Nicoletta Guerrieri

Interactions of Protein and Starch Studied Through Amyloglucosidase Action

ABSTRACT The access of amyloglucosidase to the carbohydrate molecule was taken as a measure of interactions occurring among starch, amylose, amylopectin, β-dextrin and purified gluten, gliadins, high molecular weight glutenin subunits, or bovine serum albumin when they were mixed together and gelatinized before digestion. The most relevant decrease in liberated glucose, denoting coverage of some reaction sites for amyloglucosidase, occurred when gliadins were mixed with the carbohydrates. Other proteins were not as effective as gliadins. Amylopectin was not affected by any protein. Comparison of results allows some hypothesis to be formulated about the influence of structure on molecular interactions.

VANADIUM INHIBITION OF SERINE AND CYSTEINE PROTEASES

A study was made on the effect of vanadium, in both the tetravalent state in vanadyl sulphate and in the pentavalent state in sodium meta-vanadate, and ortho-vanadate, on the proteolysis of azocasein by two serine proteases, trypsin and subtilisin and two cysteine proteases bromelain and papain. Also the proteolysis of bovine azoalbumin by serine proteases was considered. An inhibitory effect was present in all cases, except meta-vanadate with subtilisin. The oxidation level of vanadium by itself did not determine the inhibition kinetics, which also depended on the type and composition of the vanadium containing molecule and on the enzyme assayed. The pattern of inhibition was similar for proteases belonging to the same class. The highest inhibition was obtained with meta-vanadate on papain and with vanadyl sulphate on bromelain.

Involvement of the Azotobacter vinelandii Rhodanese-Like Protein RhdA in the Glutathione Regeneration Pathway

The phenotypic features of the Azotobacter vinelandii RhdA mutant MV474 (in which the rhdA gene was deleted) indicated that defects in antioxidant systems in this organism were related to the expression of the tandem-domain rhodanese RhdA. In this work, further insights on the effects of the oxidative imbalance generated by the absence of RhdA (e.g. increased levels of lipid hydroperoxides) are provided. Starting from the evidence that glutathione was depleted in MV474, and using both in silico and in vitro approaches, here we studied the interaction of wild-type RhdA and Cys230Ala site-directed RhdA mutant with glutathione species. We found that RhdA was able to bind in vitro reduced glutathione (GSH) and that RhdA-Cys230 residue was mandatory for the complex formation. RhdA catalyzed glutathione-disulfide formation in the presence of a system generating the glutathione thiyl radical (GS•, an oxidized form of GSH), thereby facilitating GSH regeneration. This reaction was negligible when the Cys230Ala RhdA mutant was used. The efficiency of RhdA as catalyst in GS•-scavenging activity is discussed on the basis of the measured parameters of both interaction with glutathione species and kinetic studies.

Enzymatic and genetic profiles in environmental strains grown on polycyclic aromatic hydrocarbons

The possible generation of oxidative stress induced by aromatic hydrocarbon degradation suggests that ancillary enzyme activities could facilitate the utilization of polycyclic aromatic hydrocarbons as sole carbon source. To investigate the metabolic profiles of low molecular weight polycyclic aromatic hydrocarbon-degrading strains of Sphingobium chlorophenolicum, Rhodococcusaetherovorans, Rhodococcus opacus and Mycobacterium smegmatis, the determination of the activity of putative detoxifying enzymes (rhodanese-like and glutathione S-transferase proteins) was combined with genetic analyses. All the studied strains were able to utilize phenanthrene or naphthalene. Glutathione S-transferase activity was found in S. chlorophenolicum strains grown on phenanthrene and it was related to the presence of the bphK gene, since modulation of glutathione S-transferase activity by phenanthrene paralleled the induction of glutathione S-transferase transcript in the S. chlorophenolicum strains. No glutathione S-transferase activity was detectable in R.aetherovorans, R. opacus and in M. smegmatis strains. All strains showed 3-mercaptopyruvate:cyanide sulfurtransferase activity. A rhodanese-like SseA protein was immunodetected in R.aetherovorans, R. opacus and in M. smegmatis strains, where increase of 3-mercaptopyruvate:cyanide sulfurtransferase activity was significantly induced by growth on phenanthrene.

