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The formation of transparent gels by 6% bovine serum albumin (BSA), pH 7.5, was examined by one- and two-step heating methods. Heating of the BSA solutions at various NaCl concentrations produced transparent gels at 25-50mM NaCl and transparent sols at 0-20 mM NaCl (one-step heating method). The transparent sol obtained by heating without NaCl was reheated after mixing with various amounts of NaCl (two-step heating method I). The result was almost identical to that obtained by the one-step heating method. However, when the first heating was done with 10 mM NaCl, transparent gels were obtained over a wide range of NaCl concentrations with a second heating (two-step heating method II). Analyses of sols obtained at various NaCl concentrations by gel permeation chromatography and transmission electron microscopy showed the presence of linear polymers in the heated BSA sol (10 mM NaCl) and gel networks formed by the linear polymers (20 mM NaCl). The mechanism of transparent gel formation in BSA may be similar to that in ovalbumin.

The alpha' and beta subunits of soybean beta-conglycinin were expressed in rice seeds in order to improve the nutritional and physiological properties of rice as a food. The alpha' subunit accumulated in rice seeds at a higher level than the beta subunit, but no detectable difference in mRNA transcription level between subunits was observed. Sequential extraction results indicate that the alpha' subunit formed one or more disulphide bonds with glutelin. Electron microscopic analysis showed that the alpha' subunit and the beta subunit were transported to PB-II together with glutelin. In mature transgenic seeds, the beta subunit accumulated in low electron density regions in the periphery of PB-II, whereas the alpha' subunit accumulated together with glutelin in high-density regions of the periphery. The subcellular localization of mutated alpha' subunits lacking one cysteine residue in the N-terminal mature region (alpha'DeltaCys1) or five cysteine residues in the pro and N-terminal mature regions (alpha'DeltaCys5) were also examined. Low-density regions were formed in PB-II in mature seeds of transgenic rice expressing alpha'DeltaCys 5 and alpha'DeltaCys1. alpha'DeltaCys5 was localized only in the low-density regions, whereas alpha'DeltaCys1 was found in both low- and high-density regions. These results suggest that the alpha' subunit could make a complex via one or more disulphide bonds with glutelin and accumulate together in PB-II of transgenic rice seeds.

α-Amylases of B. amyloliquefaciens and a mutant strain were purified through a series of four steps and the purity of enzymes was checked by electrophoresis. Differences were found among the enzymes with respect to pH, temperature, stability, Km and Vmax and binding of Ca. The degradation ability of wild type α-amylase on starch granules from various botanical sources (potato, sweet potato, corn, wheat and rice) were examined by scanning electron microscopy and by kinetic assays. Differences in enzyme activity on raw granules were noted. The gene coding mature enzyme was amplified by PCR and expressed in E. coli under trc and T7 promoter.

β-Conglycinin (7S globulin), one of the major storage proteins of soybean, is a trimeric protein composed of α (67 kDa), α′ (71 kDa) and β (50 kDa) subunits and exhibits molecular heterogeneity. It is synthesized on the endoplasmic reticulum and transported to the protein storage vacuole (PSV). In this study, we examined a vacuolar sorting determinant (VSD) of the β subunit using seeds of transgenic Arabidopsis accumulating deletion derivatives of β subunit. We found that the β subunit was transported to the matrix and the globoid compartment of the PSV through distinct pathways, and that a VSD to the matrix resides in the C-terminal 10 amino acids. We further verified that the C-terminal 10 amino acids of β subunit are sufficient to transport green fluorescent protein to the PSV matrix in soybean seed cells by using transient expression system. The results of this study show a transport mechanism for the β subunit similar to that of the α′ subunit [Plant J., 34 (2003) 647].

Summary Glycinin (11S) and β-conglycinin (7S) are major storage proteins in soybean (Glycine max L.) seeds and accumulate in the protein storage vacuole (PSV). These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the PSV by vesicles. Electron microscopic analysis of developing soybean cotyledons of the wild type and mutants with storage protein composition different from that of the wild type showed that there are two transport pathways: one is via the Golgi and the other bypasses it. Golgi-derived vesicles were observed in all lines used in this study and formed smooth dense bodies with a diameter of 0.5 to several micrometers. ER-derived protein bodies (PBs) with a diameter of 0.3–0.5 μm were observed at high frequency in the mutants containing higher amount of 11S group I subunit than the wild type, whereas they were hardly observed in the mutants lacking 11S group I subunit. These indicate that pro11S group I may affect the formation of PBs. Thus, the composition of newly synthesized proteins in the ER is important in the selection of the transport pathways.

