Myo-inositol-1-phosphatase (EC 3.1.3.25) is able to hydrolyze myo-inositol-1-phosphate in the presence of Mg(2+) ions at neutral pH, and also p-nitrophenyl phosphate in the presence of Zn(2+)-ions at acidic pH. This enzyme plays a role in phosphatidylinositol cell signalling and is a putative target of lithium therapy in manic depression. We elucidate here the kinetic mechanism of the Zn-dependent activity of myo-inositol-1-phosphatase. As part of this analysis it was necessary to determine the basicity constants of p-nitrophenyl phosphate and the stability constant of its metal-complex in the presence of zinc chloride. We find that the Zn-dependent reaction may be described either by a rapid-equilibrium random mechanism or an ordered steady-state mechanism in which the substrate binds to the free enzyme prior to the metal ion. In both models the Zn-substrate complex acts as a high affinity inhibitor, yielding a dead-end species through its binding to the enzyme-Zn-substrate in rapid-equilibrium or to the enzyme-phosphate complexes in a steady-state model. Phosphate is a competitive inhibitor of the enzyme with respect to the substrate and an uncompetitive inhibitor with respect to zinc ions.

In the germination of lipid-rich seeds, the glyoxylate cycle plays a control role in that, bypassing the two decarboxylative steps of the Krebs cycle; it allows the net synthesis of carbohydrates from lipids. The activity of isocitrate lyase, the key enzyme of the glyoxylate cycle, is an indicator of the state of seed germination: stage of germination, growth of embryo, activation and progress of protein synthesis, depletion of lipidic supplies. In order to investigate the effects of gravity on seed germination, we carried out a study on the time pattern of germination of Pinus pinea seeds that were subjected to a hypergravitational stress (1000 g for 64 h at 4 degrees C), either in a dry or in a wet environment, before to be placed in germination plates. During the whole time of germination, we monitored the state of embryo growth and the most representative enzymes of the main metabolic pathways. In treated wet seeds, we observed an average germination of only 20% with a slowdown of the enzyme activities assayed and a noticeable degradation of lipidic reserves with respect to the controls. These differences in germination are not found for dry seeds.

In the germination of lipid-rich seeds, the glyoxylate cycle plays a control role in that, bypassing the two decarboxylative steps of the Krebs cycle; it allows the net synthesis of carbohydrates from lipids. The activity of isocitrate lyase, the key enzyme of the glyoxylate cycle, is an indicator of the state of seed germination: stage of germination, growth of embryo, activation and progress of protein synthesis, depletion of lipidic supplies. In order to investigate the effects of gravity on seed germination, we carried out a study on the time pattern of germination of Pinus pinea seeds that were subjected to a hypergravitational stress (1000 g for 64 h at 4 degrees C), either in a dry or in a wet environment, before to be placed in germination plates. During the whole time of germination, we monitored the state of embryo growth and the most representative enzymes of the main metabolic pathways. In treated wet seeds, we observed an average germination of only 20% with a slowdown of the enzyme activities assayed and a noticeable degradation of lipidic reserves with respect to the controls. These differences in germination are not found for dry seeds.

Two decades of research in microgravity have shown that certain biochemical processes can be altered by weightlessness. Approximately 10 years ago, our team, supported by the European Space Agency (ESA) and the Agenzia Spaziale Italiana, started the Effect of Microgravity on Enzyme Catalysis project to test the possibility that the microgravity effect observed at cellular level could be mediated by enzyme reactions. An experiment to study the cleavage reaction catalyzed by isocitrate lyase was flown on the sounding rocket MASER 7, and we found that the kinetic parameters were not altered by microgravity. During the 28th ESA parabolic flight campaign, we had the opportunity to replicate the MASER 7 experiment and to perform a complete steady-state analysis of the isocitrate lyase reaction. This study showed that both in microgravity and in standard g controls the enzyme reaction obeyed the same kinetic mechanism and none of the kinetic parameters, nor the equilibrium constant of the overall reaction were altered. Our results contrast with those of a similar experiment, which was performed during the same parabolic flight campaign, and showed that microgravity increased the affinity of lipoxygenase-1 for linoleic acid. The hypotheses suggested to explain this change effect of the latter were here tested by computer simulation, and appeared to be inconsistent with the experimental outcome.

