Recent years have seen a great increase in the knowledge and understanding of milk proteins. Arising from several origins including the blood stream and various cellular sources, many of the proteins found in milk are products of the secretory cells directly involved in the synthesis and secretion processes of various milk components. The lactation-specific proteins present in major amounts are synthesized in the rough endoplasmic reticulum (RER) under genetic control and undergo further post-translational modifications in their secretory route from the RER through the Golgi apparatus and secretory vesicles before ejection into the lumen with other milk components. Various molecular aspects of these mechanisms and their control are now understood, but many remain to be described.

The radiolabelled primary translation products of ovine mammary mRNAs synthesized in a wheat germ cell-free system were isolated by immuno-precipitation and analysed by automated Edman degradation. The 3 ‘Ca-sensitive’ caseins (α, α andβ ), k-casein, β-lactoglobulin and α-lactalbumin were found to be synthesized as precursors with amino terminal extensions of 15, 21, 18 and 19 amino acid residues respectively. The extra pieces of these various lactoproteins are similar to ‘signal’ peptides of other secretory proteins in their length and hydro-phobicity. The occurrence of an alanyl residue at the C-termini of the extra pieces of the 6 ovine prelactoproteins suggests that the mammary proteinase responsible for the cleavage of the signal peptides may have an elastase-like specificity.

When mammary mRNAs were translated in a wheat germ cell-free system in the presence of mammary microsomal membranes, pre-β-casein was converted into authentic β-casein as demonstrated by amino terminal sequence analyses. Additionally, pre-β-casein was post-translationally converted into authentic β-casein by a specific proteinase(s) extracted from rough microsomes with Na deoxycholate.

Casein kinase from lactating bovine mammary gland catalyses the transfer of the terminal phosphoryl group of ATP to specific serine residues in dephosphorylated caseins. Best substrates for casein kinase are the dephosphorylated proteins (bovine αs1 - and β-caseins and pepsin), unphosphorylated human β-casein and the dephosphorylated peptide (residues 1–25) from bovine β-casein. Results obtained with bovine and human β-caseins indicate that the two serines underlined in the cluster Ser-Leu-Ser-Ser-Ser are particularly susceptible to the action of casein kinase. Since a similar sequence is found in dephosphorylated αs1-casein, it is probable that serines in this region of αs1-casein are also phosphorylated. The results support the concept that certain serines in casein are particularly susceptible to phosphorylation by casein kinase.

k-Casein is the main glycoprotein of cow's milk. Its polysaccharide part is O-glycosidically linked to threonine residue 133. It contains only 3 different sugars (Gal, GalNAc, NeuNAc), but a microheterogeneity has been detected at the sugar level. Two main polysaccharides have so far been characterized. The structure of the trisaccharide is NeuNAc α → 3 Gal β1 →3 GalNAc; the tetrasaccharide contains one additional sialic acid. The polysaccharide part of ovine k-casein resembles that of bovine k-casein, but contains also N-glycolyl neuraminic acid. Human k-casein contains 3 times more carbohydrate than bovine k-casein with 2 additional sugars, GlcNAc and Fuc. The various polysaccharide parts isolated from bovine colostrum k-caseinoglycopeptide are much more complex than those obtained from the normal glycopeptide, indicating an evolution of the sugar part as a function of time after parturition. Some aspects of the secondary structure of k-casein and the role of the sugar part are discussed. The carbohydrate moiety of another milk protein, human lactotransferrin, is also discussed briefly. It is comprised of 2 identical glycan groups, N-glycosidically linked to the protein, and quite different from the k-casein carbohydrate moiety.

Particles as large as several µm diam. have been observed occasionally in normal milk and commonly in prepartum and postpartum colostrum. These particles can be dissociated by EDTA and their appearance closely resembles that of normal casein micelles. However, they are often too large to have been completely formed within the Golgi vesicles of mammary epithelium and hence some degree of post-secretory aggregation of caseins is thought to occur. Two possible mechanisms of post-secretory aggregation of caseins are: (1) a continuation of the normal processes of micelle assembly in the alveolus and (2) aggregation as a result of limited proteolysis of the caseins during the time the milk is in the mammary gland. Incubation of milk with fibrinolysin, however, failed to produce aggregation of normal micelles.