Surface Properties of Gluten Investigated by a Fluorescence Approach

ABSTRACT The surface properties of glutens isolated from a durum wheat cultivar (Capeiti) and two bread wheats (Riband and Hereward) were investigated using intrinsic and extrinsic fluorescence. Intrinsic fluorescence decreased on increasing protein concentration and increased after urea addition. The extrinsic fluorescence was evaluated by a titration with 8-anilino-1-naphthalene sulphonate (ANS), an hydrophobic probe. The saturating concentration for ANS and its dissociation constant (Kd) were determined. The hydrophobicity of durum and bread wheat gluten showed a different behavior increasing the protein concentration: Capeiti was not influenced, but there was a change on the gluten surface for Riband and Hereward. The significance in understanding gluten structure and the relevance of the surface properties are discussed.

Protease activity in fractionated blood cells of the vanadium accumulating ascidian Phallusia mammillata

Proteolytic activity was studied in the fractionated blood cells of the vanadium accumulating ascidian P. mammillata by separating the cells before measuring their activity. Cells were separated to avoid vanadocyte breakdown and subsequent vanadium diffusion into the assay medium. Our study revealed activity in the morula cell extract that was obtained by sonication and Centricon concentration. After removing part of the extract for enzyme activity assay the remainder was kept at 0 degrees C; it was later found that much of the protein in this latter fraction formed a sediment whereas the protease remained in solution. The serine-protease substrate specificity of the enzyme was measured and the results are discussed in relation to serine protease involvement in immune defense.

Association and folding in legumin oligomers of lupin seed

We studied the association and conformational behavior under native or denaturing conditions in the 12S⇌7S oligomers of lupin legumin and in the modified 7S (m7S) oligomer, which has lost the capacity to make a 12S molecule. Circular dichroism (CD), gel filtration FPLC, and PAGE were used. The native m7S oligomer has more α helix and nearly the same amount of β structure as the 12S⇌7S preparation. Conditions that shift the equilibrium in the 12S⇌7S system toward the 7S oligomer also make the secondary structure more similar to that of m7S molecules: higher negative ellipticity appears to be a peculiarity of 7S assemblies, whether they contain modified or unmodified monomers. Part of the helical components show low stability and disappear in 1 M urea. The CD and the separation behavior on increasing the urea concentration, and in 6 M guanidine HCl, denote similar multistep unfolding in both preparations. The 12S oligomer disassembles progressively: however, also under highly denaturing conditions, modified and unmodified preparations are mainly present in an associated form. Small amounts of monomer and aggregates were detected at high denaturant concentrations.

PROTEOLYTIC ACTIVITY AND VANADIUM INHIBITION IN ASCIDIAN BLOOD CELLS

Abstract Proteolytic activity on azocasein was studied in the hemocytes of vanadium-accumulating ascidians (Phallusia mammillata and Ciona intestinalis) and iron-accumulating ascidians (Halocynthia papillosa and Microcosmus sulcatus). A possible inhibitory role of vanadium on proteolytic enzymes was detected. The following results were obtained: 1. 1. In the neutral range M. sulcatus and C. intestinalis had optimal activity while H. papillosa showed only an activity shoulder, its activity being strongest at alkaline pH. P. mammillata displayed no proteolytic activity at all. Two types of individuals, with high and low activities, were present in H. papillosa at both pHs, and in M. sulcatus at alkaline pH. In C. intestinalis only individuals active at pH 6.5 were observed. 2. 2. In the blood cell sonicate of H. papillosa and M. sulcatus the proteolytic activity was inhibited by ethilenediaminetetraacetate (EDTA) and N-ethylmaleinimide (NEM). 3. 3. P. mammillata blood cells sonicate inhibited proteolytic activity of H. papillosa and M. sulcatus blood cells: this is attributed to the presence of large quantities of vanadium in the blood cells of P. mammillata. 4. 4. Metavanadate, orthovanadate and vanadyl (IV) sulfate inhibited proteolytic activity of H. papillosa and M. sulcatus blood cells. The inhibitory effect showed dependence on the metal ion species, the pH, and the ascidian species assayed.

Involvement of the Azotobacter vinelandii rhodanese-like protein RhdA in the glutathione regeneration pathway

Resumen del poster presentado al 36th FEBS Congress celebrado en Torino (Italia) del 25 al 30 de Junio de 2011.-- et al.

The gluten complex studied by urea denaturation and red-ox titration

Gluten surface sites with low affinity for ANS, La low hydrophobicity, were more labile to urea than those with high hydrophobicity and disappeared nearly completely when urea was above 6 M. The surface of the gluten complex was only moderately affected by complete reduction, in which case less hydrophobic regions were more labile.