Jean-Michel Girardet

Susceptibility of equine κ- and β-caseins to hydrolysis by chymosin

Abstract Equine whole casein was hydrolysed by chymosin and some peptides generated were characterised by microsequencing after reversed-phase high performance liquid chromatography or sodium dodecyl sulphate polyacrylamide gel electrophoresis. β-Casein was readily hydrolysed into amino- and carboxy-terminal fragments after cleavage of the Leu190–Tyr191 bond. These two fragments seemed to be resistant to further chymosin hydrolysis on incubation for up to 24xa0h. Equine κ-casein was purified by affinity chromatography on immobilised wheat germ agglutinin. O-Glycosylated κ-casein was found to represent less than 6.8% of the equine casein components. Equine κ-casein was also hydrolysed and para-κ-casein and glycomacropeptide were generated after cleavage of the Phe97–Ile98 bond.

Identification and characterization of a third thioredoxin h in poplar

Abstract Three full-length sequences encoding thioredoxin h have been isolated in a leaf/root of Populus trichocarpa cv. Trichobel expressed sequence tag (EST) library. One of these, popCXXS1 exhibits the nontypical active site CXXS homologous to atCXXS1. The second one, named popTrxh4, is related to atTrxh9 which forms with several other plant thioredoxin h a distinct subgroup of thioredoxins h. The third one, named popTrxh3, displays 66% identity and also a high degree of homology (81%) vs. the previously described popTrxh1. Nevertheless, the active sites of both proteins differ, since the active site of popTrxh1 (WCPPC) is a variant of the canonical WCGPC found in popTrxh3. The cDNA sequence of popTrxh3 has been introduced in an expression plasmid (pET3d) in order to express the corresponding recombinant polypeptide. The protein has been expressed to a high level, purified from Escherichia coli cells with a high yield and its catalytic properties compared to popTrxh1. Furthermore, two mutants, popTrxh1P40G and popTrxh3G41P, have been engineered in order to explore the importance of the active site residues in the thioredoxin h catalytic properties. The results are discussed in relation with known biochemical properties of thioredoxins h.

Surface active and emulsifying properties of casein micelles compared to those of sodium caseinate

Emulsions prepared using casein micelles and sodium caseinate as the emulsifying agent are compared. It is found that the ultrastructure of the casein proteins influences the properties of the emulsion.

Role of the O-phosphoserine clusters in the interaction of the bovine milk αs1-, β-, κ-caseins and the PP3 component with immobilized iron (III) ions

Abstract α s1 - and β-Caseins have a sequence cluster -Ser( P )-Ser( P )-Ser( P )-Glu-Glu- which is not present in κ-casein and the whey PP3 component. The affinity of these phosphoproteins for the iron(III)-iminodiacetic acid (IDA) complex immobilized on Sepharose was studied a a function of pH, urea concetnration, calcium ion concentration, enzymatic dephosphorylation and temperature. The affinity of the three polyphosphorylated proteins ( α s1 - and β-caseins, PP3) was similar. The sequence cluster was not a specific recognition pattern for the iron(III) ion. These three proteins presented a site of high affinity and a site of weak affinity. κ-Casein, which had only one Ser( P ) residue, presented only the site of weak affinity. Their primary site which was absent after dephosphorylation or calcium ion addition required the presence of at least two Ser( P ) residues close in space. Their secondary site was sensitive to the presence of urea. It was sensitive to pH variation for PP3 and κ-casein. The study of the affinity of a few free amino acids towards iron(III)-IDA showed that the secondary site involved tryptophan and tyrosine residues for α s1 - and β-caseins, histidine residues for PP3 and cysteine residues for κ-casein.

Determination of reducing power and metal chelating ability of antioxidant peptides: Revisited methods

The purpose of this study was to improve two common tests used for antioxidant capacity measurements, i.e. the reducing power and chelating ability measurements, for appropriate comparisons between the molecules tested and chosen references, as the usual methods are often performed in a qualitative way rather than a quantitative way. After revision, it was then possible to determine an AERC indice (Ascorbate Equivalent Reducing Capacity) and a CECC (Carnosine Equivalent Chelating Capacity) or EECC (EDTA Equivalent Chelating Capacity) indice according to the standard chosen, by analogy to the TEAC indice (Trolox Equivalent Antioxidant Capacity) already used in many reported works to determine the free radical scavenging activity. Thus, the determination of these relative indices enables the comparison of antioxidative capacities obtained in various studies. The adaptation of these two tests to micro-scales and the calculation of AERC, EECC and CECC were performed on model peptides.

Fast protein liquid chromatography purification of hydrophobic fraction of bovine milk proteose-peptone and characterization by bidimensional electrophoresis

Bovine milk Hydrophobic fraction of proteose-peptone was prepared by hydrophobic interaction fast protein liquid chromatography. This method has several advantages such as high rapidity, simple good reproducibility and less denaturation. The proteose-peptone was eluted from a TSK-Phenyl-5PW column with a 1 M-0 M ionic strength gradient of NaH 2 PO 4 , pH 6·8, using a 6 ml/min flow rate for 56 min. The quantity of protein injected was 62·5 mg; however, it could be increased up to 100 mg. The elution order was β -CN-4P β -CN-5P β -CN-1P. The hydrophobic fraction was obtained in pure water at the end of the gradient (17·3% of total N). A proteose-peptone cartograph was achieved by bidimensional electrophoresis. This hydrophobic fraction represented three principal zones of M r 30000–28000, 19000 and 11000, which were respectively composed of 13, 4 and 2 principal spots distributed between 4·9 and 6·1 isoelectric points (IP). These spots corresponded to glycoproteins. ·7, 5·0 and 5·1 IP which migrated to M r 18000 while β -CN-1P was identified as M r 9000 in two spots of 5·1 and 5·3 IP.

