Activity of immobilised rat hepatic microsomal CYP2E1 using alumina membrane as a support.

Porous alumina membranes are attractive materials for the construction of biosensors and also have utility for the production of immobilised enzyme bioreactors. Microsomes from rat liver were adsorbed onto alumina membrane activated by silane. Microsomal membranes were pumped through the channels where they became immobilised by binding to amine groups on the surface of the alumina membrane. In an effort to gain a quantitative understanding of the effects of microsomal film growth on enzyme activity, we compared the para-nitrophenol (pNP) hydroxylase activity of the microsomes by varying the amount of microsomes fixed in alumina microchannels. The alumina membrane was placed in a fluidic device at a fast flow that afforded short residence time (seconds) to obtain transformation of pNP to 4-nitrocatechol (pNC), which was detected by LC-MS/MS. This enabled the use of this bioreactor where CYP2E1 activity is low and tissue sources are limiting. The microsomes, successfully immobilised on the alumina membranes, were used to produce stable biocatalytic reactors that can be used repeatedly over a period of 2 months.

Electrically assisted extraction of soluble matter from chardonnay grape skins for polyphenol recovery.

The objective of this study was to investigate the effects of pulsed electric field (PEF) and high-voltage electrical discharges (HVED) application on the efficiency of aqueous extraction of total soluble matter and polyphenols from grape skins ( Vitis vinifera L.) at different temperatures within 20-60 degrees C. The highest level of polyphenol concentration C was reached after about 60 min of extraction for HVED treatment: C(HVED) = 21.4 +/- 0.8 micromol of gallic acid equivalent (GAE)/g of dry matter (DM). Almost the same level of C was reached after 180 min of extraction for the PEF-treated skins. These levels exceeded the value C = 19.1 +/- 0.5 micromol of GAE/g of DM for the untreated samples. The difference between degrees Brix values for HVED-treated and untreated systems decreased with temperature increase (from 40 to 60 degrees C), but a large difference in the total amount of polyphenols was observed for HVED-treated and untreated systems. The activation energies were W(u) = 31.3 +/- 3.7 kJ/mol and W(PEF) = 28.9 +/- 5.5 kJ/mol for untreated and PEF-treated systems, respectively.

Carnitine is associated with fatty acid metabolism in plants.

The finding of acylcarnitines alongside free carnitine in Arabidopsis thaliana and other plant species, using tandem mass spectrometry coupled to liquid chromatography shows a link between carnitine and plant fatty acid metabolism. Moreover the occurrence of both medium- and long-chain acylcarnitines suggests that carnitine is connected to diverse fatty acid metabolic pathways in plant tissues. The carnitine and acylcarnitine contents in plant tissues are respectively a hundred and a thousand times lower than in animal tissues, and acylcarnitines represent less than 2% of the total carnitine pool whereas this percentage reaches 30% in animal tissues. These results suggest that carnitine plays a lesser role in lipid metabolism in plants than it does in animals.

Anti-idiotypic antibody mimics proteolytic function of parent antigen.

Functional imaging of subtilisin Carlsberg active center by the idiotypic network yielded a catalytic anti-idiotypic antibody with endopeptidase, amidase, and esterase activities. A monoclonal antibody inhibitory to subtilisin (Ab1 5-H4) was employed as the template for guiding the idiotypic network to produce the catalytic anti-idiotypic Ab2 6B8-E12. Proteolytic activity of 6B8-E12 was demonstrated by zymography using self-quenched fluorescein-BSA conjugate and in a coupled assay detecting Ab2-dependent RNase A inactivation. Cleavage of peptide substrates by 6B8-E12 revealed distinct patterns of hydrolysis with high preference for aromatic residues before or after the scissile bond. Catalytic activity of Ab2 was inhibited by phenylmethylsulfonyl fluoride, a mechanism-based inhibitor of serine hydrolases. 5-H4 and 6B8-E12 were cloned, produced in Escherichia coli as single-chain variable fragments (scFvs), and purified. Kinetic parameters for amidolytic and esterolytic activities were similar in Ab2 and its scFv derivative. Although the antigen-specific portion of 6B8-E12 possesses no primary structure similarity to subtilisin, it mimics proteolytic and amidolytic functions of the parental antigen, albeit with 4 orders of magnitude slower acceleration rates. The lack of detectable endopeptidase activity of 6B8-E12 scFv raises interesting issues concerning general evolution of catalytic activity. The in silico 3D models of Ab1 and Ab2 revealed strong structural similarity to known anti-protease antibodies and to abzymes, respectively. These results indicate that the idiotypic network is capable, to a significant extent, of reproducing catalytic apparatus of serine proteases and further validate the use of imaging of enzyme active centers by the immune system for induction of abzymes accelerating energy-demanding amide bond hydrolysis.

The peptaibol alamethicin induces an rRNA-cleavage-associated death in Arabidopsis thaliana.

The plant-metabolic response to amphipathic peptides produced by the soil fungi of the genus Trichoderma remains largely unknown. The present investigation was undertaken to examine the death process in alamethicin-treated Arabidopsis thaliana plantlets. The rapid death triggered by alamethicin (at 50 microM) was shown to be associated with protein-synthesis arrest and with specific cleavage of 18S and 25S ribosomal RNA. The use of an inhibitor of nitric oxide (NO) synthases and of an NO scavenger suggested that rRNA cleavage was suppressed by NO. Experiments conducted with a synthetic alamethicin analogue, in which all alpha-aminoisobutyric acid (Aib) residues have been replaced by leucine moieties, showed that the non-coded residues are essential for the ability of the peptaibol to induce rRNA cleavage in Arabidopsis. Our data indicate that further investigations on the mode of action of alamethicin in planta could be of great interest to study the death-signaling pathway associated with rRNA degradation in plants.

Recovery of the oxidative activity of caged bovine haemoglobin after UV photolysis.

Caging of bovine haemoglobin with increasing amounts of 1-(2-nitrophenyl)ethyl (NPE) and uncaging after a 366 nm irradiation was examined. Caged and photolysed conjugates were characterised by enzymatic assay of the ABTS oxidation, UV/Vis absorbance, and electrospray mass ionisation. Modification of haemoglobin with 50, 75, and 100 equivalents of 1-(2-nitrophenyl)diazoethane led to a progressive decrease of enzymatic activity. Photolysis at 366 nm during 5, 15, and 30 min induced the recovery of a part of the enzymatic activity. ESI analyses showed that a reversible binding of up to 6 NPE groups per alpha-chain and that the removal of most of the photolabile groups occurred rapidly after 5 min of illumination at 366 nm and reached near completion after 15 min. A variable alteration of haemoglobin after labelling could explain that the complete removal of NPE groups did not restore its full oxidative activity.