Anomalous behavior of bovine alpha s1- and beta-caseins on gel electrophoresis in sodium dodecyl sulfate buffers

Electrophoresis in the presence of sodium dodecyl sulfate (SDS) provides a relatively simple means of determining molecular weights of proteins. This technique relies on the validity of a correlation between some function of Mr and the mobility of the protein through the gel matrix. However, bovine caseins (especially alpha s1-casein) have lower mobilities than expected on the basis of their known Mr. The binding of SDS to both alpha s1-casein (Mr 23,600) and beta-casein (Mr 24,000) reached a maximum at the slightly low value of 1.3 g SDS/g protein. Gel-filtration chromatography showed, however, that the alpha s1-casein:SDS complex was larger than the beta-casein:SDS complex at pH 6.8 or 7.0, but that they were similar in size at pH 2.9 or 3.0. Circular dichroism spectra indicated that the low helical structure content of both alpha s1- and beta-casein increased with the addition of SDS and/or decreasing the pH to 1.5. 13C NMR results showed that SDS bound to alpha s1- and beta-casein in the same way as it did to bovine serum albumin. Either esterification or dephosphorylation followed by amidation of alpha s1-casein increased its mobility in SDS-gel electrophoresis, but neither modification affected beta-casein mobility. These and other results indicate that the low electrophoretic velocity of alpha s1-casein in SDS-gel electrophoresis results from its unexpectedly large hydrodynamic size. This is caused by localized high negative charges on certain segments of alpha s1-casein, which would induce a considerable amount of inter- and intrasegmental electrostatic repulsion, leading to an expanded or extended structure for portions of the alpha s1-casein molecule in the presence of SDS. It is clear that the conformation, and hence the equivalent radius, of an SDS:protein complex is determined by the sequence of amino acids in the protein and that, a priori, it cannot be anticipated that the electrophoretic mobility of such a complex will bear more than a casual relationship to the Mr of the protein.     

Using self-consistent-field theory to understand enhanced steric stabilization by casein-like copolymers at low surface coverage in mixed protein layers

We present a statistical mechanical approach to predicting the properties of mixed copolymer layers using the Scheutjens-Fleer self-consistent-field theory. Our model copolymers are based on the primary structures of the major bovine casein monomers, alpha(s1)-casein and beta-casein. Numerical calculations have been carried out to determine the polymer segment density profiles at an isolated hydrophobic surface and the interaction forces as a pair of polymer-coated surfaces is brought to close interlayer separation. For a copolymer model containing hydrophilic and hydrophobic segments, we show how the steric stabilizing capacity of a casein-like macromolecule at very low surface coverage is enhanced in the presence of a thin dense layer of shorter tethered amphiphilic chains. Using a more refined protein model, which also distinguishes between the charged and uncharged hydrophilic segments along the chain, we clearly demonstrate that the enhanced steric repulsion from beta-casein exceeds that from alpha(s1)-casein. These calculations explain how the replacement of just a few percent of beta-lactoglobulin by casein can inhibit the heat-induced thickening and flocculation behavior observed experimentally with some whey protein-stabilized oil-in-water emulsions.     

NMR studies of the phosphoserine regions of bovine alpha s1- and beta-casein. Assignment of 31P resonances to specific phosphoserines and cation binding studied by measurement of enhancement of 1H relaxation rate

(1) High-resolution 31P-NMR was used to study the environment of the phosphoserine residues of the phosphoproteins, alpha s1-casein B, beta-casein A2 and beta-casein C. For reference purposes 31P-NMR spectra of phosvitin and ovalbumin were also collected. (2) 31P resonances were assigned to specific phosphoserine residues as a result of comparisons of the high-resolution 31P-NMR spectra for alpha s1- and beta-caseins and for peptide fragments of these proteins obtained by cyanogen bromide and trypsin cleavage. (3) Measurements of the enhancement of the relaxation rate for water protons (1H) on addition of Mn2+ to alpha s1-casein B and to a fragment alpha s1-CN3, obtained by cyanogen bromide cleavage, gave approximate pK values for the binding groups and suggest the possibility of a conformational change induced by varying the concentration of divalent cation.     