The current status of knowlege of the major proteins of milks of various species is evaluated. Most of the non-bovine milk proteins are homologous with the recognized families of those of Bos taurus, αs1-caseins, αs2-caseins, β-caseins, K-caseins, β-lactoglobulins, and α-lactalbumins, each family representing a separate genetic locus specific to the mammary gland. No prominent milk protein not homologous to one of these families has yet been discovered in milk of any species. Genetic polymorphism resulting from substitutions in the polypeptide chains and various degrees of post-translational phosphorylation, glycosylation, and proteolysis have been identified in milk proteins of several species. Total protein production ranges among species from about 0.5 to 10 g/d per kg0.75 maternal weight. Proportions of the several proteins vary greatly among species, but few accurate analytical data are available except for total casein and total whey protein contents.

Using gel electrophoresis a genetic polymorphism of αs2-casein (Cn) was discovered in individual milk samples from 2 bovine breeds of the eastern part of France (Vosgienne and Montbéliarde). The 3 observed phenotypes (Plate 1) are determined by 2 co-dominant alleles at an autosomal locus. The αs2-Cn A variant was the only one known up to now in European breeds (reference variant) and ±s2-Cn D is a new variant, whose bands overlap the β-casein A band at pH 8.6, and migrate faster than αs2-Cn A at pH 3.0.

The sequence of the polypeptide chain αs2-Cn D differs from that of αs2-Cn A by the deletion of a very acidic nonapeptide, which includes a cluster of 3 phosphoseryl residues. Due to the characteristics of the reference sequence, this deletion cannot be exactly located but it involves residues 50–58, or 51–59, or 52–60.

A genetic analysis shows that locus αs2-Cn is closely linked to the cluster αsl-Cn–β-Cn-K-Cn. The 4 casein species are thus synthesized by 4 closely linked loci.

Two constituents of the proteose-peptone fraction of bovine milk have been isolated and characterized. Component 5 (PP5) has been shown to represent residues 1–105 and 1–107 of the β-casein amino acid sequence, while component 8-fast (PP8F) corresponds to residues 1–28 of β-casein. Thus, these proteose-peptones represent the N-terminal portions of the β-casein molecule, produced by proteolytic cleavages which form the γ1-, γ2- and γ3-caseins from the C-terminal part. The continuing formation of the total proteose-peptone fraction, PP5, PP8F and the γ-caseins during storage of raw milk at 18 or 37 °C has also been demonstrated

It has been shown that there is a time-dependent transfer of β-casein and the milk serine proteinase system from micelles to milk serum with change of temperature from 38 to 4 °C. It has been established that the γ-caseins can be formed by proteolytic degradation of β-casein. By a simple extraction technique, the very hydrophobic γ-casein fraction was separated from stored milks (26 and 4 °C) and estimated quantitatively. The results showed that the proteolytic degradation of β-casein is faster at 4 °C than at room temperature and this can be explained by the immobilization of enzyme and substrate at the micelle surface at higher temperatures (26 °C).

The results indicate that irreversible changes during cooling for short periods do not cause problems in milk processing, but the formation of γ-caseins and phosphopeptides may influence the technological properties of raw milk stored for more than 48 h.

The enzymic degradation of β-casein by immobilized trypsin is described and its use as a model system to study proteolytic reactions which occur in milk during processing is examined. Quantitative determination of proteolysis can be obtained by densitometric transmission measurement of patterns obtained by polyacrylamide-gel electrophoresis. Staining properties of some of the individual components in the electrophoretic patterns were determined by calibration with pure protein and peptide preparations. The isolation of pure β- and γ-caseins and the preparation of several forms of immobilized trypsin are outlined.

The proteolytic activity in UHT-sterilized milk of a proteinase-containing fraction, prepared from the milk, is reported. The proteinase fraction was isolated from casein micelles as described by Reimerdes & Klostermeyer (1974).

The fraction was added to UHT milks and the proteolysis in the samples, measured as the liberation of non-protein nitrogen, compared with that in control samples without added proteinases. The increase in proteolytic activity and deterioration in samples containing added proteinases indicated that an enzyme process occurred in UHT-sterilized milk which could influence gel formation.

