Antigenic Reactivity of Dephosphorylated αs1-Casein,Phosphopeptide from β-Casein and O-Phospho-l-serine towards the Antibody to Native αs1 -Casein

To study whether the phosphoserine residue is associated with the antigenicity of bovine α_s1- casein, we examined the antigenic reactivity of dephosphorylated α_s1-casein, peptide 1~25 from bovine β-casein and three chemical reagents with IgG antibody specific to native α_s1-casein by an enzyme-linked immunosorbent assay.

The reaction between native α_s1-casein and its IgG antibody was inhibited more strongly by native α_s1-casein than by dephosphorylated α_s1-casein. Peptide 1~25, having a phosphoserine residue-concentrated region from bovine β-casein, noticeably inhibited the reaction between native α_s1 -casein and its antibody. Furthermore, the O-phospho-l-serine residue inhibited the reaction of peptide 61~123 with anti-native α_s1-casein antibody, although l-serine and sodium phosphate showed no measurable inhibition.

These results suggest that the phosphoserine residue associated with part of an antigenic site in bovine α_sl-casein.     

Precursors to Nitrosopyrrolidine and Nitrosopiperidine in Black Pepper Treated with Nitrous Acid

The following properties of food proteins polymerized by guinea pig liver transglutaminase were investigated: (1) solubility, (2) emulsifying activity and emulsion stability, and (3) unfrozen water content by pulsed NMR. Several food proteins (α_sl- and k-caseins, and soybean 7S and 11S globulins) were polymerized by this enzyme. Solubility and emulsifying activity of polymerized α_sl-casein were higher than those of the native protein in the range of pH 4~6. Unfrozen water contents of polymerized soybean globulins were much higher than those of the native proteins. These results suggest that transglutaminase treatment may be used for the production of new food protein material with higher hydration ability.     

Photolysis of Aryl Glycosides with Ultraviolet Irradiation

Bovine casein components (α_sl-, β-, and κ-caseins) were chemically phosphorylated and the properties of the modified components were compared with those of the native to clarify the function of the intrinsic phosphate groups of casein components in casein micelle formation. The calcium binding ability of casein components increased after chemical phosphorylation. The concentrations of calcium chloride required to precipitate modified α_sl- and β-caseins were higher than those for native components. However, phosphorylation of α_sl- and β-caseins did not affect their properties of forming micelles through interaction with κ-casein. The stabilizing ability of κ-casein for α_sl- and β caseins was impaired by its phosphorylation, but the stability was recovered by treating phosphorylated κ-casein with phosphoprotein phosphatase. The results show that the phosphate content of κ-casein must be low to form a stable casein micelle. The results also explain why the specific phosphorylation of casein components in the mammary gland is required.     

Fractionation and Characterization of 9 Subcomponents of Bovine κ-Casein A

κ-casein A was fractionated into 9 subcomponents, all of which were identified as κ-casein from immunological analyses. The microheterogeneity of the subcomponents was explained by stepwise increase of their carbohydrate contents (0~4mol/mol of GalNAc, and 0~8mol/mol of NANA). The micelle-stabilizing ability of κ-casein subcomponents increased with the increase of their carbohydrate contents: the carbohydrate rich subcomponent 7 possessed twice the stabilizing ability of the carbohydrate free subcomponent 1. The sensitivity of synthetic casein micelle composed of κ-casein subcomponents and α_sl-casein to the wheat germ lectin-induced aggregation also increased with the increase of their NANA contents.     

Flavonoids in Poncirus Hybrids

α_sl-Casein can be made either soluble or insoluble by adjusting the concentration of coexisting calcium ions. In this study, we tried to make a soluble-insoluble interconvertible enzyme through the formation of a conjugate of an enzyme and α_sl-casein using a heterobifunctional crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The conjugate of phosphoglyceromutase and native α_s1-casein did not exhibit sufficient calcium-dependent precipitation. However, conjugates of enzymes (phosphoglyceromutase, enolase or peroxidase) and α_sl-casein polymerized by transglutaminase precipitated almost completely in the presence of more than 50 mM CaCl₂. Most of the enzyme conjugates precipitated as calcium caseinates could be solubilized reversibly with EDTA, without a significant loss of activity. A mixture of the enzyme ? polymerized α_s1-casein conjugates prepared with phosphoglyceromutase, enolase and pyruvate kinase could catalyze sequential reactions which convert d-3-phosphoglycerate into pyruvate with the same efficiency as a mixture of free enzymes. These results indicate that conjugates of enzymes and polymerized α_s1-casein can be useful as soluble-insoluble interconvertible enzymes.     

