Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for unveiling the dynamics of cellular life.

Polyphenol oxidase (PPO) was isolated from grapes grown in Spain and its characteristics were studied. The partially purified enzyme had both cresolase and catecholase activities. Catecholase activity had a pH optimum in a range 3.5-4.5 and was characterized by a relatively high stability to heat. The apparent KM for 4-methylcatechol was 9.5 mM. Cresolase activity presents a lag period which is modulated by different factors: enzyme concentration, substrate concentration, temperature or pH. The presence of o-diphenols in the reaction medium abolishes the lag period, these acting as co-substrates. The apparent K, towards p-cresol and the activation constant for o-diphenol for cresolase activity were 0.35 mM and 1.75 CM, respectively. 4000 x g for 15 min. The precipitate was extracted for 30 min with 1.5% Triton X-100 in 100 mM phosphate buffer pH 7.3. Two percent water-insoluble polyvinyl pyrrolidone (PVP) and calcium chloride to give a final concentration of 0.05M were added to the' medium lo precipitate phenols and pectic substances (Cash et al., 1976). The mixture was centrifuged at 15000 x g for 1 hr. An (NH&SOJ fractionation was carried out and the fraction precipitating between 45% and 95% saturation collected and rcdissolvcd in 4 mL 10 mM phosphate buffer pH 7.0. This solution, after dialysis against the same buffer, was used as enzyme source.Protein concentration of the samples at different stages of purification were determined by the method of Lowry ct al. (1951), after precipitating phenols with PVP.

An easy effective method for the activation of commercial screen-printed carbon electrodes (SPCEs) using H 2 O 2 is presented to enhance sensing performances of carbon ink. Electrochemical activation consists of 25 repetitive voltammetric cycles at 10 mVs 1 using 10 mM H 2 O 2 in phosphate buffer (pH 7). This treatment allowed us to reach a sensitivity of 0.24±0.01 µA µM-1 cm-2 for the electroanalysis of H 2 O 2 , which is 140-fold higher than that of untreated SPCEs and 6-fold more than screen-printed platinum electrodes (SPPtEs). Electrode surface properties were characterized by SEM, EIS and XPS. The results revealed atomic level changes at the electrode surface, with the introduction of new carbon-oxygen groups being responsible for improved electrotransfer properties and sensitivity. Our method was compared with other previously described ones. The methodology is promising for the activation of commercial carbon inks-based electrodes for sensor applications.

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