SDS-extractable Proteins Research Articles

Isolation of surface (S) layer protein carrying Lactobacillus species from porcine intestine and faeces and characterization of their adhesion properties to different host tissues

Surface-layer proteins (Slps) of lactobacilli have been shown to confer tissue adherence. This study aimed to isolate and identify Slps carrying Lactobacillus species from the porcine intestine and faeces and to characterize these S-layer-expressing strains for their ability to adhere to the pig and human intestinal cells and to extracellular matrix (ECM) proteins. In total 99 strains, putatively belonging to the genus Lactobacillus, were isolated as pure cultures. SDS-PAGE and a gene probe specific for the Lactobacillus brevis ATCC 8287 S-layer protein gene ( slpA) were used to screen the presence of strains possessing putative Slps. Eight of the 99 pure cultures exhibited Slps according to the SDS-PAGE analyses. In these strains the presence of genes encoding Slps was confirmed by PCR and partial sequencing. Only one isolate of the 99 strains gave a positive hybridisation signal with the L. brevis slpA probe but did not appear to produce S-layer protein. Their taxonomic identification, based on phenotyping and the 16S rRNA sequences, revealed that the eight S-layer protein-producing strains were closely related to Lactobacillus amylovorus, Lactobacillus sobrius and Lactobacillus crispatus. The strain with the slpA positive hybridisation result was identified as Lactobacillus mucosae. The SDS-extractable protein profile, the size of the putative S-layer protein and binding capability of the strains varied greatly, even among the isolates belonging to the same Lactobacillus cluster. Removal of the intact Slps from the bacterial surface by extraction with guanidine hydrochloride reduced the adhesion of some strains to fibronectin and laminin, whereas, the adhesiveness to laminin increased with some strains.

Differences in polymeric proteins among grains in spring wheat spikes

AbstractA uniform amount and size distribution of polymeric proteins within grains in a spike might determine the stability of wheat quality. Two cultivars were grown to maturity in solution culture in a climate chamber. Nitrogen (N) in the form of nitrate was added daily and replaced with 15N before harvest. Plants were harvested during grain development. Protein composition and relationships of labelled N in grains from different spikelets within the spike were determined. Higher percentages of large unextractable polymeric proteins (%‐LUPP) and total unextractable polymeric proteins (%‐TUPP) were found in the lower‐ and uppermost spikelets in the spike compared with the middle ones for cv. WL, but not for cv. Sport. Both cultivars showed variations in the percentage of large unextractable monomeric proteins (%‐LUMP) and total SDS‐extractable protein (Tote) in the spikelets within the spike. The amount of total SDS‐unextractable protein (Totu) did not vary for either of the cultivars. The spikelets within the spike showing high and low %‐LUMP and Tote at maturity showed a similar behaviour shortly after flowering in cv. WL, but not in cv. Sport. The N concentration of SDS and sonicated extracts varied along the spikelets of the spike for both cultivars. The atom‐% excess 15N decreased in cv. Sport SDS‐extractable and ‐unextractable proteins and cv. WL albumins + globulins, gliadins and glutenins from grains at different spikelet positions along the spike. Copyright © 2005 Society of Chemical Industry

Correct GPI-anchor synthesis is required for the incorporation of endoglucanase/glucanosyltransferase Bgl2p into the Saccharomyces cerevisiae cell wall

The SSU21/ MCD4 gene encodes an essential component of the glycosylphosphatidylinositol (GPI)-anchor synthesis pathway in Saccharomyces cerevisiae. Here we demonstrate that the ssu21 mutation affected the transport and the incorporation into the cell wall of the major non-GPI yeast cross-linker – endoglucanase/glucanosyltransferase Bgl2p. This mutation also led to a decrease in the levels of both known types of cell wall mannoproteins, those covalently linked with glucan and SDS-extractable proteins. Our results indicate that the precision of the GPI-anchor synthesis is essential for cell wall assembly and suggest the strong interdependence of different groups of cell wall proteins during their incorporation into the cell wall.

Correct GPI-anchor synthesis is required for the incorporation of endoglucanase/glucanosyltransferase Bgl2p into the Saccharomyces cerevisiae cell wall.

