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When egg white was heated to between 50° and 60°C, a new protein band appeared in its polyacrylamide gel electrophoretic pattern. The formation of the protein band in stored egg white was almost the same as that of fresh egg white. This protein band was shown to be induced by the interaction between macroglobulin and lysozyme during the egg white heating. The optimum temperature for its formation was between 54°C and 57°C. This band formation may be used as an indicator for heat treatment of egg white.

The thermal denaturation of ovomucoid was investigated by immunochemical methods, namely immunoprecipitation analyses and antibody-Sepharose 4B column chromatography. In the immunoprecipitation analyses, heated ovomucoid (90 degrees C, 90 min, pH 7.2) required about twice the antigen addition of the native protein to approach maximal precipitation with specific antibody, and the maximal immunoprecipitation was decreased to 80% of that by native ovomucoid. However, heated protein inhibited the binding of antibody with native ovomucoid, and 100% inhibition was attained at about 4-times the antigen addition necessary for the native protein. Heated ovomucoid (100 degrees C, 120 min) showed little immunoprecipitation and inhibition. To ovomucoid antigenicity was diminished more slowly than the trypsin inhibitory activity by heating, e.g., heated ovomucoid (90 degrees C, 120 min) retained more than 30% of the antigenicity but little trypsin inhibitory activity. By passing through the immunoaffinity column, heated ovomucoid (90 degrees C, 90 min) was separated into two fractions, either with (fraction II) or without (fraction I) antigenicity. Fraction II contained smaller fractions of ordered secondary structure than native ovomucoid, and trypsin inhibitory activity of fraction II was only 24% of the native one. These results indicated that thermally denatured ovomucoid was heterogeneous regarding the conformational damage caused by heating, and the structure around some antigenic sites in an ovomucoid molecule was retained even after the backbone conformation was partially destroyed and trypsin inhibitory activity was lost.

A protein antigenic to the IgE antibody of allergic individuals was isolated from rice grain by ion-exchange and gel-filtration chromatographies. The molecular weight of the allergenic protein was estimated to be about 16,000 by sodiumdodecylsulfate-gel electrophoresis. The protein contained 7mol of cystine residue per mole of protein and no cysteine residues, and its immunoreactivity was quite stable to heating at 100°C. This allergenic protein was mainly present in the endosperm portion of rice seeds.

Temperature-induced structural changes in chicken egg white ovomucoid were investigated by circular dichroism (CD) and ultraviolet absorption (UV). A major unfolding transition was brought about between 60 and 90°C with a rise in temperature, as monitored by variations in CD at 222 nm and UV at 287 nm. Thermal transition was reversible when there was no prolonged exposure to the denaturing condition. The Van't Hoff plot for the major unfolding process of the ovomucoid yielded the following thermodynamic parameters at pH 7.0 between 60 and 90°C: δHVH=73 kcal.mol-1 at 74°C and δSVH=209 cal.deg-1. mol-1 at 74°C, on the assumption of a two-state transition. The equilibrium CD spectra at various temperatures suggested that the secondary structure change caused by heating from 20 to 60°C was qualitatively different from that from 60 to 90°C. By prolonged exposure to high temperature of 90°C, the reversible denatured state was slowly transformed into an irreversible denatured state.

A glycopeptide was isolated from a peptic hydrolysate of chicken ovomucoid using gel filtration after reduction with 2-mercaptoethanol. The glycopeptide was thought to be derived from the sequence Gly51-Tyr73 of chicken ovomucoid and retained the immunoreactivity to IgG of mouse antiserum and IgE of human serum specific to ovomucoid. About fifty percent of the immunoreactivity to mouse antiserum was inhibited by the sera of patients allergic to ovomucoid and also by the carbohydrate chains on ovomucoid. These results suggested that the glycopeptide, connecting the first domain and second domain of chicken ovomucoid, participated in egg allergy, and the ovomucoid carbohydrate chains contributed to the immunoreactivity of the glycopeptide.