Hydrolysis of milk-derived bioactive peptides by cell-associated extracellular peptidases of Streptococcus thermophilus

The trend to confer new functional properties to fermented dairy products by supplementation with bioactive peptides is growing in order to encounter the challenge of health-promoting foods. But these functional ingredients have not to be hydrolysed by proteases of bacteria used in the manufacture of these products. One of the two yoghurt bacteria, Streptococcus thermophilus, has long been considered as weakly proteolytic since its only cell wall-associated subtilisin-like protease, called PrtS, is not always present. Nevertheless, a recent study pointed out a possible peptidase activity in certain strains. In this present study, the stability of milk-derived bioactive peptides, e.g. the anxiolytic peptide, αs1-CN-(f91-97), in the presence of two different S. thermophilus strains with PrtS+ or PrtS− phenotype was studied. Both strains appeared to be capable of hydrolysing the αs1-CN-(f91-97) and other bioactive peptides by recurrent removal of N-terminal residues. The hydrolysis was neither due to intracellular peptidases nor to HtrA protease. Results obtained showed that the observed activity originates from the presence at the surface of both strains of an extracellular aminopeptidase activity. Moreover, a cell wall-associated X-prolyl dipeptidyl peptidase activity was also highlighted when β-casomorphin-7 was used as substrate. All of these findings suggest that, in order to use fermented milks as vector of bioactive peptides, the stability of these bioactive peptides in this kind of products implies to carefully characterize the potential action of the surface proteolytic enzymes of S. thermophilus.

Action of plasmin on equine β-casein

Abstract Equine β-casein and, in a lesser extent, αs1- and κ-caseins were good substrates for plasmin in solution. The Lys47–Ile48 bond of β-casein was readily hydrolysed leading to the production of γ-like caseins of 23xa0kDa. These γ-like caseins were degraded by plasmin in solution subsequently to their formation. The presence of PP5-like peptides of 20xa0kDa associated to caseins seemed to arise from endogenous plasmin activity in equine milk. Their formation was, however, not obvious in solution, as the amino-terminal region of β-casein seemed to be readily hydrolysed by plasmin. γ-caseins were generated when a sodium caseinate solution was incubated at 37°C and pH 8.0 for 24xa0h or when caseinate was stored in a freeze-dried form at 7°C for 2 years. This might be due to the presence of low amount of endogenous plasmin associated to equine caseinate as evidenced by assays with a specific synthetic substrate.

Camel (camelus dromedarius) milk PP3: evidence for an insertion in the amino-terminal sequence of the camel milk whey protein.

The camel (camelus dromedarius) milk proteose peptone 3 (PP3) was purified successively by size exclusion fast protein liquid chromatography and reversed phase high performance liquid chromatography and then characterized by amino acid residue composition determination and chemical microsequencing after CNBr or trypsin cleavages. In comparison with the previously reported structure of camel milk whey protein, the camel PP3 contains an insertion in the N-terminal region which has approximately 24 residues, whereas the remaining C-terminal regions of these two homologous proteins are essentially identical. The camel PP3 seems to contain a potential O-glycosylation site localized in this insertion and 2 or 3 phosphorylated serine residues. PP3 belongs to the glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) family and could therefore play an immunological role in the camel or its suckling young.

Insights into ascorbate regeneration in plants: investigating the redox and structural properties of dehydroascorbate reductases from Populus trichocarpa.

Dehydroascorbate reductases (DHARs), enzymes belonging to the GST superfamily, catalyse the GSH-dependent reduction of dehydroascorbate into ascorbate in plants. By maintaining a reduced ascorbate pool, they notably participate to H2O2 detoxification catalysed by ascorbate peroxidases (APXs). Despite this central role, the catalytic mechanism used by DHARs is still not well understood and there is no supportive 3D structure. In this context, we have performed a thorough biochemical and structural analysis of the three poplar DHARs and coupled this to the analysis of their transcript expression patterns and subcellular localizations. The transcripts for these genes are mainly detected in reproductive and green organs and the corresponding proteins are expressed in plastids, in the cytosol and in the nucleus, but not in mitochondria and peroxisomes where ascorbate regeneration is obviously necessary. Comparing the kinetic properties and the sensitivity to GSSG-mediated oxidation of DHAR2 and DHAR3A, exhibiting 1 or 3 cysteinyl residues respectively, we observed that the presence of additional cysteines in DHAR3A modifies the regeneration mechanism of the catalytic cysteine by forming different redox states. Finally, from the 3D structure of DHAR3A solved by NMR, we were able to map the residues important for the binding of both substrates (GSH and DHA), showing that DHAR active site is very selective for DHA recognition and providing further insights into the catalytic mechanism and the roles of the additional cysteines found in some DHARs.