The effects of protein concentration, and heating temperature and time on the gelling properties of soybean beta-conglycinin (7S globulins) lacking the alpha or alpha' subunit were compared with those of 7S containing all three subunits (alpha, alpha', and beta) to determine whether each subunit contributes equally. In most of the conditions, the relative order of gel hardness was alpha'-lacking7Salpha-lacking. From Fourier transform infrared studies, the secondary structure change after heating was very similar among the three samples; thus, the secondary structural change is not the reason for the differences in gel hardness. By using scanning electron microscopy, we observed differences in strand thickness and the density of the gel network among the three samples. These differences correlated well with the differences in gel hardness.

In maturing seed cells, many newly synthesized proteins are transported to the protein storage vacuoles (PSVs) via vesicles unique to seed cells. Vacuolar sorting determinants (VSDs) in most of these proteins have been determined using leaf, root or suspension-cultured cells apart from seed cells. In this study, we examined the VSD of the alpha' subunit of beta-conglycinin (7S globulin), one of the major seed storage proteins of soybean, using Arabidopsis and soybean seeds. The wild-type alpha' was transported to the matrix of the PSVs in seed cells of transgenic Arabidopsis, and it formed crystalloid-like structures. Some of the wild-type alpha' was also transported to the translucent compartments (TLCs) in the PSV presumed to be the globoid compartments. However, a derivative lacking the C-terminal 10 amino acids was not transported to the PSV matrix, and was secreted out of the cells, although a portion was also transported to the TLCs. The C-terminal region of alpha' was sufficient to transport a green fluorescent protein (GFP) to the PSV matrix. These indicate that alpha' contains two VSDs: one is present in the C-terminal 10 amino acids and is for the PSV matrix; and the other is for the TLC (the globoid compartment). We further verified that the C-terminal 10 amino acids were sufficient to transport GFP to the PSV matrix in soybean seed cells by using a transient expression system.

The accumulation and degradation in the endoplasmic reticulum (ER) of a truncated ER-60 protease, from which the C-terminal 89 amino acid residues have been deleted (K 417 ochre), was examined. K 417 ochre overexpressed in COS-1 cells is not secreted into the medium, but accumulates as insoluble aggregates in non-ionic detergent without degradation in unusual clump membrane structures. K 417 ochre, stably expressed, forms soluble aggregates in non-ionic detergent and is distributed in the reticular structures of ER. Under these conditions, K 417 ochre is not secreted into the medium but is degraded with a half-life time of more than 8 h. Since K 417 ochre/C all S, in which all the Cys residues of K 417 ochre are replaced by Ser, also forms aggregates, an inter-disulfide bond appears unnecessary for aggregation. In both types of aggregates, Ig heavy chain binding protein, calnexin, glucose regulated protein 94, calreticulin, ERp72, and protein disulfide isomerase are scarcely found. Since degradation of the stably expressed K 417 ochre was not inhibited by lactacystin, leupeptin, NH(4)Cl, or cytocharasin B, but was inhibited by N-acetyl-leucyl-leucyl-norleucinal, the self-aggregated abnormal protein in the lumen of ER is assumed to be degraded by an unknown protease system other than proteasome, lysosome or autophagy.

The degradation abilities of α-amylase from Bacillus amyloliquefaciens and β-amylases from Bacillus cereus and soybean on raw starch granules from various botanical sources (potato, sweet potato, wheat, rice and corn) were examined by scanning electron microscopy. All the amylases showed different degradation patterns on starch granules. The α-amylase was more efficient than the β-amylases. α-Amylase showed both centrifugal and centripetal hydrolysis on corn, rice and wheat granules, but only centrifugal hydrolysis on potato granules. On the other hand, β-amylase moved very slowly on granules. The kinetic assays which explain the release of maltose were carried out at 12, 18 and 24 h. The rice granules were found to be the best substrate for enzymic hydrolysis by α and β-amylases. In addition, While Bacillus cereus β-amylase hydrolyzed corn granules efficiently at 45°C; soybean β-amylase was 60% less active than bacterial β-amylase at the same temperature.