Proteomic tools to characterize the protein fraction of Equidae milk

The principal components of the protein fraction in pony mare's milk have been successfully identified and partially characterized using proteomic tools. Skimmed pony mare's milk was fractionated by either reversed phase-high-performance liquid chromatography (RP-HPLC) on a C4 column or a bi-dimensional separation technique coupling RP-HPLC in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) in the second dimension (two-dimensional RP-HPLC/SDS-PAGE). The fractions thus obtained were analyzed by Edman N-terminal microsequencing and mass determination, with or without tryptic digestion, on a matrix-assisted laser desorption/ionization-time of flight spectrometer. Based on the sequence and molecular mass information obtained, identifications were achieved through a protein database search using homology or pattern research algorithms. This methodological approach was shown to be rapid, efficient and reliable in identifying the principal proteins in pony mare's milk. kappa-, alpha(s1)-, alpha(s2)-, and beta-casein, lysozyme C, alpha-lactalbumin and beta-lactoglobulin I and II were thus identified. alpha(s1) and beta-caseins displayed polymorphic patterns, probably due to alternative splicing processes leading to casual exon skipping events involving exons 7 and 14 in alpha(s1)-casein and exon 5 in beta-casein. Edman N-terminal microsequencing over 35 amino acid residues, for pony alpha(s1)-casein, clearly demonstrated the occurrence, in Equidae, of a splicing pattern similar to that reported in rodents, characterized by the constitutive outsplicing of exon 5. Pony mare's milk SDS-PAGE and RP-HPLC patterns were compared with those obtained for other milks (cow, goat and human), as were the relative levels of caseins and major whey proteins in these milks. Our results provide further evidence to support the notion that Equidae milk is closer to human breast milk than milk from bovine and caprine with respect to the casein and lysozyme C contents and casein/whey proteins ratio.     

Polymorphism of the serum proteins of wild pigs

One hunderd and ninety five wild pigs from two different regions of Poland were investigated for transferrin, amylase and ceruloplasmin polymorphism. A new transferrin phenotype Tf PB was detected. This phenotype differed from Tf AB in the electrophoretic mobility of the more anodal transferrin. Tf P is assumed to be the product of a new allele Tf P at the Tf locus. Two amylase phenotypes Am 1–2 and Am 2 were observed. The Am 1 allele was absent from the pigs in the Poznan region. Only one ceruloplasmin phenotype, Cp B, was found.     

Linkage between the loci for transferrin and ceruloplasmin in pigs

Evidence for genetic linkage between the loci for transferrin (Tf) and ceruloplasmin (Cp) in pigs was presented. The results were based on a study of a single sire family comprising 35 informative offspring. No recombinants were observed. The recombination frequency was estimated to be in the range of 0 to 8%. This indicated that the recombination frequency between Tf and Cp loci in pigs may be much lower than that reported previously between these two loci in cattle and in human.     

Linkage between the loci for transferrin and ceruloplasmin in pigs

Summary. Evidence for genetic linkage between the loci for transferrin ( Tf ) and ceruloplasmin ( Cp ) in pigs was presented. The results were based on a study of a single sire family comprising 35 informative offspring. No recombinants were observed. The recombination frequency was estimated to be in the range of 0 to 8%. This indicated that the recombination frequency between Tf and Cp loci in pigs may be much lower than that reported previously between these two loci in cattle and in human.     

Immunochemical demonstration of alpha(s1)- and beta-casein in mouse mammary glands at early stages of pregnancy.

We generated monoclonal antibodies (MAbs) against mouse alpha(s1)- and beta-casein and used them to survey casein immunochemically in mammary glands of mice at peri-coitous and pregnant stages. Two MAb-producing hybridoma cells, designated MCalpha1 cell and MCbeta1 cell, were established. Each antibody, when used in Western blotting, recognized specifically mouse alpha(s1)- and beta-casein among a wide spectrum of proteins of both a lactating mammary homogenate and mouse skim milk. Immunohistochemistry revealed alpha(s1)- and beta-casein in sections of lactating mammary glands. Staining was found in substances in the lumen and cytoplasm of duct and alveolar cells, particularly in rough endoplasmic reticulum and the Golgi apparatus. Mammary glands at Days 2, 4, 6, 8, and 14 of pregnancy showed positive staining specific to both alpha(s1)- and beta-casein in the lumen and cytoplasm of duct cells, whereas the glands at estrus and Day 0 of pregnancy were positive mainly for alpha(s1)-casein. Semiquantitative Western blotting analysis of both casein components in epithelial cell fractions from glands during pregnancy confirmed that intra-epithelial alpha(s1)- and beta-casein changed during three phases, elevated from trace levels to detectable levels during initial stages of pregnancy (Days 0, 2, and 4), declined to lower levels during mid-pregnancy (Days 6 and 8), and then rose to high levels during late pregnancy (Day 14).     

