Rheological Characteristics of Heat-Induced Custard Pudding Gels with High Antioxidative Activity

Custard pudding gels were prepared from fresh whole egg, milk and sugar. The effects of D-psicose (Psi), a non-calorie rare hexose, on the antioxidative activity and rheological properties of the custard pudding gels were investigated at different temperatures for comparison with those of control sugars (sucrose, Suc; D-fructose, Fru). The rheological behavior of the heat-induced pudding gels was evaluated by using breaking and creep tests. During the heat-induced gel formation, custard pudding containing Psi (15%, wt/wt) demonstrated a stronger breaking strength and higher viscoelasticity than those containing Fru and Suc. The thermodynamic parameters obtained from DSC indicated that the egg white (EW) proteins were made less thermally stable when heated in the presence of Psi than in the presence of Fru and Suc. These findings are consistent with enhanced aggregation of the EW solution in the presence of Psi. Furthermore, the Psi pudding gels possessed higher antioxidative activity than the control sugar pudding gels by using an analysis of the scavenging activity on DPPH radicals and the ferric-reducing antioxidative power. These results suggest that Psi favored cross-linking of Psi-protein molecules through the Maillard reaction which increased the formation of intermediate products to improve functionality. Custard pudding containing Psi could therefore be an effective functional sweet dessert with high antioxidative activity and the outstanding gelling characteristics.     

Evidence that sucrose loaded into the phloem of a poplar leaf is used directly by sucrose synthase associated with various beta-glucan synthases in the stem

Sucrose (Suc) synthase (SuSy) is believed to function in channeling UDP-Glc from Suc to various beta-glucan synthases. We produced transgenic poplars (Populus alba) overexpressing a mutant form (S11E) of mung bean (Vigna radiata) SuSy, which appeared in part in the microsomal membranes of the stems. Expression of SuSy in these membranes enhanced the incorporation of radioactive Suc into cellulose, together with the metabolic recycling of fructose (Fru), when dual-labeled Suc was fed directly into the phloem of the leaf. This overexpression also enhanced the direct incorporation of the glucosyl moiety of Suc into the glucan backbone of xyloglucan and increased recycling of Fru, although the Fru recycling system for cellulose synthesis at the plasma membrane might differ from that for xyloglucan synthesis in the Golgi network. These findings suggest that some of the Suc loaded into the phloem of a poplar leaf is used directly by SuSys associated with xyloglucan and cellulose synthases in the stem. This may be a key function of SuSy because the high-energy bond between the Glc and Fru moieties of Suc is conserved and used for polysaccharide syntheses in this sink tissue.     

Dynamic viscoelastic properties of glycinin and β-conglycinin gels from soybeans

The effects of heating temperature on gel properties and conformational changes were investigated in glycinin and β-conglycinin gels using Theological and Fourier transform ir (FTIR) methods. Solutions of 15 wt % glycinin or β-conglycinin in 35 mM phosphate buffer at pH 7.6 were heated at various temperatures for 30 min and rheological properties were measured at 20°C. The storage modulus G′ as a function of frequency changed from a monotonical decrease with decreasing frequency to a plateau in the range from 0.0018 to 40 Hz by heating at temperatures higher than 80°C for glycinin and 65°C for β-conglycinin. A band at 1618 cm^?1 (associated with the β-sheet structure) on ir spectra increased with the formation of heat-induced gels. The value of the storage modulus G′ correlated well with the increase in absorbance at 1618 cm^?1. These results suggest that the formation of a β-sheet structure may be closely related to the value of the storage modulus G′ for heat-induced gels in soybean proteins and that heat-induced gels of glycinin and β-conglycinin are formed by cross-links with intermolecular β-sheet structures. © 1994 John Wiley & Sons, Inc.     

