded DNA molecules at sites characterized by one or more sets of nucleotide pairs sequences. These digestions are essential in such procedures as DNA cloning, DNA sequencing and restriction fragment length polymorphism (RFLP) analyses. A large number of enzymes with different sequence specificities are available. To date, most choices of restriction endonucleases have been made by trial and error. A computer program, REDI, has been developed that predicts the ability of a particular restriction enzyme to detect mutations. Characteristics of both the restriction endonuclease used and the DNA being cut are incorporated as variables in the program. The program was tested using mouse mitochondrial DNA (mtDNA) and bacteriophage lambda DNA because these have been sequenced and are well characterized. REDI was strongly correlated (rs = +0.862, n = 11, P less than 0.001) with mouse mtDNA RFLP detected by Ferris et al. [1] (Genetics, 105 (1983) 681-721). Even though predictions may be altered by a non-random association of nucleotides, which varies among DNA molecules, the predictions increase the probability of selecting the most efficient enzymes for use in the analysis of a particular DNA molecule.