Casein Variants Research Articles

Effects of Polymorphic Milk Protein Genes on Milk Yield and Composition Traits in Holstein Cattle

Abstract In assessing single gene effects on a quantitative trait, contrasts between the genotypes would be of primary interest. However, only the net differences over and above the contributions from polygene effects from other loci would be of practical importance. In a population of 11015 Holstein cows, the B allele of K-casein was significantly favourable for milk and protein yields and for protein %, and the A allele of β-lactoglobulin was favourable for fat % and protein yield. The degree of dominance for their effects was shown. Also shown were the independence between effects of the casein variants and those of polygene, and between effects of casein genes. Adjustment for casein gene effects made no difference to the estimates of heritability and other genetic parameters of all five dairy production traits.

Phenotyping of bovine milk proteins by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the separation of the most common and some less common genetic variants of the bovine caseins is described. When the method is used for analysing clarified skim milk, simultaneous identification of casein variants and major whey protein variants can be effected in a single run. The potential of the method for quantitative application is discussed.

Tertiary and quaternary structural differences between two genetic variants of bovine casein by small-angle X-ray scattering

The casein complexes of bovine milk consist of four major protein fractions, α s1, α s2, β, and κ. Colloidal particles of casein (termed micelles) contain inorganic calcium and phosphate; they are very roughly spherical with an average radius of 650 Å. Removal of Ca 2+ leads to the formation of smaller protein aggregates (submicelles) with an average radius of 94 Å. Two genetic variants, A and B, of the predominant fraction, α s1 -casein, result in milks with markedly different physical properties, such as solubility and heat stability. To investigate the molecular basis for these differences, small-angle X-ray scattering was performed on the respective colloidal micelles and submicelles. Scattering curves for submicelles of both variants showed multiple Gaussian character; data for the B variant were previously interpreted in terms of two concentric regions of different electron density, i.e., a “compact” core and a relatively “loose” shell. For the submicelle of A, there was a third Gaussian, reflecting a negative contribution due to interparticle interference. Molecular parameters for submicelles of both A and B are in agreement with hydrodynamic data in the literature. Data for the micelles, for which scattering yields cross-sectional information, were fitted by a sum of three Gaussians for both variants; for these, the corresponding two lower radii of gyration represent the two concentric regions of the submicelles, while the third reflects the average packing of submicelles within the micellar cross section. Most of the molecular parameters obtained showed small but consistent differences between A and B, but for submicelles within the micelle several differences were particularly notable: A has a greater molecular weight for the “compact” region of the constituent submicelle (82,000 vs 60,000) and a much greater submicellar packing number (6:1 vs 3:1). Reasons for these and other differences are to be sought in sequence differences and in differences in calcium-binding sites and charge distribution.

The acidic peptide-specific type II protein kinases from the nucleus and the cytosol of porcine liver are distinct

The second messenger-independent acidic peptide-specific protein kinase II (casein kinase II) from the cytosol of porcine liver has been purified to apparent homogeneity by using DEAE-cellulose, hydroxyl apatite, and phosphocellulose chromatography. The native enzyme has an apparent M r of 150,000. After sodium dodecyl sulfate-gel electrophoresis a band of M r = 39,000 and a slightly diffuse band of M r = 27,000 were found indicating an α 2 β 2 structure of this protein kinase. A thorough comparison with the corresponding enzyme from the nucleus was performed. The two enzymes differ in the subunit composition, as the nuclear enzyme is composed of subunits with a M r of 95,000; they further differ in the heparin sensitivity and binding to blue dextran-Sepharose. Distinct differences in their nucleotide binding sites were found upon mapping with ATP analogs, although both enzymes utilize ATP as well as GTP. On the other hand, both enzymes phosphorylate identical sites in the casein variants βA2 and αS1B at comparable rates. These results demonstrate for the first time the existence of distinct nucleus and cytoplasm specific type II “casein kinases.”

Association of Genetic Variants of Casein and Milk Serum Proteins with Milk, Fat, and Protein Production by Dairy Cattle