Impact of incorporations of various polysaccharides on rheological and microstructural characteristics of heat-induced quinoa protein isolate gels

This study aimed to investigate the properties of heat-induced gels (85 °C for 30 min) of quinoa protein isolate (QPI) in the presence and absence of various polysaccharides including guar gum (GG), locust bean gum (LBG), and xanthan gum (XG) at pH 7. For this purpose, samples with three gum concentrations (0.05, 0.1, and 0.2 wt%) at a fixed QPI concentration (10 wt%) and a fixed ionic strength (50 mM NaCl) were studied in terms of their gelation behaviour, small and large deformation rheological properties, water holding capabilities, and microstructural characteristics. Rheological measurements revealed that all polysaccharides incorporation could improve gel strength (complex modulus, G*) and breaking stress, accelerate gel formations, and more stiffer gels were obtained at greater polysaccharide concentrations. The XG exhibited the most gel strengthening effect followed by LBG and GG. Incorporation of 0.2 wt% XG led to a 15 folds increase in G* compared to the control. Confocal laser scanning microscopy observation revealed that the polysaccharides also altered gel microstructures, with the gels containing XG showing the most compact gel structures. The findings of this study may provide useful information for the fabrication of novel QPI based food gel products with improved texture.

     

Unraveling the difference between invertases and fructan exohydrolases: a single amino acid (Asp-239) substitution transforms Arabidopsis cell wall invertase1 into a fructan 1-exohydrolase

Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases), but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the glucose (Glc)-fructose (Fru) linkage in sucrose (Suc), whereas plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the three-dimensional structures of Arabidopsis (Arabidopsis thaliana) cell wall Invertase1 (AtcwINV1) and chicory (Cichorium intybus) 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether Suc or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this article, we present site-directed mutagenesis-based data on AtcwINV1 showing that the aspartate (Asp)-239 residue fulfills an important role in both binding and hydrolysis of Suc. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of Suc. Surprisingly, a D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by a few mutational changes. In general, family 32 and 68 enzymes containing an Asp-239 functional homolog have Suc as a preferential substrate, whereas enzymes lacking this homolog use fructans as a donor substrate. The presence or absence of such an Asp-239 homolog is proposed as a reliable determinant to discriminate between real invertases and defective invertases/FEHs.     

The Effect of Elevated Concentrations of Fructose 2,6-Bisphosphate on Carbon Metabolism during Deacidification in the Crassulacean Acid Metabolism Plant Kalanchöe daigremontiana

In C₃ plants, the metabolite fructose 2,6-bisphosphate (Fru 2,6-P₂) has an important role in the regulation of carbon partitioning during photosynthesis. To investigate the impact of Fru 2,6-P₂ on carbon metabolism during Crassulacean acid metabolism (CAM), we have developed an Agrobacterium tumefaciens-mediated transformation system in order to alter genetically the obligate CAM plant Kalanchöe daigremontiana. To our knowledge, this is the first report to use genetic manipulation of a CAM species to increase our understanding of this important form of plant metabolism. Transgenic plants were generated containing a modified rat liver 6-phosphofructo-2-kinase gene. In the plants analyzed the activity of 6-phosphofructo-2-kinase ranged from 175% to 198% of that observed in wild-type plants, resulting in Fru 2,6-P₂ concentrations that were 228% to 350% of wild-type plants after 2 h of illumination. A range of metabolic measurements were made on these transgenic plants to investigate the possible roles of Fru 2,6-P₂ during Suc, starch, and malic acid metabolism across the deacidification period of CAM. The results suggest that Fru 2,6-P₂ plays a major role in regulating partitioning between Suc and starch synthesis during photosynthesis. However, alterations in Fru 2,6-P₂ levels had little effect on malate mobilization during CAM fluxes.     

Properties of fructan:fructan 1-fructosyltransferases from chicory and globe thistle,two Asteracean plants storing greatly different types of inulin