Graphical abstract, Highlights • Chicken ZP1 and ZP3 assemble through strong interactions between their ZP-C domains. • ZP-C domains of chicken ZP1 and ZP3 are deeply embedded in the egg-coat matrix. • Chicken ZP1 forms a homocomplex through non-covalent interaction between repeat domains. • Chicken ZPD is deposited on the interstices of ZP1–ZP3 matrix in the egg coat. • We propose a model for the architecture of chicken egg-coat matrix from these results., The vertebrate egg coat, including mammalian zona pellucida, is an oocyte-specific extracellular matrix comprising two to six zona pellucida (ZP) glycoproteins. The egg coat plays important roles in fertilization, especially in species-specific interactions with sperm to induce the sperm acrosome reaction and to form the block to polyspermy. It is suggested that the physiological functions of the egg coat are mediated and/or regulated coordinately by peptide and carbohydrate moieties of the ZP glycoproteins that are spatially arranged in the egg coat, whereas a comprehensive understanding of the architecture of vertebrate egg-coat matrix remains elusive. Here, we deduced the orientations and/or distributions of chicken ZP glycoproteins, ZP1, ZP3 and ZPD, in the egg-coat matrix by confocal immunofluorescent microscopy, and in the ZP1–ZP3 complexes generated in vitro by co-immunoprecipitation assays. We further confirmed interdomain interactions of the ZP glycoproteins by far-Western blot analyses of the egg-coat proteins and pull-down assays of ZP1 in the serum, using recombinant domains of ZP glycoproteins as probes. Our results suggest that the ZP1 and ZP3 bind through their ZP-C domains to form the ZP1–ZP3 complexes and fibrils, which are assembled into bundles through interactions between the repeat domains of ZP1 to form the ZP1–ZP3 matrix, and that the ZPD molecules self-associate and bind to the ZP1–ZP3 matrix through its ZP-N and ZP-C domains to form the egg-coat matrix. Based on these results, we propose a tentative model for the architecture of the chicken egg-coat matrix that might be applicable to other vertebrate ones.

Dietary proteins are degraded into peptides or amino acids in the small intestine by several pancreatic proteinases and mucous membrane peptidases. The present experiments using HPLC and fluorescence-labeling were designed to derive basic information about the behavior and digestion of oligosaccharides in stomach and small intestine of rats

Antigenicity of egg white proteins which remained in heated shell eggs was analyzed quantitatively by using rabbit antibodies specific to egg white proteins and human antibody from patients allergic to egg. The major antigenic component in heated (100°C for 20 min) and coagulated egg white was ovomucoid. A considerable amount of ovomucoid remained immunoreactive even after heating at 100°C for 15 min, though ovomucoid in stored eggs was a little less stable to heating than that of fresh eggs. Even long-time heating (100°C for 45 min) could not completely eliminate the ovomucoid immunoreactivity to human IgE antibody.

Heat-induced aggregation of whole egg proteins through various treatment combinations ranging from 70–85°C, pH 2.0–9.0, and NaCl concentrations of 0–3%, was investigated using multiple regression analysis and vertical flat-sheet polyacrylamide gel electrophoresis. The importance of single variable was in the order of temperature, pH and NaCl concentration, and the combined variable of pH and NaCl had a highly significant (p 5) and NaCl concentration increased.

Chicken egg white ovomucoid was hydrolyzed with pepsin, fractionated by gel filtration, and the reactivity to anti-ovomucoid antibody was detected in the two carbohydrate-containing fractions. Immunoreactive fragments separated from these fractions by immunoaffinity chromatography were positive to both protein and carbohydrate staining, i.e., glycopeptides. Ovomucoid glycopeptides prepared by pronase-hydrolysis did not have this immunoreactivity. These results show that some glycopeptides with immunoreactivity remained after peptic hydrolysis, suggesting that the carbohydrate moieties of ovomucoid exert a protective effect against peptic hydrolysis.

Two kinds of the third domain, either with or without a carbohydrate chain, were prepared from chicken ovomucoid. The immunoreactivity of the domain preparations to human IgE antibody directed against ovomucoid was examined by using the sera from patients of egg allergy. About 30% of the serum antibody to ovomucoid reacted with the carbohydrate-containing domain, whereas little or no antibody with reactivity to the carbohydrate-free domain was detected, suggesting that the carbohydrate chain attached to the third domain played an important role in antigenic determinants of the carbohydrate-containing third domain against the human IgE antibody.