There are several reports indicating that weightlessness can affect certain biochemical processes. Most of the data are limited to cellular level, and the microgravity effects appear to be often contrasting [1]. It was to identify the physical–chemical link between microgravity and its potential cell sensors that about ten years ago we started the EMEC (Effect of Microgravity on Enzyme Catalysis) project, with the financial support from the European Space Agency (ESA) and the Agenzia Spaziale Italiana. In 1996, we studied the isocitrate lyase (EC 3.1.3.1) reaction during the 6 min microgravity time (average value 10−5 g) obtained by the parabolic flight of the sounding rocket MASER 7 (April 16–May 3, 1996, Kiruna, Sweden). The comparison with the data of a simultaneous test performed on ground with the same experimental hardware (the EMEC module [2]) showed that microgravity does not affect the enzyme kinetic parameters: saturation velocity and Michaelis constant [3]. Recently, an experiment performed during the 28th ESA parabolic flight campaign (May 15–26, Bordeaux, France) showed that in microgravity, the Michaelis constant of soybean lipoxygenase-1 for linoleic acid is decreased to one fourth of the ground control [4]. During the same parabolic flight campaign, we studied both the cleavage and the condensation reactions of Pinus pinea isocitrate lyase. Technically, the experiment was hosted on board a A300 Zero-G aircraft which, by means of special flight maneuvers, allows 20 s microgravity periods (about 10−2 g), alternating with hypergravity phases (1.8 g) and longer standard g conditions. The cleavage reaction (isocitrate=succinate+glyoxylate) was assayed by way of chemical coupling with phenylhydrazine; the formation of glyoxylate phenylhydrazone was followed at 324 nm at varying threo-D s-isocitrate concentrations (range 0.05–0.3 mol/dm3). The condensation reaction (glyoxylate+succinate=isocitrate) was coupled with isocitric dehydrogenase (NADP+) and the reduction of the coenzyme was monitored at the same wavelength. The condensation reaction assay consisted of 16 tests at four fixed concentrations of succinate (range 0.3–2.0 mol/dm3) each at four varied glyoxylate concentrations (range 0.04–0.2 mol/dm3). One session of tests was devoted to study the product inhibition by succinate in the cleavage reaction. The values of the kinetic constants as determined in microgravity and at 1 g are compared in Table 1; the parameters for the cleavage reaction are also reported. Our results indicate that in either direction, isocitrate lyase reaction is not affected by microgravity. To explain the microgravity effect observed on lipoxygenase affinity for linoleic acid, the authors [4] suggested that gravity may affect the reaction–diffusion process which occurs in enzyme catalysis, supporting their reasoning in the same way as Papaseit et al. [6] justified the observation that microtubule self-organization is gravity dependent. Our negative results, which come out from two distinct microgravity experiments, and from the analysis of both the forward and reverse enzyme reactions, prove that such theoretical considerations do not have general validity. Therefore, it is now necessary to understand why some reactions are sensitive, whereas others appear to be totally immune from the influence of the gravitational field. Probably, there are some other critical factors to be taken into account – mass ratio, density and/or nature of the reactants, catalytic mechanism, reversibility itself of the reaction, and/or time – to explain the opposite results observed. We think this kind of experimentation deserves to be continued and intensified, because through this way we could get new insights in understanding the biophysical and molecular basis of both the catalytic mechanisms and the striking efficiency of the enzymes.