Remarkably, within the Asteraceae, a species-specific fructan pattern can be observed. Some species such as artichoke (Cynara scolymus) and globe thistle (Echinops ritro) store fructans with a considerably higher degree of polymerization than the one observed in chicory (Cichorium intybus) and Jerusalem artichoke (Helianthus tuberosus). Fructan:fructan 1-fructosyltransferase (1-FFT) is the enzyme responsible for chain elongation of inulin-type fructans. 1-FFTs were purified from chicory and globe thistle. A comparison revealed that chicory 1-FFT has a high affinity for sucrose (Suc), fructose (Fru), and 1-kestose as acceptor substrate. This makes redistribution of Fru moieties from large to small fructans very likely during the period of active fructan synthesis in the root when import and concentration of Suc can be expected to be high. In globe thistle, this problem is avoided by the very low affinity of 1-FFT for Suc, Fru, and 1-kestose and the higher affinity for inulin as acceptor substrate. Therefore, the 1-kestose formed by Suc:Suc 1-fructosyltransferase is preferentially used for elongation of inulin molecules, explaining why inulins with a much higher degree of polymerization accumulate in roots of globe thistle. Inulin patterns obtained in vitro from 1-kestose and the purified 1-FFTs from both species closely resemble the in vivo inulin patterns. Therefore, we conclude that the species-specific fructan pattern within the Asteraceae can be explained by the different characteristics of their respective 1-FFTs. Although 1-FFT and bacterial levansucrases clearly differ in their ability to use Suc as a donor substrate, a kinetic analysis suggests that 1-FFT also works via a ping-pong mechanism.     

Control of fructose 2,6-bisphosphate levels in rat macrophages by glucose and phorbol ester

The presence of fructose 2,6-bisphosphate (Fru 2,6-P2) in elicited peritoneal macrophages of rat was examined. These cells possess an active phosphofructokinase-2 which is diminished by citrate and only slightly inhibited by glycerol 3-phosphate. Phosphofructokinase-1 submaximal activity was increased 26-fold by the addition of 1 microM Fru 2,6-P2. Incubation of cells without glucose decreased the amount of Fru 2,6-P2 to zero, but further addition of 5 mM glucose increased the levels of the sugar ester 20-fold. In addition, the presence of phorbol ester potentiated the synthesis of Fru 2,6-P2. By contrast phenylisopropyladenosine or prostaglandin F2 alpha inhibited the production of Fru 2,6-P2.     

Amino Acid esters prevent thermal inactivation and aggregation of lysozyme

Small potent inhibitors of aggregation are eagerly demanded for preventing the inactivation of proteins. This paper shows that amino acid esters (AAEs) prevent heat-induced aggregation and inactivation of hen egg lysozyme. Lysozyme was completely inactivated (<1% original activity) during heat treatment at 98 degrees C for 30 min in a solution containing 0.2 mg/mL lysozyme in 50 mM Na-phosphate buffer (pH 6.5). The residual activities only slightly increased (<5%) in the presence of 100 mM commonly used additives such as arginine, guanidine, urea, and sugars. However, in the presence of 100 mM AAEs, the residual activities were >60% and no aggregates were observed during the heat treatment at 98 degrees C for 30 min. This fact provides new information on the scaffold for designing additives to prevent heat-induced aggregation.     

Overexpression of the Vacuolar Sugar Carrier AtSWEET16 Modifies Germination,Growth, and Stress Tolerance in Arabidopsis

