Jacques Raymond

Purification and some properties of polyphenoloxidase from sunflower seeds.

Abstract The polyphenoloxidase from Helianthus annuus was purified by a combination of SP-Sephadex G-50 chromatography, DEAE-cellulose chromatography, Sepha

The seed proteins of sunflower : comparative studies of cultivars

Abstract The seeds from nine sunflower cultivars were compared on the basis of hull, lipid, protein contents and amino acid composition. Comparative studies of protein composition were carried out by sodium dodecyl sulphate polyacrylamide gel clectrophoresis (SDS-PAGE), isoelectric focusing of non reduced and/or reduced samples, in two dimensional procedures. Those experiments indicated no major differences in the cultivars. The electrophoretic patterns of 11S globulin (helianthinin) and albumin fractions or oil bodies membrane proteins were qualitatively and quantitatively similar among cultivars. The similarity was particularly evident in the helianthinin composition at the level of their polypeptide constituents from the standpoint of number, molecular size and charge. As was observed in the case of minor constituents, the major two α (Mrca 38 000 and ca 32 000) and β (Mr 21 000–25 500) polypeptides families constituting the main subunits displayed the same heterogeneity among cultivars. From these comparative studies, an accurate description and nomenclature of sunflower seed storage proteins constituents is given. Breeding programmes or environmental growing conditions appeared to have little or no effect on the qualitative gene expression encoding for storage proteins.

Influence of double enzymic hydrolyses on gluten functionality

Functional properties of chemically deamidated and/or double enzymically-treated gluten were studied. Performances of the modified glutens were compared to those of egg white proteins, casein, deamidated gluten and gluten submitted to deamidation plus single enzymic treatment. Higher nitrogen solubility index (>90%) was obtained after treatment of the gluten with the sequence pepsin + alcalase and deamidation + pepsin + papain. The sequence pepsin + papain has permitted the production of a gluten with foaming properties close to those of egg white. Deamidation + papain treatment leads to interesting emulsifying properties although different from those of casein. The results show that modified glutens present thresholds in the degree of enzymic hydrolysis (DH) for foaming and emulsifying properties (respectively 1.3% and 2.0%). For higher DH the stability decreases.

Proteasome and myogenesis.

In this report, we examine the involvement of the ubiquitin-proteasome pathway during fusion and differentiation of myoblast primary cell cultures. Up-regulation of proteasome was observed at the maximum fusion rate and was preceded by an increase of unidentified ubiquitin-conjugates. Cell permeable proteasome inhibitors prevent fusion as do antisense oligodesoxyribonucleotides targetted to three proteasome subunits. Identical results were obtained using E3 ubiquitin-ligases dipeptide inhibitor. Involvement of the ubiquitin-proteasome pathway in the regulation of myogenic factors was hypothetized.

20S proteasome, hsp90, p97 fusion protein, PA28 activator copurifying oligomers and ATPase activities.

Whether hsp90 acts in an ATP‐depeadent or independent way is of crucial importance for understanding the molecular mechanism of this chaperone and, to day, the involvement of ATP hydrolysis in hsp90 function is still a contreversial subject. ATPase activities may be detected in partially purified hsp90′s preparations from rabbit muscle. We demonstrate that the major contaminant associated with hsp90 is the p97 fusion protein and that these oligomeric structures are copurifying together with the 20S proteasome and its PA28 activator. Improving the purification procedure permits to separate hsp90 and p97 to homogeneity. Then, our attempts failed to detect any significant ATPase activity in the hsp90 fraction. Thus, p97 would be principally responsible for the ATPase activity detected in partially purified hsp90 preparations from rabbit muscle.

11 S seed storage proteins fromHelianthus species (Compositae): Biochemical, size and charge heterogeneity

The seeds of 19 sunflower species were compared on the basis of their protein contents and the relative proportions of their protein fractions. The globulin content varied from 50% to about 70% and the albumin content from 18% to 35% according to the species. The level of intermediateMr polypeptides showed a great variability (9.6 to 24.3%). Comparative studies onMr polymorphism were carried out by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of non reduced and/or reduced samples using both mono- and bidimensional procedures. Polypeptide constituents of helianthinin were compared including both number and molecular size (cultivatedH. annuus was used as a standard). Studies focused on differences observed between the major two α (Mr 38 000), α′ (Mr 32 000) and β (Mr 25 500), β′ (21 000) polypeptides families constituting the main A, B, and C subunits.α and α′ polypeptides analyses permit to discriminate easilyH. petiolaris from the other species. Charge polymorphism was studied using isoelectric focusing (IEF) and IEF-PAGE in mono and bidimensional procedures in the presence or absence of 2-mercaptoethanol (2-ME). Only a specific α4 polypeptide enables an easy discrimination betweenH. petiolaris and all the other species. Detailed nomenclature of the α, α′ and β, β′ polypeptides constituting the different helianthinin globulin subunits is given via the results of pI andMr analyses. Monodimensional IEF patterns of the more basic albumins (pI > 8.0) appear to provide a more valuable approach to identifying specific protein markers.