Our results show that the phosphate ion is a nonlinear competitive inhibitor of Pinus pinea isocitrate lyase. In addition, this compound induces a sigmoidal response of the enzyme, which usually exhibits standard Michaelis-Menten kinetics. This peculiar behavior of P. pinea isocitrate lyase could be explained by a dimer (two-site) model, in which phosphate binds cooperatively, but the affinity of the vacant site for substrate (the magnesium-isocitrate complex) remains the same. As a result, the interaction of phosphate with free enzyme produces an inhibitor-enzyme-inhibitor species that is of significant importance in determining reaction rate; a possible regulatory role of the glyoxylate cycle by inorganic phosphate is suggested. The mode of phosphate inhibition is consistent with both the mechanism for magnesium ion activation of P. pinea isocitrate lyase and its site heterogeneity. Our results explain the cooperative effects observed by some authors in kinetic studies of isocitrate lyase carried out in phosphate buffers and also account for the higher K(m) values determined by using such assay systems. Phosphate buffer should be avoided in performing isocitrate lyase kinetics.

After a brief overview of the limits of the graphical methods used to determine enzyme kinetic parameters, the paper shows the results of their application to simulated velocity data, influenced by experimental errors of increasing magnitude. The comparison indicates that the best method to evaluate VmaxandKm is nonlinear regression, even in the presence of constant relative error; whereas the double reciprocal plot should be avoided, unless used with a proper weighting factor. The paper also suggests a simple method to obtain computer-simulated velocity data, with which the student may get direct practise and experience.

Although an effect has been recognized on certain cell functions, much work still remains to do to clarify which are the molecular mechanisms and parameters affected by gravity. On this subject it was born the EMEC project whose aim was to investigate the effect of microgravity on enzyme catalysis. The experiment must be considered technically successful. In the examined system, under our experimental conditions, microgravity seems to have no appreciable effect on enzymatic catalysis. From theoretical studies we obtained that the gravitational energy is always negligible if compared to energies of strong chemical bonds, but it has the same order of magnitude as energies of both thermic motions at environmental temperatures and weak chemical bonds if we consider macromolecules with high molecular weight, such as proteins. We propose to continue our studies investigating the kinetics of protein denaturation-renaturation processes in altered gravity condition since some energies involved in maintaining protein structure are of the same magnitude of the energy related with the gravitational force.

This paper deals with a microgravity experiment concerning the EMEC project (Effect of Microgravity on Enzymatic Catalysis), performed during the parabolic flight of the sounding rocket MASER 7, launched from the base of Esrange (Kiruna, Sweden) on May 3, 1996. The experiment consisted of performing, in a microgravity environment, a number of velocity measurements of an enzyme (isocitrate lyase) catalyzed reaction at different substrate concentrations, to calculate the kinetic parameters (Km and Vmax), which were compared with those obtained at standard gravity, with identical instrumentation. The experimental hardware, the EMEC module, expressly set up by Officine Galileo (Firenze, Italy) with the financial support of the European Space Agency, was a multichannel fibre-optics radiometer, equipped with an automatic injection system, that allowed to measure simultaneously the transmittance changes in 16 reaction cells. The results indicated that under the experimental conditions applied, microgravity has no appreciable effect on the enzyme kinetic constants.

The Effect of Microgravity on Enzymatic Catalysis (EMEC) is an experiment conceived by Prof. P. Vanni of the University of Firenze, aimed at attaining information about the Microgravity influence on the molecular interactions of the biological machinery (i.e. mechanisms involved in enzyme catalysis). Enzyme reactions are presently well characterised vs. chemical and physical parameters (e.g. pH, ionic strength, temperature, pressure), but almost nothing is known about how gravity influences them. In the present work we describe the philosophy and the hardware, developed by Officine Galileo, of the experiment, which has been successfully flown on the Sounding Rocket MASER 7 (3 May 1996), which allowed 370 s of reduced gravity (<10 -5 g).

