Ghislaine Brignon

A Mendelian polymorphism underlying quantitative variations of goat αs1-casein

Using SDS-polyacrylamide gel electrophoresis and rocket immunoelectrophoresis, 3 new alleles, designated a,,-Cn’-, &alpha; s1 -Cn F and &alpha; s1 -Cn o, were identified at the goat a!-Cn locus, in addition to alleles <2,j-Cn!, as!-Cn° and <2,j-Cn! previously reported by BOULANGER et al. (1984). Alleles a,,Cn’, a,,-Cn’ and <2,j-Cn! are associated with a high content of a,,-casein (approximate mean contribution of each allele being 3.6 g/I) compared to (Y,,-Cn’ with a low content (0.6 g/I) and a,,Cn° with an intermediate content (1.6 g/1) ; a,,-Cn° appears to be a true null allele. In a sample of 213 Alpine females from 49 flocks in West Central France, the frequencies of the 6 alleles

Complete amino acid sequence of bovine αS2-casein

In a preceding paper [l] it was shown that the so-called cysZ -, as3 -, as4 and as6 -caseins, which seem to occur in all individual bovine milks, have identical peptide chains and may only differ in their phosphate content (13-10 phosphate groups/mol). Thus, the primary structure of these proteins was studied on a mixture of the four, designated as asZ -casein. Five peptides, CNl to CN5, containing 49,22, 17,4 and 115 residues, respectively, were isolated from a cyanogen bromide hydrolysate of cysZ -casein. They accounted for the 4 methionyl and 207 amino acid residues of the peptide chain. Except for residues 186-187 (His, Gln) of CNl, peptides CNl, CN3 and CN4 were completely sequenced. The alignment of the 5 CNBr peptides, H.CN4-CN2-CNS-CNl-CN3.0H, was determined from sequences studies carried out on these peptides as well as on intact as2 -casein and on some of its 25 tryptic peptides. The sequence of approximately 100 residues (112,24-5 1, 138-207) was given [ 1 ] . The present communication reports the complete primary structure of oLs2 -casein and preliminary data on the location of the phosphate groups.

Preparation and amino acid sequence of human κ-casein

Human κ‐casein was prepared from whole casein by successive hydroxyapatite and thiol‐Sepharose chromatographies. The primary structure of its 99‐residue N‐terminal fragment has been determined by sequencing peptides obtained by tryptic and chymotryptic digestions of the whole protein. This fragment overlaps the known sequence of the 65‐residue C‐terminal fragment. The 158‐residue sequence of human κ‐casein was compared to those of goat, ewe, cow and rat κ‐caseins. Only 22% of the residues are identical in homologous positions. The rate of divergence of the 93‐residue N‐terminal segment (para‐κ‐casein) appears to be higher than that of the rest of the molecule.

Premiers elements de structure primaire des caseines αs2 bovines

The bovine αs2‐, αs3‐, αs4‐ and αs6‐caseins [1] were isolated. The 4 proteins had the same amino‐acid composition and C‐terminal sequence, but different phosphorus contents. From a mixture of these proteins (designated as ‘αs2‐complex’) and from αs3‐casein a single and identical N‐terminal sequence was obtained by Edman degradation. It seems therefore that the 4 proteins have the same peptide chain and only differ in their phosphorus content. For this reason we propose to modify the nomenclature of Annan and Manson [1] and to use in future the single term αs2 to designate the caseins which have been previously called αs2, αs3, αs4 and αs6 by these authors. The study of the primary structure of the peptide chain, which has confirmed these results, was undertaken on the S‐carboxymethylated αs2‐complex. From a cyanogen bromide digest and from a tryptic hydrolyzate of the αs2‐complex, 5 and 25 peptides were obtained respectively, both sets of peptides accounting for the whole peptide chain. Examination of the tryptic peptides containing methionine combined with the N‐ and C‐terminal sequences of the αs2‐complex and some CNBr peptides, gave the order of the CNBr peptides, H.CN4CN2CN5CN1CN3.OH, which contain 4, 22, 115, 49 and 17 residues respectively. A partial sequence accounting for half of the peptide chain of the αs2‐complex is given. This peptide chain is likely composed of 207 amino‐acid residues

Structure primaire de la paracaseine κ bovine

The complete amino acid sequence of bovine para‐κ‐casein is given.

Quantification of beta-casein in human milk.

A method is described for preparing immunologically homogeneous human milk beta-casein, against which monospecific rabbit antiserum was prepared. The antiserum was used to quantify beta-casein, the major human casein, by rocket immunoelectrophoresis in individual milk samples. However, it was found that in most samples beta-casein occurred together with degradation products originating from its proteolysis by plasmin. Immunological quantification of human beta-casein, treated with plasmin for various time periods, showed that rocket height was not affected by proteolysis up to degradation states clearly more advanced than those observed in all samples of fresh human milk tested. Assays of 150 individual milk samples from 80 women, covering a lactation period of up to 730 d, gave an average concentration of beta-casein (native + degraded) of 4.67 +/- 0.89 standard deviation (g/l); extremes at 2.1 and 7.3 g/l did not vary significantly during the period under study. Comparison of this average value with an accepted casein content of 4.4 g/l (Macy & Kelly, 1961) showed that the casein content of human milk is underestimated when obtained by N determinations on milk and on its supernatants at pH 4.6 (whey). Caseins other than beta-casein occurred only in minute amounts, if at all.

Does β-lactoglobulin occur in human milk?

Although beta-lactoglobulin (beta-lg) has been considered to be absent from human milk, recent results of other workers, based on immunological reactions between human milk and rabbit antiserum to bovine beta-lg, suggest that this protein may be present. Although our results show similar immunological reactions, we consider that lactoferrin is responsible for these, as it was the only reactive protein species which could be prepared to homogeneity. Indeed two types of antibodies were found by ELISA test in the antisera to bovine beta-lg. One of them would be able to bind loosely to human lactoferrin, but its binding sites would not be antigenic in the rabbit.

Localisation dans la chaine peptidique de la β lactoglobuline bovine de la substitution Glu/Gln differenciant les variants genetiques B et D

It is definitively shown that the D variant of bovine β‐lactoglobulin D differs from the B variant in having a substitution Gln → Glu which involves residue 45 in the complete sequence recently published by Braunitzer et al. [4].

A new method for human milk protein separation

Attempts at isolating individual human milk proteins showed that cross interactions made it difficult to obtain of homogeneous components. A new method was devised, based on complete precipitation of milk proteins with saturated ammonium sulphate and progressive solubilization of the precipitate on a column of Sephadex G10 with a linear gradient of ammonium sulphate (from saturation to water). Three fractions were obtained. The first contained lactoferrin, serum albumin, lysozyme and traces of alpha-lactalbumin. Lysozyme could be obtained free from contaminants by chromatography on Ultrogel AcA 54. Lactoferrin and serum albumin coeluting as a single peak, were separated by a further chromatography on DEAE-cellulose. From the other two fractions recovered on Sephadex G10, it should be possible to prepare immunoglobulins, alpha-lactalbumin and the bulk of caseins. The homogeneity of the preparations of lysozyme, lactoferrin and serum albumin was assessed by SDS polyacrylamide gel electrophoresis, acrylamide agarose electrophoresis and immunoelectrophoresis.