Bruno Ribadeau-Dumas

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.

Identification of the active site serine of the X‐prolyl dipeptidyl aminopeptidase from Lactococcus lactis

The active site serine of the X‐prolyl dipeptidyl aminopeptidase from Lactococcus lactis (PepX) was identified. The enzyme was labeled by [3H]DFP, treated by CNBr and the resulting peptides were separated by reverse‐phase‐HPLC. The main radiolabeled peptide was sequenced. Ser‐348, in the following sequence, Gly‐Lys‐Ser‐Tyr‐Leu‐Gly, was identified as the active site serine. A sequence comparison between the active site of PepX and other serine proteases was made, showing only limited sequence homologies in this area. The consensus sequence surrounding the active site serine in the three known X‐prolyl dipeptidyl aminopeptidases (mammalian DPPIV, yeast DPAB and PepX) is G‐X‐S‐Y‐X‐G, where X is a non‐conserved amino acid.

Structure primaire de la paracaseine κ bovine

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

Determination of gradient elution conditions for the separation of peptide mixtures by reversed-phase high-performance liquid chromatography: bovine β-casein tryptic digest

Taking as an example a tryptic hydrolysate of bovine β-casein, it was shown that the method used for the determination of optimal elution conditions for isocratic systems also applied to the separation of complex peptide mixtures by reversed-phase high-performance liquid chromatography with linear gradients. Using a C18 column and the same solvents, successive studies of the influence of flow rate, pH and temperature allowed a satisfactory separation of the sample in less than 30 min. Valuable information on the specificity of the action of trypsin on β-casein was deduced from the yield of the eluted peptides.

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.

A new strategy for primary structure determination of proteins: Application to bovine β‐casein

A new approach has been developed for sequencing proteins. A radioactive label is attached specifically to the C‐terminus of the protein. The labelled molecule is subjected to varying proteolysis conditions. From the electrophoretic patterns (SDS‐PAGE) of the hydrolysates, appropriate cleavage conditions are selected, giving labelled peptides of different lengths which are purified. The labelled peptides are sequenced in order of increasing size (from 1 to n), peptide (i) being sequenced until the N‐terminal sequence of peptide (i‐1) is encountered. This approach allows the determination of a complete protein sequence with a minimal number of Edman cycles. The method was successfully applied to bovine β‐casein (209 residues) which was completely resequenced with only 239 Edman cycles.

Fractionation of whole casein on hydroxyapatite. Application to a study of buffalo κ-casein

When whole caseins from cow and Italian buffalo (Bubalus arnee) were fractionated by chromatography on a column of hydroxyapatite they behaved in a similar manner. kappa-Casein was eluted with 5 mM phosphate buffer, pH 6-8, containing 0-2 M-KCl, 4-5 M-urea and 2 mM-2-mercaptoethanol, but beta- and alphas-caseins were retained and could be eluted successively by a linear gradient from 5 mM to 250 mM-phosphate buffer. Buffalo kappa-casein preparations, obtained from bulk milk or from milks of individual animals by chromatography on hydroxyapatite, produced identical electrophoretic patterns at pH 8-6. By further fractionation of these kappa-caseins on DEAE-cellulose, in each case, at least 7 components were purified; they had different electrophoretic mobilities but were all sensitive towards chymosin. The major fraction migrated like component 1 of bovine kappa-casein B.

Localisation, dans la partie NH2-terminale de la caséine αs1 bovine, d'une délétion de 13 acides aminés différenciant le variant a des variants B et C

The A variant of bovine αs1 casein is devoid of the segment of 13 amino acid residues which occupies the 14th to 26th position from the NH2‐terminal in the polypeptide chain (198 residues) of the B and C variants.

Localisation des substitutions d'acides aminés différenciant les variants a et b de la caséine × bovine

Comme le suggéraient les résultats de WOYCHIK et al. (19 66), DE KoNirrc et al. (19 66) et HILL et al. (1970 ), les caséinomacropeptides des variants A et B de la caséine x bovine diffèrent par deux substitutions d’acides aminés : Ala (xB) Asp (xA) et Ile (xB) Thr (xA). Ces substitutions affectent respectivement la 22 e et la 34 e position à partir de l’extrémité COOH-terminale du caséinomacropeptide, qui est également celle de la caséine x. D’un point de vue génétique, les deux variants de la caséine x assurent donc le marquage de la partie terminale du locus x-Cn.

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.