Purification and Characterization of a Surface-active Macropeptide Produced from Succinylated αs1-Casein by Modification with Papain

The preceding paper described that when succinylated α_s1-casein, ca. 25,000 daltons, was modified with papain in the presence of l-leucine n-dodecyl ester (Leu-OC₁₂), an approximately 20,000-dalton macropeptide was formed as the main product. In the present work we have investigated its chemical structure and surface function. A treatment for purification at the petroleum ether/water interface gave an electrophoretically homogeneous 20,000-dalton macropeptide which functioned as a surfactant to emulsify corn oil as well as n-octane. Pulsed NMR and ESR studies demonstrated that the macropeptide, when used to emulsify n-octane in water, acted to restrict the mobility of those molecules involved in the emulsion. Various data from chemical analyses coupled with knowledge about the primary structure of α_s1-casein showed that the 20,000-dalton macropeptide was structured as succinyl-Arg¹-….-Phe¹⁴⁵-Leu-OC₁₂. A discussion is included to explain the surface function of this peptide in relation to its amphiphilic structure.     

Studies on κ-Casein of Bovine Milk

κ-Caseins were prepared by the calciurn-ethanol method, the Sephadex method and the urea-sulfuric acid method. Some important properties of κ-caseins were investigated using isoelectric focusing, starch gel electrophoresis, ultracentrifugation, chemical analysis, stabilization test of α_s-casein, and rennin treatment. Isoelectric focusing established that κ-casein had its isoelectric point near pH 6.0 in 6 m urea, usually accompanied by a second peak around pH 5.6. Ultracentrifugation, however, showed a single peak having a s_20,w value of 2.6 ~ 3.8 in the presence of 6 m urea and of 14.4 in the absence of such dispersing reagents. Normal contents of hexose, sialic acid, phosphorus, and nitrogen were about 1.5, 0.8, 0.2, and 14%, respectively. Relative patterns of amino acid composition were similar in all of the κ-caseins. In addition, amino acid composition in intact κ-casein and in the further purified κ-casein which formed the second peak in DEAE cellulose chromatography were almost identical, indicating that the κ-casein of the first peak is not an impurity but is one of the components which formed the original κ-casein complexes. The ability of κ-caseins to stabilize α_s-casein in the presence of calcium increased when purified by DEAE cellulose chromatography.     

Amino Acid Sequence of Tremerogen A–10, a Peptidal Hormone,Inducing Conjugation Tube Formation in Tremella mesenterica Fr.

κ-Casein components having various carbohydrate contents were prepared by diethylaminoethyl-cellulose chromatography and the interactions of each κ-casein component both with α_s1-casein and with β-casein were examined by Sepharose 4B gel chromatography, ultra-centrifugal experiments and viscosity measurements. Each κ-casein component could form complex with α_s1- and β-casein in the absence and presence of CaCl₂. Molecular weight of complexes of unfractionated κ-casein both with α_s1-casein and with κ-casein were about 70 × 10⁴ at 37°C in the absence of CaCl₂, while those of complexes of each κ-casein component with α_s1 and β-casein were about 50 × 10⁴. Stokes radii of complexes increased with increasing calcium ion. While sedimentation coefficient at 37°C of complex with β-casein had almost the same value, those of complexes with α_s1-casein decreased with increase of carbohydrate content of κ-casein components. Intrinsic viscosity of complex of κ-casein component having much carbohydrate was almost the same among tested temperatures. It is suggested that heterogeneity of κ-casein is necessary to form large complex and that the carbohydrate moiety of κ-casein contributes the stability of casein complex.     

Detection of immunoglobulin heavy chain IgG3 polymorphism in wild mice with xenogeneic monoclonal antibodies

We have generated four xenogeneic rat antimouse IgG₃ monoclonal antibodies recognizing at least three different antigenic determinants (epitopes) on BALB/c IgG₃ molecules. These antibodies were used in solid-phase blocking radioimmunoassays for detection of the epitopes in sera of 40 inbred strains and 134 wild mice. These antibodies detect genetic polymorphism of IgG₃ isotype among wild mice even though there is no polymorphism found among 40 inbred strains tested (except X-chromosome-linked immunodeficient CBA/N strain which lacks IgG₃ molecules). An IgG₃ variant was also isolated from hybridomas derived from Mus spretus.Abbreviations Igh-C immunoglobulin heavy chain constant region - PVC polyvinyl chloride - RIA radioimmune assay - ELISA enzyme linked immunosorbent assay     