The SSU21/MCD4 gene encodes an essential component of the glycosylphosphatidylinositol (GPI)-anchor synthesis pathway in Saccharomyces cerevisiae. Here we demonstrate that the ssu21 mutation affected the transport and the incorporation into the cell wall of the major non-GPI yeast cross-linker - endoglucanase/glucanosyltransferase Bgl2p. This mutation also led to a decrease in the levels of both known types of cell wall mannoproteins, those covalently linked with glucan and SDS-extractable proteins. Our results indicate that the precision of the GPI-anchor synthesis is essential for cell wall assembly and suggest the strong interdependence of different groups of cell wall proteins during their incorporation into the cell wall.

The impact of aluminium on the distribution of cell wall glycoproteins of pea root tip and their Al-binding capacity

Pea (Pisum sativum L.) roots were exposed to various concentrations of Al and cell wall glycoproteins containing salt-extractable proteins, SDS-extractable proteins, covalently bound proteins (CBPs), and extensins were separately extracted. The changes in their levels and the amount of bound Al to glycoproteins upon Al stress were investigated. The content of CBPs, but not of others, increased significantly with the increase of Al levels. Extensins and the salt-extractable proteins, but not the others, exhibited a marked Al-binding capacity in vivo. The amount of Al bound to extensions in vivo and in vitro was highest among the glycoproteins. The high Al-binding capacity of extensins might be caused by the high content of the hydroxy groups in hydroxyproline of extensins and Al-binding capacity to glycoproteins in the root tips may be involved in the Al-binding response in the apoplast.

Conformation and toxicity of the ABRI peptide in familial British dementia

The formation of neurofibrillary tangles (NFTs) and paired-helical filaments (PHFs) in Alzheimer's disease (AD) reflects a major disorganization of the cytoskeleton. The role of the neuronal membrane skeleton in the development of these abnormalities has not previously been investigated. In this study, we used 9 antibodies raised against the erythrocyte membrane skeleton protein 4.1 (P4.1) for immunocytochemical and immunoblot analyses to investigate whether or not the brain homologues of this protein were constituents of NFTs or PHFs. Our results show that 7 of the 9 monospecific antibodies against the human and pig erythrocyte P4.1 stained NFTs in the prefrontal cortex and hippocampus of AD brains. The P4.1 antibodies used here did not cross-react with tau protein isolated from AD brain, and preabsorption of these antibodies with tau protein did not cause loss of NFT staining. In age-matched control brains, these P4.1 antibodies stained neuronal cell bodies or nuclei. Six of the antibodies also stained isolated NFTs but the SDS-insoluble NFTswwere immunostained only by two of the P4.1 antibodies. By using inositol hexaphosphate affinity chromatography and immunoblot analysis, we identified a 68-kDa protein as the most likely brain analogue of P4.1. When SDS-extracted proteins from the isolated NFTs were immunoblotted, a 50-kDa band was immunostained. The 68-kDa and 50-kDa proteins were not stained by tau protein and neurofilament subunit NF-H antibodies, that strongly stained NFTs. We conclude that brain protein 4.1 isoform(s) are constituents of NFTs in AD.

Plasma Membrane Ordering Agent Pluronic F-68 (PF-68) Reduces Neurotransmitter Uptake and Release and Produces Learning and Memory Deficits in Rats

