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Human casein. 3. DEAE-cellulose-urea chromatography of human casein and dephosphorylation of casein fractions
... Lactating breast tissue fixed in formol saline showed only very weak staining with both LICR-LON-32. Whole human casein for use in competitive inhibition studies was prepared from skim milk by acid precipitation (27), and was chromatographed in Sephadex G-100, and then in DEAE-cellulose in a buffer system of imidazole-HCl (10 mM; pH 7.0), urea (3.3 M), EDTA (1 mM), and DTT (10 mM), in a modification of the method used by Nagasawa (28). The whole human casein separated by this method was tested in a direct micro-ELISA assay (13), using a rabbit polyclonal antiserum to casein, and monoclonal antibodies LICR-LON-32.2 ...
The immunohistochemical analysis of human beta- and kappa-caseins in the human breast, benign breast disease, and breast carcinomas is reported. The monoclonal antibodies LICR-LON-32.2 and LICR-LON-14.1 which react with human beta- and kappa-casein, respectively (Earl, H. M., and McIlhinney, R. A. J. Mol. Immunol., 22: 981-991, 1985) were used for these studies. Human beta- and kappa-caseins were detected in lactating human breast tissue, lactational foci in the resting breast, and heterogeneously in the 16th-week pregnant breast, but not in normal breast tissue. In benign breast disease occasional epithelial cells were demonstrated to synthesize beta- and kappa-caseins, but this finding did not appear to correlate with the hormonal status or previous obstetric histories of the patients. However, similar studies in 45 breast carcinomas with a wide range of estrogen receptor content, demonstrated no detectable beta- or kappa-casein. These results demonstrate that the caseins, which are biochemical markers of mammary gland differentiated function, and have been previously put forward as markers of breast cancer, were not synthesized in the 45 human breast carcinomas studied here, even in tumors with high levels of estrogen receptor protein.
Two monoclonal antibodies, LICR-LON-32.2 (32.2) and LICR-LON-14.1 (14.1), are described which react with human casein. 32.2 reacts with human beta-casein and 14.1 with human kappa-casein. 32.2 also reacts with rat band 2 casein and bovine beta-casein, but 14.1 appears to be specific for human kappa-casein. These monoclonal antibodies do not cross-react with other milk proteins.
Human beta-casein was separated according to the extent of phosphorylation and the fully phosphorylated moiety was characterized. Fully phosphorylated human beta-casein makes up to 13-15% of the beta-casein fraction. It has a partial specific volume, v, of 0.754 +/- 0.008 and an absorbancy, E1(1%)cm,280 nm of 6.4 +/- 0.2. Sedimentation and viscosity data yield a solvation of 2.9 g H2O/g protein and an axial ratio of about 5 for the monomer. This would be consistent with a prolate ellipsoid of 10 nm length and 2 nm width. There is one strong binding site for Ca2+ for each organic phosphate ester in the molecule. The protein will precipitate at room temperature upon the addition of either 10 mM Ca2+ or greater than 1 M NaCl. Increasing the temperature from 4 to 37 degrees C causes an apparent conformational change and an increase in protein aggregation which is further increased by the addition of NaCl at this temperature until a limiting size is reached at about 0.25 M NaCl. This limiting size polymer contains 95-105 monomers and is nearly spherical with a radius of about 15 nm and a solvation of 3 g H2O/g protein. If this polymer were the submicelle of human casein, it could account for the abnormally high solvation of human casein micelles but their small average size would be more difficult to reconcile without additional information concerning K-casein association. The addition of Ca2+ to the system introduces association patterns which are more complex and not easily assessed.
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, s20,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.
Amino acid composition, carbohydrate composition and rennin reactivity were investigated on 6 chromatographically separated human casein isolated from DEAE-cellulose column with urea. These 6 fractions were 85% of total casein and each of them gave a single band in polyacrylamide gel electrophresis. Discussions were given to the properties of these 6 fractions in comparison with cow casein. 1. Hexose was high in all 6 fractions, but hexosamine and sialic acid were found higher in fraction 3 and 4. 2. Except for high content of threonine in fraction 3 and serine in fraction 8, amino acid composition was found similar in all 6 fractions. 3. None of the 6 fractions are identical to cow αs-, β- or κ-casein from the data of carbohydrate or amino acid composition. 4. Human casein, different from cow casein, showed no change in the patterns of polyacrylamide gel electrophoresis before and after rennin treatments. © 1969, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.