Protéinase neutre calcium dépendante de muscle squelettique de lapin: activité sur les protéines myofibrillaires

Summary Experiments have been carried out to explore the proteolytic cleavage of rabbit skeletal myofibrils by a calcium dependent neutral proteinase (CaANP). Polyacrylamide gel electrophoresis on great slabs showed the ability of CaANP to degrade myofibrils more readily than supposed. Besides the hydrolysis of troponin T and the apparition of degradation product of 30.000 molecular weight, the activity of this enzyme is obvious too on some components of the M-line and on heavy subunits of tropomyosin as well as on three unidentified proteic fractions. The variety of the degradation products which appear suggest that the specificity of CaANP is not as selective as presumed. The participation of this proteinase in the postmorten evolution of muscle and its intervention in the turnover of myofibrillar proteins is discussed.

Variability in the 11S globulin fraction of seed storage proteins ofHelianthus (Asteraceae)

The seed storage globulins from sixHelianthus and four hybrids were studied using mono and bidimensional gel SDS electrophoresis (+ 2 mercaptoethanol). The αβ polypeptide composition of each subunit was determined. Different αβ pairs are specifically expressed according to the species studied. Three typical patterns were discriminated. All the studied species exhibit five subunits: two of them are expressed in all the species (α′1β′1 and α′2β′2). The subunit corresponding to the α1β1 pair is present inH. petiolaris and in the three populations ofH. annuus studied. The α2bβ2 pair is common toH. annuus andH. argophyllus. H. petiolaris presents two specific α2aβ2 and α4β′4 pairs andH. annuus a specific α3β′3 pair. InH. argophyllus α1β1 α3β′3 or α4β′4 are never observed but are replaced by α1β′3 and α3β1 pairs. Some globulins, poorly represented, are of αβ′ forms but present α chains of higher molecular weights (in the range 54–56 kDa). Expressing variations in the banding patterns between these species by the use of a similarity index reveals complete identity between the three populations ofH. annuus. Identity between the twoH. petiolaris studied is also observed.H. annuus andH. argophyllus appear to be closer to each other thanH. petiolaris concerning the seed storage globulins.

Wheat flour proteins: isolation and functionality of gliadin and HMW-glutenin enriched fractions

The amount of glutenin subunits of high molecular weight (HMW-glutenins) appears to be closely related to breadmaking. The relationship of their specific functional properties and gluten quality has not been demonstrated. The difficulty in isolating non-denatured HMW-glutenins explains largely the lack of information. Investigations have been conducted to isolate HMW-glutenins without using denaturing agents such as SDS or urea. Using wholemeal treated in mild reducing conditions with Na2SO3, the procedure uses solubilisation in hot aqueous ethanol with the subsequent specific precipitation of HMW-glutenins at low temperature. Proteins present in the various extracts were anlaysed by SDS-PAGE and reverse-phase HPLC. The HMW-glutenins were obtained by precipitation at low temperature from ethanol extract (purification factor ∽12), whereas the supernatant mainly contained gliadins and LMW-glutenins. After addition of these fractions to meal the modifications of the rheological properties of the resulting dough were investigated. A model explaining the participation of the different fractions to the structure and the solubility of gluten is suggested.

Degradation of an ubiquitin‐conjugated protein is associated with myoblast differentiation in primary cell culture

At the early stages of myogenesis, myoblasts fuse to form multinucleated myotubes. This morphological differentiation is the result of dynamic changes in gene regulation and expression. The ubiquitin proteasome‐dependent pathway has been reported to play an important role in many aspects of cellular functions such as regulation of growth and cell cycle progression. In this study, we showed that the amount of mRNAs corresponding to the iota subunit of the 20S proteasome, the level of the S4 subunit of the 19S complex and the 20S and 26S proteasomes peptidase activities increased during myoblast fusion. Cell permeable 20S proteasome inhibitor prevented fusion with concomitant accumulation of ubiquitin‐conjugated protein. On the other hand, inhibition of ubiquitin ligase E3 enzymes prevented the formation of ubiquitin conjugate and decreased the fusion process. These results strongly support the involvement of the ubiquitin‐proteasome proteolytic pathway in the events leading to myoblast fusion.