Kunio Yamauchi

Monoclonal antibodies as probes for monitoring the denaturation process of bovine β-lactoglobulin

Five monoclonal antibodies (MAbs) of different idiotypes were produced against bovine beta-lactoglobulin (beta-LG). Among them, MAbs 61B4 and 62A6 reacted preferentially to native beta-LG, while MAbs 21B3 and 31A4 reacted more strongly to the reduced carboxymethylated (denatured) beta-LG than to the native material. These two types of MAb were used to analyze the denaturation process of a beta-LG molecule during heating. The binding affinity of MAbs 21B3 and 31A4 with beta-LG was increased by increasing the heating temperature, the transition temperature being 67-68 degrees C, while that of MAbs 61B4 and 62A6 was reduced by increasing the temperature, this transition temperature being about 80 degrees C. Epitopes recognized by MAbs 31A4 and 61B4 were shown to be included in the segments, Lys8-Trp19 (mostly in the random-coil region) and Thr125-Lys135 (helical region), respectively. The heat-induced conformational change of the beta-LG molecule is, therefore, likely to start in random-coil region as Lys8-Trp19, and to be followed by a structural change in a helical region as Thr125-Lys135. This study demonstrates that MAb is a useful probe to monitor local conformational changes of a protein molecule during denaturation.

The topography of αs1-casein adsorbed to an oil/water interface: an analytical approach using proteolysis

Abstract An α s1 -casein solution and lipid were mixed and homogenized so that an oil/water emulsion was prepared. The topography of an α s1 -casein chain adsorbed to oil droplets in the emulsion was analyzed. Trypsin and α-chymotrypsin were allowed to act on α s1 -casein in the emulsion (the emulsion system) and that dissovled in an aqueous solution (the solution system). The digests were separated by reversed-phase high performance liquid chromatography (reversed-phase HPLC), and subjected to amino acid analyses. The peaks of the HPLC patterns were assigned to peptides in the total sequence of α s1 -casein. Cleavage of thirteen peptide bonds in an α s1 -casein chain in the emulsion system was difficult, as compared with those in the solution system. The results suggest that these peptide bonds were among definite regions inaccessible to proteinases, regions which could be some definite adsorption sites of α s1 -casein to oil. A preliminary model is proposed for the structure of α s1 -casein at an oil/water interface, and it is shown that some hydrophobic regions of an α s1 chain. The neighborhood of the residues 21–25, 94, 135 and 142 from the N-terminal, might interact strongly with oil, while the other hydrophobic region, the neighborhood of the residue 165, might not.

Serum Antibody Response Elicited by a Casein Diet is Directed to Only Limited Determinants of αs1-Casein

Feeding mice with bovine whole casein as a diet constituent elicited an anti-alpha s1-casein (alpha s1-CN) systemic humoral response. The mice fed with the casein (CN) diet responded poorly to subsequent parenteral immunization with the antigen, indicating that oral tolerance was induced in the animals. The serum antibodies elicited by feeding CN were tested for binding to a panel of synthetic peptides and are shown to recognize only a limited number of potential antigenic determinants on alpha s1-CN. Our results suggest that a similar mechanism may be involved in the onset of milk allergy.

Monoclonal antibody raised against tetrodonic acid, a derivative of tetrodotoxin.

Tetrodonic acid, a relatively non-toxic derivative of tetrodotoxin, was conjugated with bovine serum albumin and injected i.p. to BALB/c mice. After several injections, spleen cells were isolated, fused with myeloma cells X63-Ag8-6.5.3. and cloned by the limiting dilution method. The monoclonal antibody produced in ascites fluid in the mouse by the cloned cell showed an increasing reactivity with tetrodotoxin at concentrations ranging from 0.03 to 100 micrograms per well.

Proteolysis in structural analysis of αs1-casein adsorbed onto oil surfaces of emulsions and improvement of the emulsifying properties of protein

Abstractαs1-Casein has a unique amphiphilic structure which may provide excellent emulsifying properties. Structural analysis of αs1-casein adsorbed to emulsified oil globules and improvement of the emulsifying properties of αs1-casein were undertaken by using proteolysis. Enzymic cleavage of αs1-casein adsorbed to oil globules of the emulsion was compared with that dissolved in an aqueous solution by using trypsin and chymotrypsin. Thirteen peptide bonds of the adsorbed αs1-casein in the emulsion were hardly cleaved and were probably among definite regions inaccessible to the proteases. Based on the results, a preliminary model is proposed for the structure of αs1-casein at an oil/water interface. Emulsifying activity (EA) of αs1-casein was increased by pepsin digestion. A peptide fraction (PF) separated from the peptic hydrolysate, being composed of αs1-CN(fl-23) and a small amount of other peptides, showed an EA similar to that of αs1-casein at neutral pH. Removal of the small amount of the peptides from PF resulted in a marked decrease of EA. However, the addition of the removed peptides to αs1-CN(fl-23) restored the EA. Some synergistic effect between αs1-CN(fl-23) and the other peptides in emulsification was suggested.