Here, we report that SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER (SWEET16) from Arabidopsis (Arabidopsis thaliana) is a vacuole-located carrier, transporting glucose (Glc), fructose (Fru), and sucrose (Suc) after heterologous expression in Xenopus laevis oocytes. The SWEET16 gene, similar to the homologs gene SWEET17, is mainly expressed in vascular parenchyma cells. Application of Glc, Fru, or Suc, as well as cold, osmotic stress, or low nitrogen, provoke the down-regulation of SWEET16 messenger RNA accumulation. SWEET16 overexpressors (35S_Pro:SWEET16) showed a number of peculiarities related to differences in sugar accumulation, such as less Glc, Fru, and Suc at the end of the night. Under cold stress, 35S_Pro:SWEET16 plants are unable to accumulate Fru, while under nitrogen starvation, both Glc and Fru, but not Suc, were less abundant. These changes of individual sugars indicate that the consequences of an increased SWEET16 activity are dependent upon the type of external stimulus. Remarkably, 35S_Pro:SWEET16 lines showed improved germination and increased freezing tolerance. The latter observation, in combination with the modified sugar levels, points to a superior function of Glc and Suc for frost tolerance. 35S_Pro:SWEET16 plants exhibited increased growth efficiency when cultivated on soil and showed improved nitrogen use efficiency when nitrate was sufficiently available, while under conditions of limiting nitrogen, wild-type biomasses were higher than those of 35S_Pro:SWEET16 plants. Our results identify SWEET16 as a vacuolar sugar facilitator, demonstrate the substantial impact of SWEET16 overexpression on various critical plant traits, and imply that SWEET16 activity must be tightly regulated to allow optimal Arabidopsis development under nonfavorable conditions.Sugars are of enormous importance for plant properties and the agronomic values of most crop species (John, 1992). In plants, they serve as energy reserves, as building blocks for carbohydrate polymers like starch or cellulose, as precursors for amino and carboxylic acids, and as osmolytes required for the molecular antifreezing program initiated after exposure to cold temperatures (Nägele et al., 2010).Sugars in leaves are synthesized either during the day via photosynthesis or in the night as a product of starch degradation. The major sugar synthesized in most plants during the day is Suc, which, after the export of triose phosphates from the chloroplast, is synthesized in the cytosol. During nocturnal starch degradation, maltose leaves the chloroplast and serves as a substrate for the cytosolic synthesis of heteroglycans (Fettke et al., 2005). Subsequent to this, heteroglycans are degraded by phosphorylases (Fettke et al., 2005) and act as a carbon source to synthesize Suc, which can be hydrolyzed by cytosolic or vacuolar invertases to monosaccharides (Roitsch and González, 2004). These processes, in sum, enable leaf mesophyll cells to synthesize Glc and Fru, in addition to Suc, during the day and at night.Besides these metabolic processes, sugars are transported between different intracellular compartments and between different cells in order to serve as a long-distance carbon supply for sink organs. Due to their large size and hydrate shell, the movement of neutral sugars like Suc, Glc, or Fru across membranes requires the presence of membrane-bound carriers. For example, in the plant plasma membrane, a wide number of monosaccharide- and Suc-specific carriers were identified and have been analyzed with biochemical and molecular approaches. The Arabidopsis (Arabidopsis thaliana) genome harbors more than 50 isoforms of putative monosaccharide carriers, most of which belong to the sugar transport protein subfamily (Büttner and Sauer, 2000), while about 20 putative disaccharide carriers sucrose transporters (named SUT and SUC) are present in this plant species (Lalonde et al., 2004). Most of the sugar transport protein, SUT, or SUC carriers analyzed so far reside in the plasma membrane and import, as proton-coupled transporters, apoplastic sugars against a concentration gradient (Lalonde et al., 2004). This proton-driven sugar import allows a substantial accumulation of Suc in phloem sieve elements, building the driving force for interorgan long-distance sugar transport (Turgeon and Wolf, 2009). All monosaccharide and disaccharide carriers mentioned above exhibit 12 predicted transmembrane domains and group into the large major facilitator superfamily of carriers (Marger and Saier, 1993).In both photosynthetic active mesophyll cells as well as storage tissues, the large central vacuole represents the internal storage compartment for sugars (Martinoia et al., 2007, 2012), leading, in sugar beet (Beta vulgaris) or sugarcane (Saccharum officinarum), up to even 20% sugars per fresh biomass (John, 1992). Suc import into the vacuole occurs either via facilitated diffusion (Kaiser and Heber, 1984) or electrogenically via antiport against protons (Willenbrink and Doll, 1979). The latter process is driven by the significant proton-motive force across the vacuolar membrane (Schumacher and Krebs, 2010) and allows a substantial Suc accumulation in storage organs of high-sugar species (Getz, 1987; Getz and Klein, 1995). However, no Suc importer at the vacuolar membrane (tonoplast) has been identified on the molecular level yet, while tonoplast-located Suc exporters have been identified. This vacuolar Suc export is mediated by members of the SUT4-type clade of carriers, in cereals named SUT2 (Endler et al., 2006; Eom et al., 2011), procuring a proton-driven Suc export into the cytosol (Schulz et al., 2011). Loss of function of this type of carrier in Arabidopsis, poplar (Populus spp.), or rice (Oryza sativa) leads to an accumulation of Suc in leaves (Eom et al., 2011; Payyavula et al., 2011; Schneider et al., 2012), elegantly proving that this type of carrier fulfills an export function under in vivo conditions.In contrast to vacuolar Suc import, the import of monosaccharides into this compartment has been deciphered on the molecular level. In the Arabidopsis tonoplast, two different monosaccharide importers have been identified, namely the vacuolar Glc transporter protein and three isoforms of the tonoplast monosaccharide transporter (TMT; Wormit et al., 2006; Aluri and Büttner, 2007). While vacuolar Glc transporter loss-of-function plants do not show significant changes in monosaccharide levels (Aluri and Büttner, 2007), decreased TMT activity correlates with impaired vacuolar sugar import and low levels of both Glc and Fru in leaves (Wormit et al., 2006). This fact and the observations that (1) TMT1 is a sugar/proton antiporter (Schulz et al., 2011), (2) increased TMT activity provokes improved seed biomass (Wingenter et al., 2010), and (3) TMT activity is highly regulated via protein phosphorylation (Wingenter et al., 2011) clearly underline the superior function of TMT for monosaccharide loading into the plant vacuole.So far, two carriers, ESL1 and ERDL6, have been found to be responsible for Glc export from the plant vacuole (Yamada et al., 2010; Poschet et al., 2011). ESL1 (for early responsive to dehydration6-like1) represents a carrier majorly expressed in pericycle and xylem parenchyma cells and is known to be induced by drought stress (Yamada et al., 2010). Loss-of-function mutants of the ERDL6 (for early responsive to dehydration6-like6) carrier show increased leaf Glc levels and decreased seed weight, indicating that controlled Glc export via this carrier is critical for interorgan movement of sugars in Arabidopsis (Poschet et al., 2011). ESL1 seems to transport Glc in a facilitated diffusion, while in contrast to the plasma membrane-located sugar carriers and to TMT, the transport mode of ERDL6 has not been identified so far.In marked contrast to the carriers mentioned above, the recent identification of the so-called SWEET proteins opened our understanding of how cellular sugar export is achieved. SWEET proteins occur in plants as well as in animals and humans and consist of only seven predicted transmembrane domains (Chen et al., 2010). The observation that the expression of several plant SWEET proteins is strongly induced by various pathogens indicated that they serve as sugar exporters. That hypothesis has been proven for some SWEET isoforms by heterologous expression in Xenopus laevis oocytes (Chen et al., 2010), and detailed analysis revealed that Arabidopsis SWEET11 and SWEET12 catalyze Suc export from source leaves and are critical for interorgan sugar transport (Chen et al., 2012).In a recent quantitative trait locus analysis, we identified SWEET17 as a novel determinant of leaf Fru content, especially under cold conditions and conditions of low nitrogen supply (Chardon et al., 2013). In fact, a detailed molecular-physiological analysis revealed that SWEET17 is the first vacuole-located SWEET protein and that it serves as a Fru-specific exporter, connecting the vacuolar lumen to the cytosol. In contrast to SWEET17, the subcellular localization of its closest homolog, SWEET16, is elusive. Moreover, transport properties of SWEET16 are unknown, and the effect of increased SWEET16 activity (or any other SWEET proteins) on plant properties has not been determined. The latter aspect is of particular interest, since most genes coding for SWEET proteins are only comparably weakly expressed or are only expressed in certain cell types (Chen et al., 2010; Chardon et al., 2013).In this report, we analyzed the intracellular localization of SWEET16 and studied its transport properties in X. laevis oocytes. Moreover, we constructed constitutive SWEET16-overexpressing Arabidopsis lines and report the impact of this overexpression of a vacuolar SWEET protein on plant development and stress tolerance. Our results support the hypothesis that the activity of a SWEET facilitator has to be controlled in planta to cope with altering environmental and developmental conditions.     