Heating white sauce with bovine serum albumin (BSA) at 90 °C caused the sauce to separate into aggregates and liquid phase, although this did not occur at 75 °C. Data from differential scanning calorimetry of BSA solution suggested that heat denaturation of BSA was insufficient at 75 °C but was complete at about 90 °C. Larger aggregates of BSA were formed by heating at 90 °C compared with 75 °C, as shown by gel permeation chromatography combined with a multi-angle laser light-scattering detector. Dynamic viscoelasticity measurement showed a higher storage modulus of BSA solution formed by heating at 90 °C than at 75 °C. Scanning electron microscopy revealed a random agglomerate structure of aggregates (spherical aggregates) obtained by 75 °C heating, while a well-developed network structure containing voids was observed after heating at 90 °C. These findings suggest that sauce liquid separation induced by heating at 90 °C is due to encapsulation and restriction of white sauce components by large structured aggregates of BSA containing voids. Aggregates generated at 75 °C were not sufficiently developed and structured; therefore there was no sauce liquid separation due to encapsulation and restriction of components. © 2000 Society of Chemical Industry

Gelation of ovalbumin (OVA) was induced by the addition of fatty acid salts (FAS), such as sodium caprate (NaC10:0), to the protein solution at ambient temperature. The rheological and structural p...

Polymerization caused by defective folding of heat-denatured ovalbumin was examined. A compactly misfolded ovalbumin that was produced by cooling heat-denatured protein rapidly tended to aggregate in the presence of salt. Two different forms of aggregates were observed as the concentration of salt was varied: a linear polymer at a physiological concentration and a massive agglomerate at a higher concentration. Salt-induced polymerization depended on the species of anion and the order of effectiveness followed the lyotropic series of Hofmeister. Defective folding of heat-denatured ovalbumin induced the exposure of cysteine residues in sequences located in the interior of the native protein. The misfolded ovalbumin, but not the native protein, bound to bovine BiP and stimulated its ATPase activity with the K(m) of 64 microM and the V(max) of 0.5 nmol/min per milligram. Measurement of surface plasmon resonance revealed that only the misfolded ovalbumin was recognized with the K(d) of 4.12 X 10(-8) M by the Fab fragment of a monoclonal antibody raised against hen ovalbumin, and its epitope was determined to be a hydrophobic segment in the beta-strand of central sheet A. Transmission electron microscopy showed that the linear polymerization was inhibited by the addition of bovine BiP and the Fab fragment. These results demonstrated that the compactly misfolded ovalbumin polymerized through hydrophobic interaction occurring among the areas exposed as a result of defective folding of the heat-denatured protein. Exposure of the region of, or adjacent to, the central beta-sheet A was required for axial contact among the misfolded molecules, suggesting that this process may be explained by reference to the mechanism proposed for loop-sheet polymerization in the Z type variant of a serpin alpha1-antitrypsin.

The physicochemical properties of heat-induced ovalbumin (OVA) gels containing fatty acid salts (FAS) were investigated. Water-holding ability and transparency markedly increased in the presence of sodium caprate or sodium laurate. At 9% of the protein concentration transparent and hard gels formed; at 7%, transparent and soft gels formed. Scanning electron microscopy showed that the transparent gels had a more homogeneous structure than turbid gels without FAS. Differential scanning calorimetry indicated that denaturation temperature of OVA was decreased and the peaks became broader after addition of FAS.

Treating bread dough by a high-voltage electric field (HVEF) during the first fermentation enabled the bread dough to retain water in the gluten fibers. The microstructure of the gluten fibers was ...

The microstructures of transparent gels from ovalbumin, hen egg white lysozyme, and bovine serum albumin were examined by high-resolution scanning electron microscopy. Three transparent gel matrices were clearly found to be composed of a common structural unit in which heat-denatured molecules formed fine networks of linear aggregates. The molecular conformation of heat-denatured proteins in linear aggregates produced after heat treatment in the absence of salt was studied by measuring circular dichroism spectra, intrinsic tryptophan fluorescence, and adsorption of the dye 1-anilinonaphthalene-8-sulfonate. These experiments showed that all three heat-denatured proteins did not take on a random-coiled state but rather remained partially folded, with some hydrophobic regions becoming exposed to the solvent environment. This conformation could thus be categorized as the molten globule state.

Previous studies have shown that selenium (Se) deficiency is associated with hematological abnormalities, which may result in an increased distribution of iron in various tissues. This report describes histological studies of the location of excess iron deposits in tissues. Male Wistar rats were fed a Torula yeast-based Se-deficient [Se(-)] or Se-adequate [Se(+); containing 0.1 mg Se/kg as sodium selenite] diet for 8 or 82 weeks. Excised tissues were embedded in either paraffin or epoxy resin. A dramatic increase was observed in iron deposition in the liver and kidneys of rats on the Se(-) diet. Prussian blue-stained sections under the light microscope showed iron deposits in the parenchymal cells and Kupffer cells of liver and in the proximal tubules of kidneys. The liver and kidneys of Se(-) rats had considerably altered morphology: lysosomes were enlarged and contained electron-dense areas. X-Ray microanalysis showed that the areas that corresponded to the lysosomes contained iron. No iron deposits were observed in sections of kidney and liver from rats fed the Se(+) diet. Thus, these studies identified subcellular sites of iron deposition in the liver and kidneys of Se(-) rats. These iron deposits may be an important factor in the pathogenesis of Se deficiency.