The chaperone action of bovine milk αS1- and αS2-caseins and their associated form αS-casein

α_S-Casein, the major milk protein, comprises α_S1- and α_S2-casein and acts as a molecular chaperone, stabilizing an array of stressed target proteins against precipitation. Here, we report that α_S-casein acts in a similar manner to the unrelated small heat-shock proteins (sHsps) and clusterin in that it does not preserve the activity of stressed target enzymes. However, in contrast to sHsps and clusterin, α-casein does not bind target proteins in a state that facilitates refolding by Hsp70. α_S-Casein was also separated into α- and α-casein, and the chaperone abilities of each of these proteins were assessed with amorphously aggregating and fibril-forming target proteins. Under reduction stress, all α-casein species exhibited similar chaperone ability, whereas under heat stress, α-casein was a poorer chaperone. Conversely, α_S2-casein was less effective at preventing fibril formation by modified κ-casein, whereas α- and α_S1-casein were comparably potent inhibitors. In the presence of added salt and heat stress, α_S1-, α- and α_S-casein were all significantly less effective. We conclude that α_S1- and α-casein stabilise each other to facilitate optimal chaperone activity of α_S-casein. This work highlights the interdependency of casein proteins for their structural stability.     

The production of Non-H-2 histocompatibility alloantibodies in the mouse

Alloantibodies specific for non-H-2 histocompatibility antigens of the mouse have been produced. Immunization (BALB/cJ×DBA/2J)F₁ anti-B10.D2/n was conducted, followed by hemagglutination, immunofluorescence, and mixed hemabsorption tests on absorbed and unabsorbed sera. The results indicate that antibodies specific for H-3^a and H-8^a antigens are present. In addition, H-8^a antigenic determinants were detected on erythrocyte membrane surfaces, as well as on cells of other body tissues.     

Conformation of αs-Casein in Various Concentrations

Conformation of α_s-casein and its association were investigated from behaviors of tyrosyl and tryptophyl residues and hydrophobic sites. The chromophoric residues and ANS binding sites were buried into a region inaccessible to solvent with increasing concentration of α_s-casein. It is considered that the association of α_s-casein with concentration is proceeded by the hydrophobic sites to be able to bind ANS and the hydrophobic segments in which tyrosyl and tryptophyl residues exist. Below 0.04% of α_s-casein, α_s-casein exists in the monomer state and 80% of tyrosyl and tryptophyl residues are accessible to aqueous solvent. The hydro-phobic sites of α_s-casein may be exposed to solvent in the monomer state.     

Structure of Bovine αs-Casein

The secondary structure of bovine α_s-casein and chemically modified α_s-casein in various solvents was investigated by infrared absorption spectrum and optical rotatory dispersion measurements. Amino groups of α_s-casein were either succinylated or acetylated, and carboxyl groups were either methylated or ethylated. Acetylated- and ethylated-α_s-caseins are insoluble in water. Water-soluble samples have unordered structure in water. In organic solvents, such as 2-chloroethanol and ethylene glycol, they have about 50% α-helical fraction. On the other hand, it was found that methylated-α_s-casein had two infrared absorption peaks centered at 1625 and 1643 cm^?1 in D₂O-CH₃OD mixed solvent. This fact may be connected with the presence of β-structure. In the case of solid film of this sample, cast from solution containing CH₃OH, the presence of β-structure was indicated, too. The authors attempted to explain the formation of β-structure in methylated-α_s-casein in terms of the electrostatic interactions due to the differences in the net charge between methylated and unmodified α_s-caseins.     

Interaction between Casein and Carboxymethyl Cellulose in the Acidic Condition

The stabilizing action of carboxymethyl cellulose (CMC-1 and CMC-2) on caseins was studied in the acidic pH region. CMC-1 stabilized 1% whole, α-, α_S- and β-casein at pH 4.6 and 5.0, and at 5°C. But CMC-2 could not completely stabilize these caseins at pH 5.0. Interaction between κ-casein and CMC-1 commenced when pH was adjusted to 6.3, but CMC-2 interacted with κ-casein below pH 5.6. An α_S- and κ-casein mixture (4 : 1) with CMC-2 was destabilized by the addition of 0.02 m NaCl or NaH₂PO₄ at pH 5.0. The α_S/κ ratio of the precipitated casein was about 10. But the same system with CMC-1 was not destabilized by the salts.     