Antigenic Identity between the Soluble Glycoprotein of Milk Fat Globule Membrane and a Heat-stable Protein Fraction of Whey

The protein which is antigenically equivalent to the soluble glycoprotein (SGP) isolated from milk fat globule membrane was immunologically found to be contained in whey protein. The identical antigeneicity between the SGP and an unknown protein component of whey was certified by double immunodiffusion and immunoelectrophoretic assays, using the antiserum of the SGP and its antiserum absorbed with whey protein, and using the whey protein fractions fractionated by gel filtration or ion exchange chromatography. The concentration of this protein in whey (anti-SGP reacting protein) was estimated to be about 15% of total whey protein by simple radial immunodiffusion assay. The major whey protein components, such as ƒÀ-lactoglobulin, ƒ¿-lactalbumin, bovine serum albumin, and IgG-immunoglobulin were confirm-d to be not the anti-SGP reacting

Presence of Heparan Sulfate in the Fat Globule Membrane of Bovine and Human Milk

Glycosaminoglycans were isolated from bovine and human milk fat globule membrane (MFGM) preparations and characterized. The amount of glycosaminoglycans in human MFGM protein fraction was 5 ~ 10 times higher than that in bovine MFGM protein fraction. On Dowex-1 column chromatography most of the glycosaminoglycans were eluted with 1 M and 3 M NaCl. Cellulose acetate electrophoresis, chondroitinase digestion and nitrous acid degradation indicated that the major glycosaminoglycan in both bovine and human MFGM was heparan sulfate. Bovine MFGM was shown to contain chondroitin sulfate A/C in addition to heparan sulfate. The glycosaminoglycan composition of bovine colostral MFGM was similar to that of bovine normal MFGM. The glycosaminoglycan fraction prepared from MFGM of human colostrum, however, did not contain heparan sulfate, suggesting some compositional changes in glycosaminoglycans during the lactation period.

Decomposition of β-Casein by Milk Protease: Similarity of the Decomposed Products to Temperature-sensitive and R-Caseins

Comparative studies of temperature-sensitive (TS-) and R-caseins with two decomposed products (F-II and III) of β-casein by milk protease were performed. In polyacrylamide gel electrophoresis at pH 9.1 and 3.0, F-II and III had the mobilities which were the same as TS- and R-caseins, respectively. Amino acid composition, molecular weight and sedimentation coefficient of F-II almost coincided with those of TS-casein. F-III and R-casein also appeared to coincide each other in these properties. It is suggested that TS- and R-caseins are possibly the decomposed products of β-casein by milk protease.

Conformation and some properties of β-casein-like fraction of human casein

A β-casein-like fraction was isolated from human casein by Sephadex G-150 gel filtration. The temperature-dependent polymerization and conformation of the β-casein-like fraction has been examined by analytical ultracentrifugation, viscometry, optical rotatory dispersion, and circular dichroism. The human β-casein-like fraction exists as a monomer, s°20,w 1.3, with a molecular weight of 24 100, at 4 and 10 °C; it begins to polymerize partially at 20 °C. At 24 °C its polymerization increases and a polymer, s 20,w 13.9, coexists with a monomer. It was made clear by means of viscometry that the most part of the protein molecule has the characteristic conformation of a random coil. Moreover, it was shown by means of optical rotatory dispersion and circular dichroism that the protein molecule contains the α-helical and poly-l-proline II-like helical conformation known in β-casein of bovine milk. The human β-casein-like fraction changes its structural conformation with the increase of temperature. This behavior is very similar to that of bovine β-casein.

Identification of α-lactalbumin and β-lactoglobulin in cynomolgus monkey (Macaca fascicularis) milk

1. 1. An electrophoretic analysis of whey protein from cynomolgus monkey milk revealed that its consituents are more similar to bovine milk than human milk, i.e. cynomolgus monkey milk whey contains, besides α-lactalbumin-like protein (LaP), another predominant component similar to bovine β-lactoglobulin (LgP), in its electrophoretic behavior on both disc- and SDS-polyacrylamide gel electrophoreses. 2. 2. The amino acid composition of LaP shows close similarity to that of human α-lactalbumin, and LaP forms an immunoprecipitin line with anti-human α-lactalbumin rabbit antiserum. The homology between LaP and α-lactalbumin was further confirmed by an alaysis of the N-terminal amino acid sequence. 3. 3. LgP is not immunologically identical to bovine β-lactoglobulin, but its amino acid composition is similar. The result of the N-terminal amino acid seuence analysis of LgP (up to the 26th residue) strongly suggests homology between this protein and β-lactoglobulin.