A method for the preparation of αs0-casein, and for the preparation of αs0-casein fractions 1 and 2 from buffalo casein are described. The amino acid composition and phosphorus content of αs0-casein are given and compared with those of other αs0-caseins.

Human β-casein occurs in multiphosphorylated forms having the same amino acid composition but with 0–5 phosphate groups/molecule. Sequence analysis was used to determine whether each of the phosphorylated forms is a mixture of species having a certain number of phosphate groups randomly distributed or whether each form contains phosphate groups on specific seryl or threonyl residues. It was found that forms containing 2, 4 and 5 phosphate groups/molecule are homogeneous with respect to their phosphorylation sites. The monophosphorylated form, however, is a mixture of equal amounts of species phosphorylated at residues 9 or 10.

A review is presented of the various inter molecular forces important in stabilizing protein structure and association. Emphasis is put on entropic as well as enthalpic contributions to the free energy. The factors affecting size and size distribution in protein aggregates are discussed. A theory is presented for the size distribution of casein micelles, taking into account the spherical nature of the micelles and the importance of surface repulsive forces in limiting size.

In reconstituted casein systems, complete sub-micelles were previously observed only under extreme conditions of ionic strength, namely a NaCl concentration of 2 M or greater. However, studies with the analytical ultracentrifuge on phosphate-containing casein systems revealed that under certain conditions, a stable sub-micelle-micelle equilibrium was established. Conditions which were standard throughout were 7.5 mg/ml protein, 0.04 M-NaCl, pH 6.6 and 37 °C. The concentrations of added CaCl2 (Ca2+) and inorganic phosphate (P1) were variable. With no P1 present, Ca-sensitive caseins precipitated or formed micelles when k-casein was present, between 6 and 7 mM-Ca2+. With 10–20 mM-P1 precipitation or micelle formation began at about 4 mM-Ca2+ and was complete at about 5 mM-Ca2+. Over this interval, during micelle formation, a sub-micelle peak with s20, w of about 13 S was observed in equilibrium with the broad micelle peak. In a system containing a 2:2:1 weight ratio of ±sI-:²-:k-casein, this sub-micelle was isolated at 4 mM-Ca2+ and 2.5 mM-P1. The mol. wt was 760000 and it thus contained approximately 33 monomer caseins. The reconstituted micelle system formed in the absence of P1 was quite temperature sensitive, forming at 37 °C but disappearing upon cooling. In the presence of P1 the micelles formed at 37 °C but were stable to cooling as are natural micelles. Evidently, a combination of hydrophobic and electrostatic interactions are involved in natural micelle formation, probably with the production of salt bridges of Ca and phosphate ions between sub-micelles.

A model for the coagulation of Ca2+ αsl-caseinate is described wherein the casein molecules are considered to aggregate as polyfunctional units. The functionalities on the protein molecules are produced as a consequence of the binding of Ca2+, and the multiple equilibria which are established during this binding produce a distribution in which not all casein molecules have the same functionality. The theory is compared with experimental data of the precipitation reaction, and is shown to provide an adequate quantitative description of the precipitation.

The aggregation of αs1-casein by Ca is viewed as resulting from a reduction in the net negative charge by the binding of positively charged Ca2+. This reduction in charge leads to the equivalent of isoelectric precipitation. A quantitative relationship between the rate of aggregation of the casein and the monomer net charge is predicted in this isoelectric precipitation model. Precipitation results are presented for αs1-casein labelled with dansyl chloride. Providing the changes in charge brought about by the modification are taken into account, the precipitation behaviour of these caseins can be reconciled with that of native αs1-casein. This emphasises the role of electrostatic repulsion in this process and lends further support to the isoelectric precipitation model.

The self-association of SH-κ-casein at pH 7·0 in 0·01 m-EDTA, 0·001 m-dithiothreitol buffer, containing 0·1, 0·2, 0·5 or 1·0 m-NaCl is of a monomer–polymer type. The polymer is a spherical particle, diam. 23 nm and mol. wt 570000 (30 monomers). These parameters are not greatly influenced by variations in ionic strength above 0·1. The critical micelle concentration, which is a property of a monomer–polymer equilibrium, decreases with increasing ionic strength. The standard free energy of association is about – 36 kJ/mol. monomer at 20 °C.