RESUMEN
AIMS: The alkaliphilic haloarchaeon Natrialba magadii secretes a halolysin-like protease (Nep) that is active and stable in high salt and in organic solvents, which represents a potential resource for biocatalysis in low water activity conditions. In this study, the effect of the growth stage on Nep biosynthesis was examined. METHODS AND RESULTS: Nep mRNA and extracellular protease activity were measured by RT-PCR and azocaseinolytic activity determination, respectively. Increased abundance in Nep mRNA was observed in Nab. magadii cells with culture age, which correlated with accumulation of extracellular protease activity. Moreover, a 'stationary phase behavior' on synthesis of Nep was evidenced in low-density cultures incubated with stationary phase medium. CONCLUSIONS: nep gene expression is up-regulated during the transition to the stationary phase in response to 'factors' (metabolite and/or regulatory molecule) occurring in high-density cultures of Nab. magadii. Although the identity of these molecules remains to be determined, preliminary evidence suggests that they are hydrophobic and stable in high salt and high pH values (3.5 mol l(-1) NaCl, pH 10). SIGNIFICANCE AND IMPACT OF STUDY: This study contributes to gain insight into the regulation of haloarchaeal protease biosynthesis, facilitating the large-scale production of this extremozyme for basic studies or potential applications.
Asunto(s)
Proteínas Arqueales/biosíntesis , Regulación de la Expresión Génica Arqueal , Halobacteriaceae/enzimología , Péptido Hidrolasas/biosíntesis , Proteínas Arqueales/química , Caseínas/metabolismo , Estabilidad de Enzimas , Perfilación de la Expresión Génica , Concentración de Iones de Hidrógeno , Péptido Hidrolasas/química , ARN de Hongos/biosíntesis , ARN de Hongos/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sales (Química)RESUMEN
The allosteric behaviour of the p-nitrophenyl-phosphatase (E.C.3.1.3.1.) from membrane erythrocytes was investigated in the following multisystemic diseases: myotonic dystrophy, limb-girdle muscular dystrophy, Charcot-Marie-Tooth and juvenile spinal muscular atrophy; in myotonia congenita, which is not a multisystemic disease, and in healthy controls. The Hill coefficient in F inhibition in controls was different from that in multisystemic diseases patients but not from that in myotonia congenita patients. Changes in the cooperative type kinetics would suggest that the interaction membrane-enzyme in controls and in patients with neuromuscular disorders is only different for multisystemic diseases.
Asunto(s)
Fosfatasa Alcalina/análisis , Membrana Eritrocítica/enzimología , Distrofias Musculares/enzimología , Humanos , Distrofias Musculares/sangre , Enfermedades Neuromusculares/sangre , Enfermedades Neuromusculares/enzimologíaRESUMEN
Plants respond to pathogen infection and environmental stress by regulating the coordinate expression of many stress-related genes. In plants, the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced under environmental stress. This work was aimed at investigating whither the expression pattern of cytosolic GAPDH is also modulated upon infection of potato plants (Solanum tuberosum L.) with the late blight fungal agent Phytophthora infestans. Northern blot analysis showed the accumulation of the GAPDH gene transcripts in leaves and stems of inoculated potato plants. When tuber discs were treated with eicosapentaenoic acid (EPA), an elicitor found in P. infestans, GAPDH gene transcripts level increased. The increase was parallel to that of the hydroxymethyl glutharyl coenzyme A reductase (HMGR), an enzyme involved in pathogen defense reactions. Glucans obtained from P. infestans cell wall acts synergistically with EPA on GAPDH and HMGR gene induction. Salicylic acid, an endogenous signal for inducing systemic acquired resistance, was also effective in stimulating the GAPDH transcript accumulation in potato leaves. These experiments suggest that related multi-component factors, which are part of both primary and secondary metabolism, are probably regulated by similar signal transduction pathways when they are induced under biotic or abiotic stress conditions.