A substantial body of evidence indicates that aged-related changes in the fluidity and lipid composition of the plasma membrane contribute to cellular dysfunction in humans and other mammalian species. In the CNS, reductions in neuronal plasma membrane order (PMO) (i.e., increased plasma membrane fluidity) have been attributed to age as well as the presence of the beta-amyloid peptide-25-35, known to play an important role in the neuropathology of Alzheimer's disease (AD). These PMO increases may influence neurotransmitter synthesis, receptor binding, and second messenger systems as well as signal transduction pathways. The effects of neuronal PMO on learning and memory processes have not been adequately investigated, however. Based on the hypothesis that an increase in PMO may alter a number of aspects of synaptic transmission, we investigated several neurochemical and behavioral effects of the membrane ordering agent, PF-68. In cell culture, PF-68 (nmoles/mg SDS extractable protein) reduced [3H]norepinephrine (NE) uptake into differentiated PC-12 cells as well as reduced nicotine stimulated [3H]NE release. The compound (800-2400 microg/kg, i.p., resulting in nmoles/mg SDS extractable protein in the brain) decreased step-through latencies and increased the frequencies of crossing into the unsafe side of the chamber in inhibitory avoidance training. In the Morris water maze, PF-68 increased the latencies and swim distances required to locate a hidden platform and reduced the time spent and distance swam in the previous target quadrant during transfer (probe) trials. PF-68 did not impair performance of a well-learned working memory task, the rat delayed stimulus discrimination task (DSDT), however. Studies with 14C-labeled PF-68 indicated that significant (pmoles/mg wet tissue) levels of the compound entered the brain from peripheral (i.p.) injection. No PF-68 related changes were observed in swim speeds or in visual acuity tests in water maze experiments, rotorod performance, or in tests of general locomotor activity. Furthermore, latencies to select a lever in the DSDT were not affected. These results suggest that PF-68 induced deficits in learning and memory without confounding peripheral motor, sensory, or motivational effects at the tested doses. Furthermore, none of the doses induced a conditioned taste aversion to a novel 0.1% saccharin solution indicating a lack of nausea or gastrointestinal malaise induced by the compound. The data indicate that increases in neuronal plasma membrane order may have significant effects on neurotransmitter function as well as learning and memory processes. Furthermore, compounds such as PF-68 may also offer novel tools for studying the role of neuronal PMO in mnemonic processes and changes in PMO resulting from age-related disorders such as AD.

Role of NaOH-extractable cell wall proteins Ccw5p, Ccw6p, Ccw7p and Ccw8p (members of the Pir protein family) in stability of the Saccharomyces cerevisiae cell wall.

The Saccharomyces cerevisiae cell wall contains more than 20 identified mannoproteins. Some of them can be released from the wall by hot SDS/mercaptoethanol treatment and are, therefore, considered as disulphide-linked or non-covalently attached to wall structural components. A number of covalently linked cell wall proteins are released after SDS extraction. They can be divided into these extractable by glucanases and those which can be released with 30 mM NaOH. The SDS-extractable proteins either possess enzymatic activities or are homologues of enzymes, mainly glucanases. Nothing is known, however, about the function of covalently linked proteins. In order to investigate the role of NaOH-extractable cell wall proteins, genes encoding all four identified members of this family of Pir proteins, CCW5, CCW6, CCW7 and CCW8, were disrupted and the phenotype of the mutants obtained was examined. They grew somewhat more slowly, were larger and irregularly shaped, and showed pronounced susceptibility to cell wall synthesis inhibitors like Calcofluor white and Congo red. In addition, the triple and the quadruple deletants had a decreased mating ability. All these properties were more obvious the more of these genes were disrupted, indicating that probably all members of this protein family are at least functionally equivalent in the cell wall.

Factors Governing Levels and Composition of the Sodium Dodecyl Sulphate-Unextractable Glutenin Polymers During Straight Dough Breadmaking

The effect of several additives (1·215 μmol KIO3, 0·892 μmol cysteine, endo-xylanase and 0·5% (w/w) rye-water-extractable arabinoxylans) on changes in the level and glutenin subunit composition of the sodium dodecyl sulphate (SDS)-unextractable protein during breadmaking was investigated. Protein extractability drastically increased during dough mixing and was enhanced both by cysteine and KIO3. The mixing-induced increase in protein extractability was partly reversed during fermentation. Fermenting doughs containing endo-xylanase had a higher level of SDS-unextractable protein than control doughs, while with KIO3the amount of SDS-unextractable protein remained very low. During baking most protein became SDS-unextractable. Bread baked from doughs with added KIO3contained a significantly higher level of SDS-extractable protein. Changes in subunit composition of the SDS-unextractable glutenin polymers, determined with RP-HPLC, coincided with changes in protein extractability during dough processing. Mixing decreased the ratio of high to lowMrglutenin subunits. Simultaneously, the relative proportions of the different highMrglutenin subunits in the unextractable glutenin polymers changed. During fermentation changes in subunit composition of the SDS-unextractable glutenin were opposite to those during mixing.