Scanning electron microscopy (SEM) shows remarkable morphological surface changes in Sphingopyxis sp. 113P3 cells grown in polyvinyl alcohol (PVA) but not in Luria-Bertani medium (LB) (Hu et al. in Arch Microbiol 188: 235-241, 2007). However, transmission electron microscopy showed no surface changes in PVA-grown cells and revealed the presence of polymer bodies in the periplasm of PVA-grown cells, which were not observed in LB-grown cells. The presence of polymer bodies was supported by low-vacuum SEM observation of PVA- and LB-grown cells of strain 113P3, and the presence of similar polymer bodies was also found when Sphingopyxis macrogoltabida 103 and S. terrae were grown in polyethylene glycol (PEG). The extraction of PVA and PEG from the periplasmic fraction of cells using a modified Anraku and Heppel method and their analysis by MALDI-TOF mass spectrometry strongly suggested that the polymer bodies are composed of PVA and PEG, respectively, in Sphingopyxis sp. 113P3 (PVA degrader) and Sphingopyxis macrogoltabida 103 or S. terrae (PEG degraders). PEG-grown S. macrogoltabida 103 and S. terrae showed higher transport of (14)C-PEG 4000 than LB-grown cells. Recombinant PegB (TonB-dependent receptor-like protein consisting of a barrel structure) interacted with PEG 200, 4000 and 20000, suggesting that the barrel protein in the outer membrane contributes to the transport of PEG into the periplasm.

Application of cryoultramicrotomy to immunoelectron microscopy showed the localization of the chitin synthase of Absidia glauca in situ by using anti-chitin synthase IgG. Mild fixation with a mixture of 0.25% glutaraldehyde and 4% formaldehyde did not destroy the antibody-binding capacity of chitin synthase and preserved the ultrastructure of organelles. Infusion with a mixture of polyvinylpyrrolidone and sucrose as a cryoprotectant before freezing provided adequate sectioning plasticity. Immunolabeling of colloidal gold was detected both at the plasma membrane and the vesicles, and more so in the latter. The results of immunoelectron microscopy were consistent with the fractionation profile of isopycnic sedimentation of the microsomal fraction on a linear sucrose gradient. The profile also demonstrated two peaks of chitin synthase, one of these cosedimented with vanadate-sensitive ATPase and the other with lower buoyant density populations. The former corresponded to the plasma membrane and the later, ve...

Chimeric genes composed of the class I patatin promoter, A1aB1b preproglycinin cDNA and its modified versions (IV+4Met and V+4Met) containing four contiguous methionine codons and nopaline synthase gene terminator were inserted into the genome of potato by Agrobacterium-mediated transformation. Transcripts of the chimeric genes were detected specifically in tubers. Translational products were observed in parenchyma cells of the tubers of potato plants transformed with the normal and modified glycinin cDNAs. This distribution is consistent with the expression specificity of the patatin promoter. The expression levels of the normal and the modified glycinins were 0.2–1.0% of the total soluble tuber proteins. Both the normal and the modified glycinins expressed in the tubers were present as proglycinin trimers. These results indicate that the modifications in IV+4Met and V+4Met did not disrupt any stage in the biosynthetic process of glycinin in potato tubers.

精白した新米と古米の微細構造を比較すると, 米穀粒内部の胚乳組織には顕著な差異は認められなかった。しかし, 精白米の表面は, 古米では糊粉粒が米の表面を完全に覆っているのが観察されたが, 新米では糊粉細胞の膜だけが残り, 糊粉粒は精白によって脱落してしまっていた。新米米飯の表面や内部のデンプン貯留体に小さな穴状の構造が観察された。これは新米米飯に特徴的に認められた構造で, 古米米飯では認められなかった。一方, 高圧電場処理した古米米飯にも, 部分的ではあるが, 新米米飯と同様の構造が米飯表層部に観察された。そして, 精白米に直接処理するより, 精白米を水に浸漬した状態で処理したほうが効果が顕著に現れた。このことから高圧電場処理によって吸水された水の状態に依存して, 未処理古米米飯には観察されなかった特徴的な構造が高圧電場処理古米米飯の表層, 表面に観察されたと考えられる。

The effects of ionic strength and dithiothreitol concentration, a reducing agent, on the heat-induced gelation of a lysozyme solution were examined at pH 7. Two different heating methods were used, one involving single heating, and the other, a two-step heating process in which a sample solution was heated twice under different medium conditions. With 7.5 mM dithiothreitol, a 5% lysozyme solution gave a transparent sol, a transparent gel, a translucent gel and a turbid gel after a single heating with 0, 40, 50, and 60mM NaCI, respectively. When the transparent sol obtained without NaCI was reheated with 50mM NaCI, it produced a firm and transparent gel. This gel was at least 6 times harder than the transparent lysozyme gel obtained by the one-step heating method. High-resolution scanning electron microscopy showed that the transparent gel produced by the two-step heating method had a fine network of linear polymers composed of heat-denatured lysozyme molecules.