Pseudouridylation of yeast U2 snRNA is catalyzed by either an RNA-guided or RNA-independent mechanism

Yeast U2 small nuclear RNA (snRNA) contains three pseudouridines (Psi35, Psi42, and Psi44). Pus7p and Pus1p catalyze the formation of Psi35 and Psi44, respectively, but the mechanism of Psi42 formation remains unclear. Using a U2 substrate containing a single (32)P radiolabel at position 42, we screened a GST-ORF library for pseudouridylase activity. Surprisingly, we found a Psi42-specific pseudouridylase activity that coincided with Nhp2p, a protein component of a Box H/ACA sno/scaRNP (small nucleolar/Cajal body-specific ribonucleoprotein). When isolated by tandem affinity purification (TAP), the other protein components of the H/ACA sno/scaRNP also copurified with the pseudouridylase activity. Micrococcal nuclease-treated TAP preparations were devoid of pseudouridylase activity; however, activity was restored upon addition of RNAs from TAP preparations. Pseudouridylation reconstitution using RNAs from a Box H/ACA RNA library identified snR81, a snoRNA known to guide rRNA pseudouridylation, as the Psi42-specific guide RNA. Using the snR81-deletion strain, Nhp2p- or Cbf5p-conditional depletion strain, and a cbf5 mutation strain, we further demonstrated that the pseudouridylase activity is dependent on snR81 snoRNP in vivo. Our data indicate that snRNA pseudouridylation can be catalyzed by both RNA-dependent and RNA-independent mechanisms.     

Passive nitrate transport by root plasma membrane vesicles exhibits an acidic optimal pH like the H(+)-ATPase

Previous work has indicated that sugar sensing may be important in the regulation of fructan biosynthesis in grasses. We used primary leaves of barley (Hordeum vulgare cv Baraka) to study the mechanisms involved. Excised leaf blades were supplied in the dark with various carbohydrates. Fructan pool sizes and two key enzymes of fructan biosynthesis, sucrose (Suc):Suc-1-fructosyltransferase (1-SST; EC 2. 4.1.99) and Suc:fructan-6-fructosyltransferase (6-SFT; EC 2.4.1.10) were analyzed. Upon supply of Suc, fructan pool sizes increased markedly. Within 24 h, 1-SST activity was stimulated by a factor of three and 6-SFT-activity by a factor of more than 20, compared with control leaves supplemented with mannitol (Mit). At the same time, the level of mRNA encoding 6-SFT increased conspicuously. These effects were increased in the presence of the invertase inhibitor 2, 5-dideoxy-2,5-imino-D-mannitol. Compared with equimolar solutions of Suc, glucose (Glu) and fructose stimulated 6-SFT activity to a lesser extent. Remarkably, trehalose (Tre; Glc-alpha-1 and 1-alpha-Glc) had stimulatory effects on 6-SFT activity and, to a somewhat lesser extent, on 6-SFT mRNA, even in the presence of validoxylamine A, a potent trehalase inhibitor. Tre by itself, however, in the presence or absence of validoxylamine A, did not stimulate fructan accumulation. Monosaccharides phosphorylated by hexokinase but not or weakly metabolized, such as mannose (Man) or 2-deoxy-Glc, had no stimulatory effects on fructan synthesis. When fructose or Man were supplied together with Tre, fructan and starch biosynthesis were strongly stimulated. Concomitantly, phospho-Man isomerase (EC 5.3.1.8) activity was detected. These results indicate that the regulation of fructan synthesis in barley leaves occurs independently of hexokinase and is probably based on the sensing of Suc, and also that the structurally related disaccharide Tre can replace Suc as a regulatory compound.     

Thermally induced protein gelation: gelation and rheological characterization of highly concentrated ovalbumin and soybean protein gels

In order to optimize the use of proteins as functional ingredients in foods, one needs more insight into the effects of environmental conditions (pH, ionic strength, and temperature) on the functional properties of protein. This paper summarizes the results of an extensive study on heat-induced gelation of ovalbumin (egg-white protein) and soybean protein in the concentration range from 10 to 35 g/100 g. It was the aim of the study to relate the rheological properties of thermally induced protein gels to the microstructure of the gel and the physicochemical properties of the constituent protein. The gelling behavior of the protein was quantified with rheological techniques, and the physical properties of the gels were determined, at small and large deformations. From the swelling/dissolving behavior of the gels in various media, the nature of the crosslinks was determined qualitatively. The microstructure of the gels was determined with electron microscopy. Nmr-spectroscopy was applied in order to elucidate changes in conformation during heating. It was found that the formation of a continuous covalently crosslinked network is not a prerequisite for thermally-induced protein gelation. The properties of a gel strongly depend on the pH at which the gel is formed. When heat-set at high pH(pH～10), a homogeneous, strong, and almost transparent gel is formed, consisting of flexible crosslinked protein gels. Heat-setting at low pH (pH 5) leads to the formation of a heterogeneous and weak gel, which easily exudes water. This gel consists of crosslinked aggregated protein. The ionic strength of the solvent in which the protein is dissolved and heat-set has a much lower effect on gel properties.     