RP–HPLC and Capillary Electrophoresis of Subunits from Glutenin Isolated by SDS and Osborne Fractionation

Abstract The polypeptide subunits present in SDS-unextractable glutenin, the glutenin macropolymer (GMP) and the 70% (v/v) ethanol unextractable protein, the Osborne glutenin fraction, of various cultivars were separated by RP–HPLC and capillary electrophoresis (CE) under denaturing (urea and SDS, respectively) and reducing conditions. In addition, the SDS-extractable protein was separated by CE. High M r glutenin subunits were well separated by CE, while the separation of low M r glutenin subunits was better by RP–HPLC. High M r glutenin subunits separated by RP–HPLC were collected and separated by CE. The subunits were identified unequivocally using the combined information from these two techniques and from SDS–PAGE patterns using the cvs. Spring and Troy Spring. By both RP–HPLC and CE it could be demonstrated for flour from three wheat cvs. (Camp Remy, Obelisk and Rektor) and a blend of flour from two of those cvs. (Camp Remy/Obelisk) that the high M r glutenin subunit content of the GMP was 29–31%. In contrast, the SDS-extractable protein consisted of 4–6% high M r glutenin subunits, which accounted for 14–23% of the high M r glutenin subunits in flour. Interestingly, the SDS-extractable high M r glutenin subunits consisted mainly (90–96%) of x-type subunits whereas, in the GMP, only 70–75% of the high M r subunits were x-type subunits. Although the SDS extractable protein was not separated by RP–HPLC, results similar to those obtained by CE could be inferred from the subtraction of the contents of glutenin subunits of the GMP from the contents in the Osborne glutenin fraction. The results suggest that x- and y-type high M r glutenin subunits may have a different role in the structure (size and composition) of glutenin polymers.

Comparative biochemical and morphological changes in imbibed cotton seed hypocotyls and radiclesin situandin vitro– Protein breakdown and elongation growth

AbstractAxes, hypocotyls and radicles excised from dry cotton seeds (Gossypium hirsutumL. cv. M-8, a double haploid) were imbibed for 24 h and compared with axial segments (excised sections of embryos below the cotyledons) of imbibed, intact seeds. Radicles of excised axes had a 7.4-fold increase in length compared with only 5.2- and 5.7-fold increases, respectively, in radicles of intact seeds and in those isolated when dry. Change in hypocotyl length was not as extensive. EM data for hypocotyl and radicle cortical cells from dry and imbibed seeds revealed a major reduction in matrix protein in protein storage vacuoles along with significant organelle development at 24 h from the start of imbibition. This occurred in parallel with a reduction in salt-extracted proteins and an increase in 2% SDS-extractable proteins. SDS-PAGE of protein from low (0.2 M NaCI) and high (1.0 M NaCI) salt extracts showed a reduction in amount of the major storage proteins (53 and 48 kDa), these bands being almost totally absent in gels of protein extracts from imbibed radicles and significantly reduced in hypocotyls, within 24 h from the start of imbibition. These results indicate that initial elongation of hypocotyls and radicles in intact seeds or of excised axes, after 24 h imbibition, involves breakdown of storage proteins in these axial parts to supply nutrients for growth, with very limited contribution from the cotyledons.

Evidence for the association of protein 4.1 immunoreactive forms with neurofibrillary tangles in Alzheimer's disease brains

The formation of neurofibrillary tangles (NFTs) and paired-helical filaments (PHFs) in Alzheimer's disease (AD) reflects a major disorganization of the cytoskeleton. The role of the neuronal membrane skeleton in the development of these abnormalities has not previously been investigated. In this study, we used 9 antibodies raised against the erythrocyte membrane skeleton protein 4.1 (P4.1) for immunocytochemical and immunoblot analyses to investigate whether or not the brain homologues of this protein were constituents of NFTs or PHFs. Our results show that 7 of the 9 monospecific antibodies against the human and pig erythrocyte P4.1 stained NFTs in the prefrontal cortex and hippocampus of AD brains. The P4.1 antibodies used here did not cross-react with tau protein isolated from AD brain, and preabsorption of these antibodies with tau protein did not cause loss of NFT staining. In age-matched control brains, these P4.1 antibodies stained neuronal cell bodies or nuclei. Six of the antibodies also stained isolated NFTs but the SDS-insoluble NFTswwere immunostained only by two of the P4.1 antibodies. By using inositol hexaphosphate affinity chromatography and immunoblot analysis, we identified a 68-kDa protein as the most likely brain analogue of P4.1. When SDS-extracted proteins from the isolated NFTs were immunoblotted, a 50-kDa band was immunostained. The 68-kDa and 50-kDa proteins were not stained by tau protein and neurofilament subunit NF-H antibodies, that strongly stained NFTs. We conclude that brain protein 4.1 isoform(s) are constituents of NFTs in AD.