Polymorphism of the serum proteins of wild pigs

One hundred and ninety five wild pigs from two different regions of Poland were investigated for transferrin, amylase and ceruloplasmin polymorphism. A new tranferrin phenotype Tf PB was detected. This phenotype differed from Tf AB in the electrophoretic mobility of the more anodal transferrin. Tf P is assumed to be the product of a new allele Tf P at the Tf locus. Two amylase phenotypes Am 1-2 and Am 2 were observed. The Am 1 allele was absent from the pigs in the Poznan region. Only one ceruloplasmin phenotype, Cp B, was found.     

A linkage study of protein-coding loci in Macaca mulatta and Macaca fascicularis

Genealogical and gene marker data from the closely related species Macaca mulatta and Macaca fascicularis have been used to search for linkage between genes coding for the blood proteins albumin, carbonic anhydrase 1 and 2, diaphorase 1 and 2, group-specific component, glucose phosphate isomerase, hemoglobin alpha chains, isocitrate dehydrogenase, prealbumin, and transferrin. The results are consistent with conservation of the linkage between the loci coding for albumin and group-specific component and loci coding for the two carbonic anhydrase isozymes, as observed in other species. Among the 38 possible pairwise comparisons, no new linkage groups were identified. Tight linkage can be excluded for most pairs of loci.     

RFLP and linkage analysis of the porcine casein loci—CASAS1, CASAS2, CASB and CASK

Restriction fragment length polymorphisms (RFLPs) were revealed at the porcine casein loci with the following combinations of restriction endonucleases and porcine cDNA clones: α_s1,-casein (TaqI); α_s2-casein (BamHI); and ß-casein (Sacl). These RFLPs were shown to be under simple monogenic control by segregation analysis of two- and three-generation families. The CASAS1, CASAS2 and CASB casein loci were also shown to be linked with no recombinant haplotypes observed amongst 77 meioses in Large White and Meishan F₁ and F₂ crosses. No recombinants were observed in a further 106 meioses that were informative for linkage between CASAS1 and CASAS2.     

Substrate specificity of the cell envelope-located proteinase of Lactococcus lactis subsp. lactis NCDO 763.

1. The specificity of the cell envelope-located proteinase of Lactococcus lactis subsp. lactis NCDO 763 towards caseins has been submitted to a statistical study. Positive and negative relations have been evidenced between several amino acids and positions P6 to P'2 of the cleaved bonds. 2. Fragment 1-23 of alpha s1 and oxidized B chain of insulin are well cleaved by the proteinase while CMP (fragment 106-169 of kappa-casein) is a poor substrate. 3. Comparison with other cell envelope-located proteinase has been done. The enzyme of the strain 763 hydrolyses alpha s1-casein and fragment 1-23 of alpha s1-casein as the enzyme of the strain Sk11 and beta-casein as the enzyme of the strain Wg2. 4. The specificity of these proteinases and the comparison of their amino acid sequences let us postulate a more complex substrate binding area for these lactococcal proteinases than for the subtilisin.     

Analysis of the human casein phosphoproteome by 2-D electrophoresis and MALDI-TOF/TOF MS reveals new phosphoforms

Mammalian breast milk contains an array of proteins and other nutrients essential for the development of the newborn. In human milk, the caseins (alpha S1, beta and kappa) are a major class of proteins; however, the dynamic range of concentrations in which the various isoforms of each casein exist presents challenges in their characterization. To study human milk casein phosphoforms, we applied traditional two-dimensional polyacrylamide gel electrophoretic (2-DE) separation combined with matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) tandem mass spectroscopic analysis. The abundant beta-casein was resolved as a train of 6 spots differing in phosphorylation level with 0-5 phosphates attached. To study the less abundant alpha S1-casein, a cysteine-tagging enrichment treatment was used prior to 2-DE. A train of 9 spots with 4.4 < p I < 5.3 were identified as alpha S1-casein. This included five previously uncharacterized phosphoforms with up to 8 phosphate groups located in two serine-rich tryptic phosphopeptides ( (27)L-R (51), (69)N-K (98)) consistent with alpha-caseins from various ruminant species. MS/MS analysis of the phosphopeptides released by tryptic digestion enabled identification of the residue-specific order of phosphorylation among the 6 beta-casein and 9 alpha S1-casein phosphoforms. Deamidation of N (47) of alpha S1-casein was also a feature of the MS analysis. This study represents the first comprehensive analysis of the human casein phosphoproteome and reveals a much higher level of phosphorylation than previously recognized. It also highlights the advantages of 2-DE for examining the global pattern of protein phosphoforms and the limitations of attempting to estimate phosphorylation site occupancies from "bottom-up" studies.     