The secondary structure of chicken egg white ovomucoid and its domains was studied by means of circular dichroism (CD). The various domains (domain I, domains II . III, domains I . II, and domain III with and without carbohydrate) were prepared from the ovomucoid by cyanogen bromide cleavage and Staphylococcus aureus protease V-8 digestions. The carbohydrate moiety in the ovomucoid was isolated after its extensive pronase and endo-beta-N-acetyl-glucosaminidase H digestions. On the net CD spectra of polypeptide chain in the ovomucoid and domain preparations, which were obtained by subtracting the spectrum of the carbohydrate moiety from their spectra, the fractions of secondary structure were calculated by the method of Chang et al. (Chang, C.T., Wu, C.S.C. and Yang, J.T. (1978) Anal. Biochem. 91, 13-31). The estimated composition of the secondary structure in the ovomucoid was as follows: alpha-helix, 26%; beta-structure, 46%; beta-turn, 10%; and random coil, 18%. The secondary structure compositions estimated for the three domains suggested that domains I and II were homologous to one another but not to domain III in regard to the proportion of secondary structure content.

An antibody subpopulation, anti high molecular weight (anti-HMW) kininogen-Ca2+ antibody able to bind specifically to the HMW kininogen-Ca2+ complex, was isolated from anti-HMW kininogen antiserum. Partially purified anti-HMW kininogen antibody was applied to a HMW kininogen-Sepharose column equilibrated with 40 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5, containing 1.0 M NaCl and 1 mM CaCl2, and anti-HMW kininogen-Ca2+ antibody was eluted with 5 mM ethylenediaminetetraacetic acid. As a result of characterization by enzyme-linked immunosorbent assay, this antibody specifically recognized the cyanogen bromide cleaved fragment 1 (CB-1) region (1-160 amino acid sequence) of the heavy chain of kininogen molecules in the presence of Ca2+ or Mg2+. Furthermore, circular dichroism (CD) experiments showed that the conformational changes of HMW kininogen and heavy chain were induced by metal ions such as Ca2+ and Mg2+ and that these changes were due to the conformational change of the CB-1 region of the heavy chain. The dissociation constant (Kd) for the heavy chain-Ca2+ measured by CD analysis at 214 nm was found to be 0.33 +/- 0.09 mM (mean +/- SD). The number of Ca2+-binding sites of heavy chain calculated from the Hill plot was 1.15 +/- 0.04 (mean +/- SD). Then, a possible Ca2+-binding site was found in the amino-terminal portion of the heavy chain of kininogen molecules.

Ovomucoid and ovoinhibitor were isolated from chicken egg white. Enzyme-linked immunosorbent assay (ELISA) and affinity chromatography of ovoinhibitor-coupled Sepharose 4B showed that about 25% of rabbit anti-ovomucoid antibody reacted with ovoinhibitor. Ovoinhibitor required about 1000 times the concentration of ovomucoid to give 50% inhibition of anti-ovomucoid antibody binding to ovoinhibitor in the plate-binding ELISA. These results suggested that ovoinhibitor possesses antigenic determinants which are conformationally homologous with those of ovomucoid and cross-react with anti-ovomucoid antibodies.

In order to measure muscle stiffness changes with a high time resolution and with minimal disturbance to the contractile mechanism per se, we constructed an apparatus with which the propagation velocity of ultrasonic waves (MHz region) in the longitudinal or transverse direction was measured to serve as a measure of muscle stiffness. The longitudinal muscle stiffness started to increase on stimulation before the onset of isometric force, and reached a maximum before the peak twitch force. Analysis of experimental data indicated that, during an isometric tetanus, the increment of muscle longitudinal stiffness was about 6×107N/m2, a value similar to those obtained by Truong (1974) and Ford et al. (1981) with sinusoidal vibrations (3 kHz) and length steps respectively. This suggests that the increment of muscle longitudinal stiffness during the activation of the contractile system results from the recruitment of an almost non-dispersive elastic component.

The maize response regulator genes ZmRR1 and ZmRR2 respond to cytokinin, and the translated products seem to be involved in nitrogen signal transduction mediated by cytokinin through the His-Asp phosphorelay. To elucidate the physiological function of the proteins, we examined the temporal and spatial distribution in maize leaves by immunochemical analysis and use of transgenic plants. ZmRR1 and ZmRR2 polypeptides could be distinctively detected by western blotting. The polypeptides accumulated in leaves within 5 h of the supply of nitrate to nitrogen-depleted maize, and the accumulation was transient. The extent of induction was larger in the leaf tip, which is rich in photosynthetically matured cells, than elsewhere. In leaves, the polypeptides accumulated mostly in mesophyll cells. Histochemical analyses of transgenic maize harboring a ZmRR1 promoter-beta-glucuronidase fusion gene also showed most of the expression to be in these cells. These results suggest that ZmRR1 and ZmRR2 are induced in mesophyll cells and function in nitrogen signal transduction mediated by cytokinin.