Expression of the Cytoskeletal-Associated Protein Filamin in Adult Rat Organs

Filamin is a well-characterized actin-associated protein first isolated from chicken smooth muscle. Subsequently, this polypeptide and its nonmuscle homolog actin-binding protein have been shown to be expressed in avian muscle tissue, mammalian smooth muscle, mammalian macrophages and other blood cell types, as well as several cultured cell lines. In this report, the occurrence of this polypeptide in adult mammalian organs has been investigated. Immunoblot analysis using three anti-filamin monoclonal antibodies showed that this protein was largely detected in adult rat organs that possess a substantial smooth muscle component. Furthermore, the limited expression of filamin in smooth muscle tissue was corroborated by immunohistochemical analysis. In contrast to avian systems, filamin was never found in detectable quantities in either mammalian cardiac or skeletal muscle. Quantitative immunoblot analysis demonstrated that filamin amounts roughly correlated with the abundance of the smooth muscle component of a given organ, comprising as much as 16.5% of the total SDS-extractable protein in bovine aorta. Work in avian systems and cells in culture has suggested that filamin is a rather ubiquitous cytoskeletal element. By contrast, this work demonstrates that filamin is highly restricted in its expression in mammalian organ systems, in situ.

Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte‐like Caco‐2 and mucus‐secreting HT29 cells in culture

Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco-2 cells and the human mucus-secreting HT29-MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well-defined apical microvilli of differentiated Caco-2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29-MTX cells. The binding of C. difficile to Caco-2 cells developed in parallel with the differentiation features of the Caco-2 cells, suggesting that the protein(s) which constitute C. difficile-binding sites are differentiation-related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte-like Caco-2 cells and the human mucus-secreting HT29-MTX cells. A labile surface-associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface-associated proteins of C. difficile subjected to different culture conditions was conducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS-extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27 kDa surface-associated proteins in the adhesion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27 kDa proteins. Indeed, adhesion to Caco-2 cell monolayers of C. difficile strains grown in the presence of blood, without or with heat-shock, was blocked. Taken together, our results suggest that C. difficile may utilize blood components as adhesins to adhere to human intestinal cultured cells.

103 PURINE 5′-NUCLEOTIDASE—ITS REESTIMATED SUBUNIT MOLECULAR MASS AND IMMUNOCYTOCHEMICAL LOCALIZATION IN CHICKEN LIVER

Purine 5′-nucleotidase, formerly termed cytosol 5′-nucleotidase (Tsushima, K. (1986) Adv Enzyme Regul 25, 181), is one of soluble nucleotidases including pyrimidine 5′-nucleotidase and deoxyribo-nucleotidase, and deferentially hydrolyzes IMP, GMP, and AMP in the presence of Mg++. The enzyme has been investigated to have allosteric properties characterized by activation by ATP, ADP, 2, 3-diphosphoglycerate, and diadenosine tetraphosphate. Purine 5′-nucleotidase, has been purified from various sources, but structural studies seem to be incomplete. The subunit molecular mass of chicken liver enzyme, which was earlier reported to be 51 kDa upon SDS-PAGE, was reinvestigated. By immunoblot analyses after SDS-PAGE, a crude fraction from the liver homogenized in the presence of leupeptin showed multiple bands around 57 kDa, and SDS-extracted proteins directly from the liver exhibited a single immunoreactive 70-kDa band. In vitro translation products using chicken liver polysomes also showed a radioactive 70-kDa band after immunoprecipitation. Immunocytocheraical study showed that the antigen was exclusively located in the cytoplasmic matrix of chicken liver sinusoidal and parenchymal cells, suggesting that physiological processing might not be the case with chicken liver enzyme. These results indicate that the subunit molecular mass of chicken liver purine 5′-nucleotidase might be 70 kDa, and the enzyme is the first case to be morphologically located in the cytosol among several known cytosolic nucleotidases.