Polyacrylamide gel electrophoresis methods were used to phenotype caseins for 2045 cows and milk serum proteins for 3870 cows distributed in 63 Quebec dairy herds. Frequencies were: αs1-casein A .003, αs1-casein B .970, αs1-casein C .027; β-casein A1 .561, β-casein A2 .421, β-casein A3 .011, β-casein B .007; κ-casein A .744, κ-casein B .256; β-lactoglobulin A .387, β-lactoglobulin B .613; α-lactalbumin B 1.00. Overall unadjusted means for 305-day production in first lactation were: 5530±26.6kg, 197±1.0kg, 172±1.0kg, 3.58±.009, 3.12±.009 for milk yield, fat yield, protein yield, fat percentage, and protein percentage for 1687 cows for casein systems. Similar data for 2906 cows phenotyped for milk serum proteins were: 5412±20.6kg, 193±.8kg, 170±.7kg, 3.57±.007, and 3.13±007. Least squares analysis of variance showed that herd location, month of calving, age of cow at first calving, and protein variants had significant effects on production traits. αs1-Casein B and β-casein A phenotypes were associated with higher milk, fat, and protein yields than other variants in the two casein systems. Milk from κ-casein BB and β-lactoglobulin AA phenotypes contained .13 and .05% more protein than the AA and BB phenotypes with the AB phenotype intermediate.

Substrate specificity of the nuclear protein kinase NII from porcine liver. Studies with casein variants.

The substrate specificity of the nuclear protein kinase NII from pig liver was studied using purified casein variants alpha s1B and beta A2 both in the phosphorylated and dephosphorylated form. Chymotryptic peptides of the phosphorylated casein were analyzed by ascending thin-layer chromatography followed by thin-layer electrophoresis. Phosphorylated peptides were detected by autoradiography. The determination of a phosphorylated site in a cluster of three serinees was performed by solid phase Edman degradation of the whole beta A2 casein. Phosphorylated beta A2 casein was by far the best substrate. A threonine at position 41 in the amino acid sequence was found to be predominantly phosphorylated. Dephosphorylated beta A2 casein became rephosphorylated at serine-17 whereas phosphorylated at threonine-41 was greatly reduced. In both sites acidic amino acid side chains are present at the C-terminal side though at different positions suggesting that acidic residues in the vicinity of the serin or threonine residue to be phosphorylated play an important role in the recognition by the nuclear protein kinase NII.

Heterogeneity of guinea-pig caseins synthesized and sequestered by cell-free protein-synthesizing systems.

1. Individual mRNA species encoding guinea-pigs caseins A, B and C, and alpha-lactalbumin, were purified by hydridization to recombinant milk-protein plasmid DNA immobilized on diazobenzyloxymethyl-paper or diazobenzyloxymethyl-cellulose. Addition of the purified mRNA species to a reticulocyte-lysate cell-free system, in the presence or absence of a dog pancreas microsomal membrane fraction, established a precursor-product relationship between the primary translation products and those sequestered within microsomal vesicles, as determined by polyacrylamide-gel analysis in one and two dimensions. 2. Three sequestered variants of sequestered casein A were identified, but only single forms of sequestered casein B and alpha-lactalbumin. Sequestered variants of casein C proved to be unexpectedly basic, and did not focus on the pH gradient utilized. 3. Comparative analysis of milk proteins synthesized in the reticulocyte-lysate and wheat-germ cell-free systems by two-dimensional gel electrophoresis demonstrated both quantitative and qualitative differences. In particular, marked but variable heterogeneity was apparent within the primary translation products of casein A and casein B. Pre-casein C did not focus. Limited N-terminal processing of the primary translation products was also evident. These observations are discussed in relation to (i) unscheduled post-translational modifications by cell-free protein-synthesizing systems and (ii) multiplicity of signal sequences. 4. Overall we demonstrate that complex precursor-product relationships between primary translation products and their sequestered variants, programmed in vitro by a mixed mRNA population, may be readily analysed by using individual mRNA sequences purified by hybridization to immobilized cloned complementary-DNA sequences.

Cyclic Nucleotide‐Independent Protein Kinases from Rabbit Reticulocytes

Purified variants of αs1 casein and β casein have been used to examine the substrate‐specificity of two adenosine‐3′:5′‐monophosphate‐independent protein kinase activities purified from rabbit reticulocytes. These enzymes, identified as casein kinase I and II were stimulated by monovalent cations. Casein kinase I had a pH optimum between 6.8 and 8.0, used ATP as the phosphate donor and preferentially phosphorylated serine. Casein kinase II had optimal activity at pH 8.5 to 10, utilized both ATP and GTP in the phosphotransferase reaction and modified predominantly threonine residues. The chymotryptic peptides of the phosphorylated casein variants were analyzed by thin‐layer electrophoresis and ascending chromatography and the phosphorylated peptides were identified by autoradiography. In the β caseins, the phosphorylated peptides observed with casein kinase I were different from those obtained with casein kinase II. With the αs1 caseins, the same phosphorylated peptide was observed with both protein kinases. The phosphorylation sites in the αs1 and β caseins have been assigned on the basis of the amino acid analyses of the phosphorylated chymotryptic peptides and the phosphoserine and phosphothreonine determinations in conjunction with the known primary sequences. Casein kinase I phosphorylated Ser‐22 in β‐A2 casein and Ser‐41 in αs1‐A casein, suggesting that this protein kinase recognized the primary sequence Glu‐X‐Ser. Thr‐41 in β‐A2 casein and Thr‐49 in αs1‐A casein were the primary sites phosphorylated by casein kinase II. The recognition determinants for casein kinase II appeared to be Thr‐Gou‐Asp. When endogenous phosphate was removed from the caseins, the rate of phosphorylation of β casein was diminished by greater than six‐fold indicating that negative charge near the phosphorylation site facilitated phosphorylation.

