Pascal Baret

Enzyme kinetics by mid-infrared spectroscopy: β-fructosidase study by a one-step assay

An alternate method for enzyme study is proposed. Multidimensional statistical analysis applied on mid-infrared attenuated total reflectance spectra (Cadet et al. (1991) Appl. Spectrosc. 42, 166-172) collected during a kinetic allows a direct and fast quantification of the remaining substrate, as well as a one step enzymatic assay. Furthermore, the combination of these techniques may be used as a structural tool. The method applied to the study of beta-fructosidase is developed in this paper as an example. With appropriate calibration, the method may be extend to any enzyme.

Purification and characterization of a multienzymatic complex in sugarcane, a C4 plant

We have discovered a multienzymatic complex in fresh young sugarcane leaves. This complex is constituted of three enzymes: PEPcase, NADP-MDH and malic enzyme. After successive molecular sieving chromatography, we have obtained a highly purified sample of the complex which has a molecular weight of 711 kDa. Its functional interest has been evaluated by comparing the kinetic properties of the enzymes in their free forms to those in their complexed form. We show that the association of the three enzymes leads to important changes in their respective kinetic properties.

Microquantification of Proteins by Spectrophotometry. Part I: From 190 nm to 1100 nm, Selection of Wavelengths

Abstract Several methods for protein determination have been described. In almost all cases, a detection reagent is involved (Coomassie Blue reagent, bicinchoninic acid, Folin reagent). Proteins quantification determination by measurements in the U.V. region1 have been abandonned because of the weak sensitivity of the apparatus (concentrations of the order of 100 μg/ml) and because of the lack of precision at low wavelengths (far U.V.). Owing to the progress in the performance of equipments, (i) we have been able to show that the reliability of current equipments allowed measurements in the more sensitivity range of proteins (190 to 220 nm) and beyond (220 to 1100 nm) and (ii) we have hence calculated the correlation factors between absorbance values and wavelengths for 17 proteins. It was found that 190 nm and 277 nm were the best wavelengths in far U.V. region for the protein quantification (correlation factor respectively of 0.69 and 0.62). The concentrations of 16 proteins were predicted at 190 nm and...

Regulation of photosynthetic enzymes via redox systems

Introduction In the process of photosynthesis complex enzymatic reactions are involved particularly in the Benson-Calvin cycle which occurs in the chloroplasts. Some of these enzymes are inactive in darkness but are activated by light, hence, the cycle functions only during the day. Examples of such enzymes are fructose bisphosphatase (FBPase) and sedoheptulose bisphosphatase (SBPase) which are inactivated in darkness. Several mechanisms have been proposed that account for the activation and deactivation of the Benson-Calvin cycle. For example, upon illumination, the pH of the stroma increases from pH 7 to pH 8 owing to the active pumping of protons into the intrathylakoid space. This is accompanied by a transfer of Mg 2+ ions via the thylakoid membrane into the stroma. As the envelope of the chloroplast is impermeable to Mg 2+ ions, the concentration of Mg 2+ in the stroma increase from 1 to 2-5mm. Most of the enzymes of the Benson-Calvin cycle are sensitive to pH and to changes in Mg 2+ concentration but these changes, by themselves, do not wholly account for the activation of the cycle. Two other types of activation mechanisms have been shown: (1) the photoregulation of these enzymes in which light-regulated redox systems such as ferredoxins are involved, and (2) regulation via energy load and via the concentration of metabolites from the cycle.

Diabetes-induced hepatic oxidative stress: a new pathogenic role for glycated albumin.

Abstract Increased oxidative stress and advanced glycation end‐product (AGE) formation are major contributors to the development of type 2 diabetes. Here plasma proteins e.g. albumin can undergo glycoxidation and play a key role in diabetes onset and related pathologies. However, despite recent progress linking albumin‐AGE to increased oxidative stress and downstream effects, its action in metabolic organs such as the liver remains to be elucidated. The current study therefore investigated links between oxidative perturbations and biochemical/structural modifications of plasma albumin, and subsequent downstream effects in transgenic db/db mouse livers and HepG2 cells, respectively. Our data reveal increased oxidative stress biomarkers and lipid accumulation in plasma and livers of diabetic mice, together with albumin glycoxidation. Purified mouse albumin modifications resembled those typically found in diabetic patients, i.e. degree of glycation, carbonylation, AGE levels and in terms of chemical composition. Receptor for AGE expression and reactive oxygen species production were upregulated in db/db mouse livers, together with impaired proteolytic, antioxidant and mitochondrial respiratory activities. In parallel, acute exposure of HepG2 cells to glycated albumin also elicited intracellular free radical formation. Together this study demonstrates that AGE‐modified albumin can trigger damaging effects on the liver, i.e. by increasing oxidative stress, attenuating antioxidant capacity, and by impairment of hepatic proteolytic and respiratory chain enzyme activities. Graphical abstract Figure. No Caption available. HighlightsHigh oxidative stress and lipid accumulation in plasma and livers of diabetic mice.Albumin modifications in diabetic mice resembled those found in diabetic patients.Exposure of human hepatoma cells to glycated albumin elicited oxidative damages.

Microquantification of Proteins by Spectrophotometry. Part II : Application Procedure for Complex Mixture Containing Interfering Substances

Abstract In a previous paper we have shown that it was possible to quantify protein solutions at very weak concentrations directly by UV-visible spectroscopy. Nevertheless the protein quantification could not be possible if there is any trace of interferents left in the solution. So it is necessary to eliminate all the interferents to make the measure at 190 and/or 277 nm possible. We have developped a method based on the use of Microcon membranes and centrifugation. Interferents could be eliminated from protein solutions after four centrifugations at 13000 g during 5 min. This procedure allowed the recovering of proteins, with 80 to 99 % yield, and thus making microquantification possible. This method is particularly interesting for enzymatic solutions after a purification procedure by HPLC where very tiny quantities of protein are recovered. This protocol has been tested on three enzymes; enzymatic activity recovered after four centrifugations was quite high for PEPcase and malic enzyme (71 and 64 % res...

Contribution to the study of the Hatch and Slack photosynthetic cycle. Part II: Kinetic properties of the PEPcase-MDH-Malic enzyme multienzymatic complex purified from a C4 plant (sugarcane)

We found out the existence in fresh young leaves of sugarcane of a PEPcase-MDH(NADP)-Malic enzyme multienzymatic complex. We have been able to purify this complex. Its functional advantage has been investigated by comparing the kinetic properties of the constitutive enzymes in their free state with those in their complexed state.

Contribution to the study of the Hatch and Slack photosynthetic cycle. Part I: Purification of a PEPcase-MDH-malic enzyme multienzymatic complex in a C4 plant (sugarcane)

We found out the existence in fresh young leaves of sugarcane of a PEPcase-MDH(NADP)-Malic enzyme multienzymatic complex. The enzyme was purified as follows: