Environmental influences on bovine kappa-casein: reduction and conversion to fibrillar (amyloid) structures |
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Authors: | Farrell Harold M Cooke Peter H Wickham Edward D Piotrowski Edwin G Hoagland Peter D |
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Affiliation: | (1) United States Department of Agriculture, ARS, Eastern Regional Research Center, Wyndmoor, Pennsylvania |
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Abstract: | The caseins of milk form a unique calcium–phosphate transport complex that provides these necessary nutrients to the neonate. The colloidal stability of these particles is primarily the result of -casein. As purified from milk, this protein occurs as spherical particles with a weight average molecular weight of 1.18 million. The protein exhibits a unique disulfide bonding pattern, which (in the absence of reducing agents) ranges from monomer to octamers and above on SDS-PAGE. Severe heat treatment of the -casein (90°C) in the absence of SDS, before electrophoresis, caused an increase in the polymeric distribution: up to 40% randomly aggregated high–molecular weight polymers, presumably promoted by free sulfhydryl groups (J. Protein Chem.17: 73–84, 1998). To ascertain the role of the sulfhydryl groups, the protein was reduced and carboxymethylated (RCM-). Surprisingly, at only 37°C, the RCM--casein exhibited an increase in weight average molecular weight and tendency to self-association when studied at 3000 rpm by analytical ultracentrifugation. Electron microscopy (EM) of the 37°C RCM sample showed that, in addition to the spherical particles found in the native protein, there was a high proportion of fibrillar structures. The fibrillar structures were up to 600 nm in length. Circular dichroism (CD) spectroscopy was used to investigate the temperature-induced changes in the secondary structure of the native and RCM--caseins. These studies indicate that there was little change in the distribution of secondary structural elements during this transition, with extended strand and turns predominating. On the basis of three-dimensional molecular modeling predictions, there may exist a tyrosine-rich repeated sheet-turnsheet motif in -casein (residues 15–65), which may allow for the stacking of the molecules into fibrillar structures. Previous studies on amyloid proteins have suggested that such motifs promote fibril formation, and near-ultraviolet CD and thioflavin-T binding studies on RCM--casein support this concept. The results are discussed with respect to the role that such fibrils may play in the synthesis and secretion of casein micelles in lactating mammary gland. |
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Keywords: | Casein structure molecular weight disulfide interactions amyloid fibrils |
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