Characterization of antigenic properties of horseradish peroxidase with monoclonal antibodies

Monoclonal antibodies (mcAbs) specific to alkaline isoenzymes of horseradish peroxidase were used to characterize the antigenic properties of horseradish peroxidase. The results of a competitive binding assay indicated that monoclonal antibodies can be divided into three groups directed against distinct parts of the protein. The interaction of monoclonal antibodies with native and modified horseradish peroxidase showed also three different patterns of reactivity. Antibodies from groups I and II are directed against epitopes which are conformational and formed by tertiary structure elements. Epitopes recognized by these antibodies are sensitive to heme removal or partial denaturation of peroxidase. Antibodies from group III bind specifically with epitopes consisting of primary or secondary structure elements. The antigenic determinants recognized by antibodies from group III PO ₁ and 36F ₉ were shown to be linear (continuous) and formed by amino acid residues 261-267 and 271-277, respectively, as determined by the peptide scanning method (PEPSCAN). The location of revealed linear antigenic determinants in the molecular structure of peroxidase is analyzed.     

The Primary Structure of Water Buffalo αs1- and β-Casein: Identification of Phosphorylation Sites and Characterization of a Novel β-Casein Variant

The primary structure of water buffalo α_s1-casein and of β-casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a β-elimination/thiol derivatization. Water buffalo α_s1-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine α_s1-casein C variant, the water buffalo α_s1-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine βA₂-casein variant, the two water buffalo β-casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo β-casein variants seem to be homologous to bovine βA₂-casein.     

Calcium-Binding Property of Dephosphorylated Caseins

Whole casein, α_s-casein and k-casein were dephosphorylated with a phosphoprotein phosphatase prepared from beef spleen and their calcium-binding capacities were compared with those of respective native caseins by a ultracentrifugal method.

The bindings of the calcium to 94% dephosphorylated whole casein and to 97 % dephosphorylated α_s-casein at neutral pH were approximately one third of those to respective native caseins. The decrease of calcium-binding capacity of k-casein due to dephosphorylation was also significant.

The effect of pH on the state and the calcium-binding capacity of dephosphorylated caseins was also examined and the role of organic phosphate groups of casein as calcium-binding sites was discussed.     

Studies on the Interaction between αs1- and β-Caseins

The interaction of α_s1-casein with β-, dephosphorylated β-,γ- and R-caseins was studied. It was proved by the sedimentation velocity experiments that α_s1-casein formed a complex with each of these components at 25±C in the presence of 3 mm CaCl₂.

In the presence of 10 mm CaCl₂, β- and dephosphorylated β-casein prevented the precipitation of α_s1-casein and gave micelle-like turbid solutions. However, γ- and R-caseins, fragments of β-casein, did not stabilize α_s1-casein. It was concluded from these results that α-casein interacted with α_s1-casein through its hydropholic region corresponding to R-casein and that hydrophilic region of β-casein was responsible for the stabilization of α_s1-casein.     

Thermodynamics of the Interaction between αs1- and κ-Caseins

Pyrenebutyrate-conjugated α_s1-casein was prepared and the complex formation between α_s1- and κ-casein polymers was investigated by fluorescence polarization. The complex formation was also investigated by a microcalorimetric technique. The positive enthalpy and entropy changes and endothermic nature suggested the hydrophobic interaction between α_s1- and κ-casein polymers.

The degree of polarization of κ-casein polymer decreased with the addition of 1-anilino-8-naphthalenesulfonate (ANS), while that of α_s1-casein polymer and α_s1-κ-casein complex was invariant. Moreover the reaction of κ-casein polymer and ANS was exothermic. These facts suggested that the intermolecular hydrophobic regions in κ-casein polymer were disrupted by the adsorption of ANS. The rotational relaxation time of pyrenebutyrate conjugated complex between cyanoethyl-κ-casein and α_s1-casein polymer was smaller than that of cyanoethyl-κ-casein alone. From these results, it was postulated that the dissociation of κ-casein polymer by the complex formation with α_s1-casein polymer might be caused by the disruption of the intermolecular hydrophobic bonds in κ-casein polymer.     

Conformational Changes of αs-Casein by Heating

Conformational changes of α_s-casein by heating were investigated by measuring ultraviolet difference spectra. The ultraviolet difference spectra at elevated temperature against 5.5°C were measured in various ionic strengths and pHs. Thermal effects of the difference spectra were cancelled by comparing with the spectra of model compounds such as lysozyme and ribonuclease, and the blue shift of α_s-casein spectra was observed at above 30°C in these all experimental conditions. This shift was considered to mean unfolding of the α_s-casein molecule. The aggregation of α_s-casein was observed above ionic strength of 0.4 by heating. These heat-induced changes were reversible until the aggregation was observed.