In this work on Rhodotorula gracilis, we studied the level of the representative enzymes of foundamental metabolic ways (HK, PK, G6PDH and IDH) and those of ICL and MS key enzymes of glyoxylate cycle with different carbone sources. The glucose appears the best source of carbone for all the tested enzymes except ICL and MS which are practically absent in the cells developed with this carbone source. In presence of acetate and ethanol we have obtained the expression of the glyoxylate cycle enzymes (ICL and MS). Moreover we obtained the operativity of the glyoxylate cycle in the lipid-rich cells or cells developped on neutral detergents or anionics similar to the fatty acids.

We measured the level of second messengers, the activity of carbohydrate metabolism enzymes, and the resistance to ionizing radiations in normal 32D hematopoietic cells, in v-erbB transformants and in spontaneous transformants. v-erbB and spontaneous transformants were resistant to radiations as compared with their normal counterpart. The second messenger diacylglycerol was elevated in radioresistant clones. Only v-erbB transformants showed increase of the activities of enolase and glucose-6-phosphate dehydrogenase. v-erbB-transformed NIH/3T3 cells, selected as control, showed identical correlation between radioresistance, increase of diacylglycerol, and of enolase and glucose-6-phosphate dehydrogenase activity. These results indicate that increase of diacylglycerol is correlated with resistance to the killing effect of ionizing radiations and could be proposed as a marker of radioresponse.

We describe a continuous coupled spectrophotometric assay for alkaline phosphatase which uses alpha- or beta-glycerophosphate as substrate, and glycerol dehydrogenase as ancillary enzyme. The glycerol liberated by alkaline phosphatase is determined by measuring the increase in absorbance at 340 nm caused by NADH formation that is combined with glycerol oxidation by the ancillary enzyme. The assay procedure was optimized using a bovine bone extract as alkaline phosphatase source.

We found that the fern leaf crystallisation of human saliva is a non-specific phenomenon, independent on the action of estrogenic hormones. Salivary ferning can be obtained in practice during the whole menstrual cycle and occurs in the saliva of postclimateric, pregnant and prepuberal women. Even the saliva of male subjects gives invariably the ferning reaction. In conclusion, salivary ferning does not seem a reliable method to establish woman fertile period.

We describe a simple method for the analysis of activation systems in which a metal ion modifier may combine with either the enzyme or the substrate (or both) and the metal ion-substrate complex is the true substrate of the enzyme reaction. The suggested approach is essentially a ‘graphical’ method that both provides unbiased criteria for the choice of the activation mechanism and yields good rough estimates of the kinetic parameters. The procedure, tested on a variety of simulated models, produces appropriate and reliable results. Applying this treatment to isocitrate lyase, we confirmed the data previously reported for Mg2+ [Giachetti, Pinzauti, Bonaccorsi & Vanni (1988) Eur. J. Biochem. 172, 85-92], and we found that Mn2+ functions with the same mechanism as does Mg2+, but with quite different kinetic constants. In particular, its ratio of the Vmax, values of the activated and the non-activated enzyme is less than 1, and thus Mn2+ is to be considered an inhibitor rather than an activator.

It is well-established that mitochondria are the powerhouses of the cell, producing adenosine triphosphate (ATP), the universal energy currency. However, the most significant strengths of the electron transport chain (ETC), its intricacy and efficiency, are also its greatest downfalls. A reliance on metal complexes (FeS clusters, hemes), lipid moities such as cardiolipin, and cofactors including alpha-lipoic acid and quinones render oxidative phosphorylation vulnerable to environmental toxins, intracellular reactive oxygen species (ROS) and fluctuations in diet. To that effect, it is of interest to note that temporal disruptions in ETC activity in most organisms are rarely fatal, and often a redundant number of failsafes are in place to permit continued ATP production when needed. Here, we highlight the metabolic reconfigurations discovered in organisms ranging from parasitic Entamoeba to bacteria such as pseudomonads and then complex eukaryotic systems that allow these species to adapt to and occasionally thrive in harsh environments. The overarching aim of this review is to demonstrate the plasticity of metabolic networks and recognize that in times of duress, life finds a way.