Heat-induced ovalbumin gels prepared under given conditions were melted with a second heating to below 90°C. The sample melted was gelled again on cooling. This gel-sol transition, which was detected both by eye and by the change in the dynamic viscoelastic parameters G' and G", was reversible. Differential scanning calorimetry showed that the endothermic heat flow that accompanied the gel-sol transition was much lower than that accompanied the denaturation of ovalbumin. With the repetition of the gel-sol transition, intermolecular disulfide bridges were formed, detected by sodium dodecyl sulfate-polyacrylamide electrophoresis done without a reducing agent.

Glycinin (11S) and beta-conglycinin (7S) are major storage proteins in soybean (Glycine max L.) seeds and accumulate in the protein storage vacuole (PSV). These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the PSV by vesicles. Electron microscopic analysis of developing soybean cotyledons of the wild type and mutants with storage protein composition different from that of the wild type showed that there are two transport pathways: one is via the Golgi and the other bypasses it. Golgi-derived vesicles were observed in all lines used in this study and formed smooth dense bodies with a diameter of 0.5 to several micrometers. ER-derived protein bodies (PBs) with a diameter of 0.3-0.5 microm were observed at high frequency in the mutants containing higher amount of 11S group I subunit than the wild type, whereas they were hardly observed in the mutants lacking 11S group I subunit. These indicate that pro11S group I may affect the formation of PBs. Thus, the composition of newly synthesized proteins in the ER is important in the selection of the transport pathways.

In maturing seed cells, many newly synthesized proteins are transported to the protein storage vacuoles (PSVs) via vesicles unique to seed cells. Vacuolar sorting determinants (VSDs) in most of these proteins have been determined using leaf, root or suspension-cultured cells apart from seed cells. In this study, we examined the VSD of the alpha' subunit of beta-conglycinin (7S globulin), one of the major seed storage proteins of soybean, using Arabidopsis and soybean seeds. The wild-type alpha' was transported to the matrix of the PSVs in seed cells of transgenic Arabidopsis, and it formed crystalloid-like structures. Some of the wild-type alpha' was also transported to the translucent compartments (TLCs) in the PSV presumed to be the globoid compartments. However, a derivative lacking the C-terminal 10 amino acids was not transported to the PSV matrix, and was secreted out of the cells, although a portion was also transported to the TLCs. The C-terminal region of alpha' was sufficient to transport a green fluorescent protein (GFP) to the PSV matrix. These indicate that alpha' contains two VSDs: one is present in the C-terminal 10 amino acids and is for the PSV matrix; and the other is for the TLC (the globoid compartment). We further verified that the C-terminal 10 amino acids were sufficient to transport GFP to the PSV matrix in soybean seed cells by using a transient expression system.

Three different types of proteinase inhibitors, I, II and III, were fractionated from Japanese radish seed by repeated column chromatographies on SE- and CM-cellulose. The finally purified preparation of inhibitor III was found to be homogeneous by both chromatographic and electrophoretic analyses. All three of them strongly and stoichiometrically inhibited trypsin whereas they showed weak inhibition on other proteinases, such as chymotrypsin, Nagarse and Pronase. From nitrogen content and ultraviolet absorption spectra, each of the inhibitors I and III was confirmed to be a protein. The molecular weights of inhibitors I and III were calculated to be 8000 and 12,000, respectively. These inhibitors were stable at temperatures above 90°C in an acidic pH.

Three different types of proteinase inhibitors, I, II and III, were fractionated from Japanese radish seed by repeated column chromatographies on SE- and CM-cellulose. The finally purified preparation of inhibitor III was found to be homogeneous by both chromatographic and electrophoretic analyses. All three of them strongly and stoichiometrically inhibited trypsin whereas they showed weak inhibition on other proteinases, such as chymotrypsin, Nagarse and Pronase. From nitrogen content and ultraviolet absorption spectra, each of the inhibitors I and III was confirmed to be a protein. The molecular weights of inhibitors I and III were calculated to be 8000 and 12,000, respectively. These inhibitors were stable at temperatures above 90°C in an acidic pH.