Eugenio Giachetti

Isocitrate lyase from higher plants

Abstract Work on isocitrate lyase, the first enzyme unique to the glyoxylate cycle, is reviewed.

What students must know about the determination of enzyme kinetic parameters

Abstract 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 V max and K m 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.

An isocitrate lyase of higher plants: analysis and comparison of some molecular properties

A new purification procedure for isocitrate lyase from Pinus pinea is reported. The final preparation shows charge homogeneity and a purity degree higher than 95%. It is possible to remove catalase completely by exploiting the high hydrophobicity of isocitrate lyase. The enzyme has a Mr of 264,000 and is likely composed of four subunits, each with a Mr of 66,000. The binding of radioactively labeled oxalate revealed four catalytic sites per oligomer. These data suggest that isocitrate lyase subunits are similar, if not identical. The Michaelis constant for isocitrate is equal to 33 microM; molecular activity is about 2670 mol X min-1 X mol of enzyme-1. The amino acid composition of the enzyme was also determined. Isocitrate lyase appears resistant to proteolysis by carboxypeptidase A. Hydrazinolysis, Edman degradation, and dansyl chloride treatment indicate that both carboxy and amino terminals are probably inaccessible or blocked.

Isocitrate lyase of conifers (Pinus pinea

1. Isocitrate lyase has been purified about 60 times from the conifer Pinus pinea. A first characterization was made. 2. The high instability is an important feature of this enzyme from higher plants, this causes serious problems in the purification and characterization. 3. A substantial agreement with the data from the literature was found for what concerns pH dependence of Vmax and pKm, the effect of bivalent cations and the requirement of Mg2+. 4. Kinetic studies gave evidence for a mechanism ordered uni-bi with glyoxylate being the last product released, kinetic constants were calculated, no evidence for cooperative effects was found. 5. Equilibrium constant by Haldane method calculation agrees with value calculated with isocitrate lyase from the bacterium Pseudomonas indigofera.

A new continuous optical assay for isocitrate lyase

A new continuous optical assay method for isocitrate lyase is reported. This is a coupled assay which requires lactate dehydrogenase as an ancillary enzyme. The method yields linear data up to 0.12 units/ml. The assay is also suitable for crude extracts.

Isocitrate lyase: Artifacts and multiple enzyme forms

Multiple enzyme forms of isocitrate lyase from various sources have been frequently reported. Protease action after cell rupture was sporadically claimed to explain the observed multiple enzyme forms. In this communication studies which are consistent with a protease action in vitro on isocitrate lyase of Pinus pinea germinating seeds are reported. Moreover, changes in DEAE-Sephacel patterns, mainly related to the age of germination, were observed. Differences regarding the heat stability of the detected enzyme forms were also found. The results indicate that isocitrate lyase from P. pinea may be detected in at least three different forms, one of which is heat stable and may be obtained only at the early stages of germination.

Gravitational stress on germinating Pinus pinea 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.

Proteolytic activity in pure preparations of Pinus pinea isocitrate lyase

Abstract A proteinase was copurified with Pinus pinea isocitrate lyase. Its proteolytic action, consisting of isocitrate lyase irreversible inactivation and the appearance of enzyme forms with lower M r s, became evident when Mg 2+ was removed from pure preparations or when EDTA or SDS was added. This action was greatly reduced by Mn 2+ or oxalate, and partially by PMSF, but not by several other protease inhibitors. Some data suggest that the proteolytic activity may be adsorbed on isocitrate lyase. A model for isocitrate lyase inactivation, involving Mg 2+ dissociation, is proposed.

Enzyme catalysis in microgravity: steady-state kinetic analysis of the isocitrate lyase reaction

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.

A continuous spectrophotometric assay for alkaline phosphatase with glycerophosphate as substrate

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.