Substrate specificity of the cyclic AMP-dependent protein kinase.

The protein substrate specificity of the catalytic subunit of rabbit skeletal muscle cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) has been studied using genetic variants of beta casein. It was found that beta casein-B was phosphorylated at a much greater rate than beta caseins A1, A2, A3, or C. The enhanced phosphorylation of beta casein-B, as compared with the most common variant A2, was attributed to an arginine substitution for a serine at position 122, which caused a nearby residue, serine 124, to become a phosphorylation site for the protein kinase. These results further support the concept that the local primary structure is important in specificity and that arginine may be a specific determinant common to all the local phosphorylation site sequences recognized by the cyclic AMP-dependent protein kinase.

Polymorphism in human casein.

MANY human proteins exist in more than one genetically determined variety1, but no polymorphism has been reported in human milk proteins, although some components have been sufficiently characterized from the biochemical standpoint2,3 and extensive polymorphism has been detected in cow's milk. We therefore decided to investigate the possible occurrence of polymorphic variants of human casein, since electrophoresis has now given a clear picture of bovine casein genetics, which can be used as a model for study of the situation in man4,5. Such variants would obviously be more easily used as genetic markers at the population than at the family level. Moreover, data reported by Grosclaude et al.6 suggest the existence of a supergene controlling bovine αs1, β and k-caseins. For this reason, the possibility of a similar genetic control of human polymorphism (if any) was also tested.

Genetic variants of casein in Indian and African Zebu cattle

1. 1. The caseins present in the milk of cows of five Indian and two East African Zebu breeds were examined by gel-electrophoretic procedures. 2. 2. Like those of Western breeds, the α s1 -, β- and κ-casein systems were polymorphic. 3. 3. Most of the genetic variants appeared to be identical to those seen in Western breeds, but a variant (β-casein B Z) of the same electrophoretic mobility as the β-casein B of Western cattle proved to be different in amino acid composition. 4. 4. A rare new variant, β-casein D, was found in the milk of Indian Deshi and East African Boran cows. 5. 5. Zebu breeds had a very high gene frequency of α s1 -casein C, in contrast to Western breeds in which the B-variant predominates. 6. 6. The East African milks were additionally examined for, but did not contain, β-lactoglobulin C, a variant characteristic for the Jersey breed.

Isolation and Polyacrylamide Gel-Urea Electrophoretic Characterization of αS1-Casein

Abstract An improved method for the isolation of pure α S1 -casein from pooled milk is described. The method involves chemical isolation followed by DEAE-cellulose column-chromatographic fractionation. Alpha S1 -casein was characterized by SGUE, PGUE, chemical, and sedimentation analyses. The chromatographically pure α S1 -casein was found to contain α S1 -B and α S1 -C casein variants. These variants cannot be separated by DEAE-cellulose column chromatography. The preparation contained 1.10%P, equivalent to 10 phosphorus atoms per mole of 28,500. The molecular weight by approach to sedimentation equilibrium was 25,500. Alpha S1-casein in 0.1 M KCl, pH 5.6, showed random aggregation of 14S, while in tris-citrate buffer, pH 8.6, it showed 1.5 S, and in Na-PO4 buffer, pH 7.0, 4.2 S species.

Inherited casein variants in cow's milk: II. Breed differences in the occurrence of β-casein variants

SummaryA survey was made of the frequency of β-casein variants in the milk of cows of five major British breeds. Three variants A, B and C differing in electrophoretic mobility were found to occur either singly or in pairs, as expected if each were determined by a single allele. The A variant was the most common and found in all breeds. Ayrshires and Shorthorns produced no other variant, whilst the B variant occurred in Jerseys and, at low frequency, in Guernseys and Friesians. Production of the C variant was restricted to the Guernsey breed in which it occurred at low frequency. The technique of paper electrophoresis, used for β-casein ‘typing’ of some 600 cows, is described in detail.