Localization of kappa-casein on thin sections of casein micelles by the gold method

The ultrastructural location of kappa-casein in bovine casein micelles was investigated by the protein A-gold method. Casein micelles, fixed in glutaraldehyde, were embedded at low temperature to enhance immunocytochemical marking of thin sections. kappa-Casein was found distributed throughout the micelles of all sizes with a higher concentration in the smaller micelles. No peripheral location of kappa-casein was observed, even in the larger micelles. These results do not agree with "coat-core" structures proposed for casein micelles. However they favor models where kappa-casein is distributed uniformly throughout the micelles.     

Composition and size distribution of bovine casein micelles

Chromatography of glutaraldehyde-fixed skim-milk on controlled-pore glass (CPG-10, 300 nm) gave three micellar fractions whose averaged diameters, measured by electron microscopy, decreased progressively with increasing elution volume. Casein micelles with diameters up to 680 nm were detected. The casein composition of the same fractions from unfixed skim-milk was determined. As the fraction elution volume increased, κ-casein varied from 7.7 to 11.4% of total casein, giving α_s/κ ratios of 6.1, 4.7 and 3.3.A plot of κ-casein content versus micelle surface-to-volume ratio for skim-milk and the column fractions approximated to a straight line. Re-calculation of the published results from two other studies also gave linear relationships between κ-casein content and surface area for artificial micelles. The three regression lines thus obtained had small intercepts. It was concluded that the data indicated the same fundamental structure for casein micelles, with a pre-dominant surface location for κ-casein, whether the micelles are natural or artificial and whether they are aggregated or by Ca²⁺ alone oy Ca²⁺ together with calcium phosphate-citrate complex.     

Localization of glycosylated kappa-casein on thin sections of casein micelles by lectin-labelled gold markers

The location of the glycosylated part of kappa-casein in bovine casein micelles was investigated using gold particles (6 nm in diameter) labelled with Ricinus communis lectin and Limulus polyphemus lectin. The pattern of marking of thin sections of micelles was similar with both lectins. Glycosylated kappa-casein was distributed uniformly throughout most micelles of all sizes. Peripheral location of glycosylated kappa-casein was observed only occasionally in some of the largest micelles. Quantitative data indicated that the concentration of the glycosylated protein was constant in micelles of increasing sizes. As larger micelles contain less total kappa-casein than smaller ones, these data indicated that a greater proportion of their kappa-casein is glycosylated. These results support models for casein micelle structure where kappa-casein is distributed throughout the micelles. They do not agree with "coat-core" structures.     

Small-angle neutron scattering study of bovine casein micelles and sub-micelles

Whole micelles and sub-micelles of cows' milk casein have been studied in solution by small-angle neutron scattering. Sub-micelles were found to have a mean molecular weight of approximately 3.0 × 10⁵ and a radius of gyration (R_g) in ²H₂O of 65 Å. R_g² appeared to decrease slightly with increasing reciprocal contrast (1/-?) whereas most globular proteins show the opposite behaviour. Estimated voluminosity (hydrated specific volume) of sub-micelles was 4.0 to 5.5 ml/g, which is much higher than values from electron microscopic measurements on whole micelles.Scattering from whole micelles showed an inflection at $\text{Q((}\text{4π sin θ)}\text{λ}\text{) = 0.035}\text{A}\text{?}{}^{\mathrm{?1}}$), which was attributed to inter-sub-micelle interference within the whole micelle.     

A multinuclear, high-resolution NMR study of bovine casein micelles and submicelles

High-resolution, natural abundance 13C[1H] (100.5 MHz), 31P[1H] (161.8 MHz) and 1H (400.0 MHz) NMR spectroscopy was used to identify the calcium-binding sites of bovine casein and to ascertain the dynamic state of amino acid residues within the casein submicelles (in 125 mM KCl, pD = 7.4) and micelles (in 15 mM CaCl2/80 mM KCl, pD = 7.2). The presence of numerous, well-resolved peaks in the tentatively assigned 13C-NMR spectra of submicelles (90 A radius) and micelles (500 A radius) suggests considerable segmental motion of both side chain and backbone carbons. The partly resolved 31P-NMR spectra concur with this. Upon Ca2+ addition, the phosphoserine beta CH2 resonance (65.8 ppm vs DSS) shifts upfield by 0.2 ppm and is broadened almost beyond detection; a general upfield shift (up to 0.3 ppm) is also observed for the 31P-NMR peaks. The T1 values of the alpha CH envelope for submicelles and micelles are essentially identical corresponding to a correlation time of 8 ns for isotropic rotation of the caseins. Significant changes in the 31P T1 values accompany micelle formation. Data are consistent with a loose and mobile casein structure, with phosphoserines being the predominant calcium-binding sites.     