Amyloid fibril formation by bovine milk alpha s2-casein occurs under physiological conditions yet is prevented by its natural counterpart, alpha s1-casein

The calcified proteinaceous deposits, or corpora amylacea, of bovine mammary tissue often comprise a network of amyloid fibrils, the origins of which have not been fully elucidated. Here, we demonstrate by transmission electron microscopy, dye binding assays, and X-ray fiber diffraction that bovine milk alpha s2-casein, a protein synthesized and secreted by mammary epithelial cells, readily forms fibrils in vitro. As a component of whole alpha s-casein, alpha s2-casein was separated from alpha s1-casein under nonreducing conditions via cation-exchange chromatography. Upon incubation at neutral pH and 37 degrees C, the spherical particles typical of alpha s2-casein rapidly converted to twisted, ribbon-like fibrils approximately 12 nm in diameter, which occasionally formed loop structures. Despite their irregular morphology, these fibrils possessed a beta-sheet core structure and the ability to bind amyloidophilic dyes such as thioflavin T. Fibril formation was optimal at pH 6.5-6.7 and was promoted by higher incubation temperatures. Interestingly, the protein appeared to be less prone to fibril formation upon disulfide bond reduction with dithiothreitol. Thus, alpha s2-casein is particularly susceptible to fibril formation under physiological conditions. However, our findings indicate that alpha s2-casein fibril formation is potently inhibited by its natural counterpart, alpha s1-casein, while is only partially inhibited by beta-casein. These findings highlight the inherent propensity of casein proteins to form amyloid fibrils and the importance of casein-casein interactions in preventing such fibril formation in vivo.     

Caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate

Artificial casein micelles were prepared by adding 30 mM calcium, 22 mM phosphate and 10 mM citrate to sodium caseinate solutions, and the content of the casein aggregates cross-linked by colloidal calcium phosphate was determined by high-performance gel chromatography on a TSK-GEL G4000SW column in the presence of 6 M urea. The content of the casein aggregates cross-linked by colloidal calcium phosphate in artificial whole casein micelles was 48% of total casein, and their relative casein composition determined by high-performance ion-exchange chromatography was 53.1% for alpha s1-casein, 15.8% for alpha s2-casein, 31.1% for beta-casein and 0% for kappa-casein. The order of cross-linking by colloidal calcium phosphate agreed with that of the ester phosphate content of casein constituents. The content of the casein aggregates cross-linked by colloidal calcium phosphate was higher in alpha s1-kappa-casein micelles than in beta-kappa-casein micelles. kappa- and gamma-caseins and dephosphorylated alpha s1-casein were not cross-linked by colloidal calcium phosphate. Although kappa-casein was not cross-linked, chemically phosphorylated kappa-casein, of which the average phosphate content was 8.5 per molecule, was cross-linked. It is concluded that caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate.     

Genetic polymorphism of hemoglobin, protein systems, blood enzymes and their relationship to reproducibility]

The genetic polymorphism of haemoglobin, transferrin, amylase, alkaline phosphatase, ceruloplasmin, beta-lactoglobulins and casein were studied. The relationship between the level of the heterozygosity of the blood polymorphic protein and enzyme systems was determined.     

Phosphorylation of K-casein by glycogen synthase kinase-3 from rabbit skeletal muscle

Glycogen synthase kinase-3 (ATP:protein phosphotransferase, EC 2.7.1.37) phosphorylated K-casein 20-fold more rapidly than beta-casein, while alpha S1-casein was not a substrate. This distinguished it from casein kinase-I and casein kinase-II, which phosphorylate the beta-casein variant preferentially. Glycogen synthase kinase-3 phosphorylated a serine residue(s) in the C-terminal cyanogen bromide fragment on K-casein. In contrast, cyclic AMP-dependent protein kinase phosphorylated the N-terminal fragment, and phosphorylase kinase the N-terminal and intermediate cyanogen bromide fragments. The results emphasize the potential value of casein phosphorylation as a means of classifying protein kinases.     

Genetic structure of local Ukrainian cattle breeds

Analysis of 6 cattle breeds (5 local Ukrainian breeds and the Holstain breed) on the 9 polymorphous molecular-genetic markers (transferrin, ceruloplasmin, amylase-1, posttransferrin, receptor to vitamin D, haemoglobin, leptin, kapa-casein) was carried out. The rare allele of transferrin was revealed in two local breeds and the rare allele of leptin--in two another local breeds. Associations between syntenic loci (transferrin, ceruloplasmin, kappa-casein, receptor to vitamin D) were observed only in the local breeds but not in the Holstain one. Locus-specific conservation of the ancestor allelic variants in the local breeds and effect of selection on interloci associations are discussed.