Intermolecular Forces Involved in the Gelation and Gel Stability of Sesame 13S Globulin

The effect of various reagents on the formation and stability of heat-induced gels of sesame 13S globulins were investigated. Electrostatic interaction, the hydrophobic bond and the disulfide bond were important for forming the network structure of gels, and the hydrogen bond also had an influence on the formation of the gel. Hydrophobic bonds mainly contributed to the stability of the gel. Subunit analyses of the proteins solubilized from the gels showed the presence of a free acidic subunit (AS) and basic subunit (BS), a polymer of AS, a dimer of BS and the dimer of a fragment from AS or BS. From the results, sulfhydryl-disulfide exchange reactions during gelation are suggested.     

Enzymatic and fluorescence studies of four single-tryptophan mutants of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase.

In order to determine environments around four tryptophan residues, located in the N-terminus, in the kinase and in the phosphatase domains of rat testis Fru 6-P,2-kinase:Fru 2,6-bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site-directed mutagenesis. The kinetic constants of these mutant enzymes were similar to those of the wild-type enzyme. The sum of the fluorescence intensities of the enzymes was 1.5 x that of the wild-type enzyme, and Trp 299, Trp 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respectively. The fluorescence polarization of the wild-type enzyme was significantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild-type enzyme. The polarization in the presence of Fru 6-P affected only Trp 15, which suggested that it is located near the Fru 6-P binding site, but Trp 64 is not. Inactivation of both enzyme activities and unfolding of these enzymes in guanidine were monitored by activity assays and fluorescence intensities and maxima. Both Fru 6-P,2-kinase and Fru 2,6-bisphosphatase activities of all these enzymes were inactivated between 0.7 and 1 M guanidine. Enzymes containing Trp 64 or Trp 15 showed biphasic fractional unfolding curves, but those of Trp 299 or Trp 320 showed gradual steady changes. Fluorescence quenching by iodide indicated that Trp 64 was not accessible and that other Trp residues were only slightly accessible to solvent. These results suggest that all the Trp residues are in heterogeneous environments and that none are exposed on the protein surface.     

Production of starch with antioxidative activity by baking starch with organic acids

A starch ingredient with antioxidative activity, as measured by the DPPH method, was produced by baking corn starch with an organic acid; it has been named ANOX sugar (antioxidative sugar). The baking temperature and time were fixed at 170 °C and 60 min, and the organic acid used was selected from preliminary trials of various kinds of acid. The phytic acid ANOX sugar preparation showed the highest antioxidative activity, but the color of the preparation was almost black; we therefore selected L-tartaric acid which had the second highest antioxidative activity. The antioxidative activity of the L-tartaric acid ANOX sugar preparation was stable against temperature, light, and enzyme treatments (α-amylase and glucoamylase). However, the activity was not stable against variations in water content and pH value. The antioxidative activity of ANOX sugar was stabilized by treating with boiled water or nitrogen gas, or by pH adjustment.     

Lysozyme activity in animal extracts after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

1. Lysozyme activity was detected after electrophoresis in sodium dodecyl sulfate-polyacrylamide gels containing 0.2% (W/V) autoclaved Micrococcus lysodeikticus cells as substrate. 2. Lysozyme activity appeared as clear lysis zones after incubation of opaque gels at 37 degrees C in buffered Triton X-100. 3. As low as 0.1 pg of purified hen egg white lysozyme could be detected after 16 hr incubation at pH 6.5. 4. Bands with lytic activity from kidney and pancreas acetone powders, bird's egg whites and vitelline membranes, animal sera and human saliva corresponded to c-type (Mr 14,500), g-type (Mr 20,500) or both lysozymes as far as molecular weight is concerned. 5. Some extracts, like porcine kidney, exhibited more than two bands. 6. Bands with lytic activity migrating at the level of g-type lysozymes were detected in some kidney and pancreas extracts.