Three kinetically different inorganic phosphate entities in bovine casein micelles revealed by isotopic exchange method and compartmental analysis

Much controversy exists concerning the way calcium phosphate is linked to milk phosphoproteins including caseins. Homoionic exchange of inorganic phosphate between micellar calcium phosphates (MCP) of casein micelles and solute phosphates in cows' milk was investigated using H(32)PO(4)(2-) as radiotracer. Compartmental analysis and modelling revealed the presence of three MCP-related inorganic phosphate compartments each representing a separate phosphate entity. The relative phosphate quantities per compartment, i.e. the quantities of kinetically identical phosphate ions per MCP-ion cluster, and their mean residence times are 2:1:1 and 818, 0.24 and 23 h, respectively. Hence each MCP-ion cluster comprises four inorganic phosphate ions divided over three intra-MCP binding sites each characterised by a mean residence time for homomolecular phosphate exchange at solution/MCP interface.     

The Bos taurus casein genes. Isolation and characterization of the kappa-casein gene

A region spanning 25 kb of genomic DNA containing the kappa-casein gene, has been isolated from two genomic libraries in EMBL3 and EMBL4 phage vectors. Five phage clones containing kappa-casein gene have been found. Gene organisation has been determined using restriction mapping and a partial sequencing the 5' and 3' flanking regions. The kappa-casein gene includes 5 exons, the first of them coding for 64 nucleotides from the 5' untranslated mRNA zone. The gene is 12.5 kb long, which is almost 16 times longer than the corresponding mRNA. The first intron spans 2.5 kb, the second is the largest one and spans 5.5 kb. The 5' flanking region sequence has been analysed; it contains a TATA box from -30 to -25 bp, somewhat different from the canonic sequence, and a CAAT box at -80 bp.     

The multimeric structure and disulfide-bonding pattern of bovine kappa-casein.

Bovine kappa-casein was analyzed by SDS/PAGE, MS and amino acid sequence analysis in order to determine its multimeric composition and disulfide-bonding pattern. SDS/PAGE revealed that kappa-casein in the native state can range in size from a monomer to a multimeric structure larger than a decamer. Three types of interchain disulfide linkage, Cys11-Cys11, Cys11-Cys88 and Cys88-Cys88, were all assigned in multimers purified from [14C]carboxymethylated and untreated bulk milk, as well as a milk sample from a kappa-casein-variant-B homozygote Co20. These results indicate that multimerization occurs in a random or at present unpredictable disulfide-bonding pattern regardless of the size of the multimer or the genotype.     

A comprehensive study of the relationship between size and protein composition in natural bovine casein micelles

Casein micelles of bovine skimmed milk were fractionated by permeation chromatography on porous glass (CPG-10, 50 nm followed by CPG-10, 300 nm) at 30 degrees C. Micelles were pooled in eight eluant fractions and their size distribution was determined by electron microscopy. The composition of casein in the eight fractions was determined by quantitative hydroxyapatite chromatography. Micelle size decreased progressively with increasing elution volume, and volume-to-surface average diameter ranged from 154 nm in fraction 1 to 62 nm in fraction 8. Concurrently there was a decrease in relative proportions of alpha s- and beta-caseins and a large enrichment of kappa-casein, which changed from 4.1% total casein in fraction 1 to 12.1% total casein in fraction 8. At least half the decrease in alpha s-casein proportions was attributed to the alpha s1-casein component, but the data also suggested a decline in proportions of alpha s2-casein in the smallest micelle fractions. A plot of kappa-casein fractional content versus micelle surface-to-volume ratio gave a straight line (correlation coefficient from linear regression 0.98) from which an average kappa-casein surface coverage of 1.5 m2/mg or 47.3 nm2/molecule was obtained. If a constant surface coverage for kappa-casein is assumed, the parameters of the linear equation predict that micelle voluminosity is inversely related to micelle diameter, being approximately 30% larger in fraction 8 compared to fraction 1.