Jean-Louis Azanza

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

Subunit composition of the globulin fraction of rapeseed (Brassica napus L.)

Abstract Multidimensional gel electrophoretic procedures have been employed to studythe polypeptide composition of the 12 S globulin (cruciferin) from rapeseed ( Brassica napus L. cv. Tandem). Cruciferin was shown to consist of three main groups of proteins with M r in the range of 60-52 (A group), 37-35 and 31-29 (respectively B 1 and B 2 groups) and 24-22 (C group). When reduced the A protein group gave rise to two classes of polypeptide constituents. The larger had M r and isoelectric points (6.4–7.1) corresponding to those of B protein groups whereas the smaller were essentially composed of M r 22 and a specific 25 kilodaltons (kD) polypeptide with basic isoelectric points. The initial B and C protein groups were unaltered by reducing conditions. These results support the notion that native cruciferin is composed of subunits with large and small polypeptides linked by disulphide bonds and of similar or closely-related polypeptides which are not covalently bonded.

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.

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.

Relationship between immunological properties and structural model of 11s rapeseed globulin

Abstract The immunodetection of rapeseed ( Brassica napus ) 11S globulin (cruciferin) constituents was investigated using two different rabbit immune sera raised against native cruciferin and against α and β free polypeptides constituting the cruciferin subunits. After two dimensional SDS-PAGE separation and transfer on immobilon membrane, the immunoblotting showed that only the α polypeptides reacted with anti-native cruciferin antibodies. The β as well as a polypeptides displayed intensive immunoreactivity using anti-free polypeptides antibodies. These results are discussed with regard to the proposed model for the quaternary structure of the hexameric cruciferin molecule. The data were in agreement with the surface localization of the a polypeptides; β polypeptides would be buried within the cruciferin core.

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.

Comparative studies of 11S globulin constituents of Brassica napus L. and of its related species Brassica campestris L. and Brassica oleracea L.

Abstract Like Brassica napus L. cv. Darmor (rapeseed) cruciferin, the seed 11S globulin of diploid species, B. oleracea L. cv. Valentine (cauliflower) and cv. Proteor (fodder cabbage) and B. campestris L. cv. Chicon (fodder turnip), had legumin-like subunit consisting in large (αs) and small (βs) disulfide-linked polypeptide components and, in addition, α and β free (f) polypeptides not covalently bonded. Free and subunit component polypeptides of the 11S cruciferin from different species were compared using two dimensional gel electrophoresis analysis, CNBr and V8 protease peptide mapping experiments and immunological studies. It has been established that (i) αf and αs polypeptides had high degree of homology. (ii) βs polypeptides were similar. (iii) βf polypeptides were different, not only from βs polypeptides but also between them. (iv) In spite of these differences, the βf and βs polypeptides were immunologically related as were the αf and αs polypeptides.

La cathepsine D de rate de cheval: II. - Étude de quelques propriétés enzymatiques

Summary This work reports some enzymatic properties of highly purified horse spleen cathepsin D. Hydrolysis rate of several proteins are compared. The Kinetic constants (Km = 4.95 10 −5 M and Vm = 1,76 ΔDO/mn/μg) have been determined in the presence of a denatured haemoglobin substrate. Stability of the enzymatic preparation is discussed according to the pH, concentration and time of storage. Some investigations concerning the active site are described. Enzymatic and chemical results show that dicarboxylic and tryptophanyl residues seem to be involved in the hydrolytic process. Catalysis does not depend on sulfhydryl or seryl residues. Different salts, particulary nitrate, nitrite and polyphosphate are potent inhibitors of enzymatic activity.