A reappraisal of the subunit molecular mass of chicken liver cytosol 5′-nucleotidase

The subunit molecular mass of chicken liver cytosol 5′-nucleotidase was earlier reported to be 51 kDa upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Naito, Y. and Tsushima, K. (1976) Biochim. Biophys. Acta 438, 159–168). By immunoblot analyses after SDS-gel electrophoresis, however, a fraction from the liver homogenized in the presence of leupeptin showed multiple bands with molecular masses around 57 kDa, and SDS-extracted proteins directly from the liver exhibited a single 70 kDa component. These results indicate that the 51 kDa peptide observed in the cytosol 5′-nucleotidase preparation might, in fact, be a proteolytic artifact.

Effect of gibberellin on protein and non-structural carbohydrate in dormant Avena fatua caryopses

Application of gibberellic acid (GA 3) to dormant Avena fatua L. caryopses resulted in the termination of dormancy within 24 h as indicated by germination between 24 and 48 h. During the period of imbibition from 0 to 24 and 24 to 48 h changes occurred in protein and carbohydrate metabolism in GA-treated and untreated caryopses. Germination did not occur in untreated caryopses, therefore physiological changes in these caryopses were not associated with the termination of dormancy. GA-treatment increased the concentration of soluble and SDS-extractable protein in the endosperm tissue by 4 and 5%, respectively, over the 24 h untreated material; no changes were apparent when the protein profiles of GA-treated and untreated tissues were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 0, 24 and 48 h after imbibition. The concentration of hexose and sucrose in the GA-treated endosperm tissue increased 189 and 151 μmol, respectively, over the untreated material at 24 h. Gibberellic acid had no effect on starch metabolism in the endosperm tissue in the first 24 h, the period associated with the termination of dormancy. The concentration of hexose increased by 57 μmol and starch decreased by 80 μmol in the GA-treated embryo tissue within 24 h. Our results demonstrate that exogenously applied GA influences sucrose and hexose metabolism in the endosperm tissue. The specific effect of GA on starch and hexose metabolism in the dormant A. fatua caryopsis embryo tissue may be associated with the termination of dormancy.

The transneuronal transport of proline within the mouse visual system: Some characteristics of the [ 3H]-proline containing material

Following the intraocular injection of tritiated proline in the mouse, the progressive transport of radioactivity in the brain and the nature of the cortical material(s) to which the label is bound was examined. About 35–40% of the radioactivity that was present in the cerebral cortex at four weeks post-injection was extractable with either distilled water or various buffers. By using 0.1% SDS this value can be increased up to 94%. Polyacrylamide gel electrophoresis of the extracted proteins showed that a major part of the radioactivity is accumulated in one band of proteins. This heavily labeled band could not be identified in the homolateral parietooccipital cortex or in the frontal cortex. Similarly, SDS electrophoresis of the SDS-extracted proteins also demonstrated the presence of a major band of proteins whose molecular weight was estimated at approximately 68,000 daltons. The protein was purified by ammonium sulfate precipitation and DEAE-Sephadex separation.

The effect of heat on wheat gluten and the involvement of sulphydryl-disulphide interchange reactions

The baking performance of gluten declined progressively on heating and most of its functionality was destroyed by 75°C. This was accompanied by a decreased extractability of the gluten proteins in an SDS buffer, and chromatographic examination of the SDS-extracted proteins showed that glutenin proteins were affected predominantly. Extractability of gliadin proteins in aqueous propan-l-ol was unaffected by heating at temperatures up to 75°C but decreased markedly after heating at 100°C. Electrophoresis showed that the gliadin patterns were essentially unaltered up to 75°C but at 100°C ω-gliadins dominated the patterns. Both the change in propanol extractability at 100°C and the alteration in the gliadin pattern were reversed by including dithioerythritol in the extradant. Total free sulphydryl groups were measured and these were esssentially constant irrespective of temperature. However, there was a progressive shift of free sulphydryl groups from an SDS-extractable to an SDS-insoluble form. Chromatographic examination showed that free sulphydryl groups were found predominantly in glutenin aggregates of lowest molecular weight and in gliadins; sulphydryl groups in these glutenin species were particularly involved in the changes occurring at 55–75°C. These data indicate that there are heat-induced alterations in gluten proteins at temperatures above 55°C, which appear to be involved in the loss of functionality (baking performance) on heating. It is postulated that the glutenin proteins are unfolded on heating up to 75°C and that this facilitates sulphydryl/disulphide interchange between exposed groups. The protein is then ‘locked’ into the denatured state on cooling due to this disulphide bond rearrangement. At temperatures above 75°C the gliadin proteins are also affected, involving similar mechanisms.