SummaryUsing SDS-polyacrylamide gel electrophoresis and rocket immunoelectrophoresis, 3 new alleles, designated a,,-Cn'-, α s1 -Cn F and α 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 B OULANGER 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 were : <2,j-Cn! = 0.14 ; a,,-Cn° = 0.05 ; <2,j-Cn! = 0.01 ; ot,,-Cn' = 0.34 ; a,,-Cn F = 0.41 ; and a, i -Cn° = 0.05. In a sample of 159 Saanen females from 52 flocks of the same region, the frequencies were : <2,j-Cn! = 0.07 ; <2,j-Cn! = 0.06 ; < 2 ,j-Cn<' = 0 ; a 'I -CnB-= 0.41 ; a,,-Cn' ! 0.43 ; <2,j-Cn'" = 0.03. Additional data confirm that loci a, I -Cn and a,,-Cn are closely linked.Preliminary investigations indicated a significant superiority in casein content of milks from goats possessing the allele <2,j-Cn!, as compared to that of milks from goats of genotypes (xs,-Cn F / / <2,j-Cn! and <2,j-Cn! /a 'I -Cn F and, in a large herd (N = 251), a strong correlation was observed between the a,,-casein content and the rennet-casein content of milk (r = 0.68 ; b = 0.64).

In a previous report [1], we have given the complete primary structure of ϰB1‐caseinomacro‐peptide which is the soluble COOH‐terminal fragment split from bovine ϰB1‐casein by rennin. We also reported on the COOH‐terminal sequence of the NH2‐terminal fragment, the so‐called para‐ϰ‐casein. The present paper deals with the complete amino acid sequence of bovine ϰB‐casein, which has now been achieved by establishing the primary structure of para‐ϰB‐casein of which we discuss the salient features. This work has been reported briefly in a short communication [2]. SCM‐para‐ϰB‐casein and maleyl ϰBCN1, the NH2‐terminal cyanogen bromide fragment of ϰB‐casein [1], were used as starting material. The tryptic and peptic peptides of SCM‐para‐ϰB‐casein and the chymotryptic peptides of ϰBCN1 were isolated on Dowex 50 and Sephadex G‐50 or G‐25, and their sequences were determined either partially or completely by using classical methods and in some cases mass spectrometry. All these peptides and a NBS fragment of SCM‐para‐ϰB‐casein have provided all the overlaps needed for the completion of the amino acid sequence of para‐ϰB‐casein. Para‐ϰB‐casein is a single polypeptide chain containing 105 amino acid residues: Asp3, Asn4, Thr3, Ser7, PyroGlu1, Glu4, Gln12, Pro12, Gly1, Ala9, Val5, 1/2 Cys2, Met1, Ile6, Leu7, Tyr9, Phe4, Lys6, His3, Trp1, Arg5, and its molecular weight has been calculated to be 12269. The average hydrophobicity, calculated according to Bigelow [3], is 5.48 kJ (1.310 kcal) per residue, and para‐ϰB‐casein can be therefore considered to be a very hydrophobic molecule. Its net positive charge at pH of native milk (about 6.8) is very close to 4.5. The high content (11.5%) and rather uniform distribution of prolyl residues are incompatible with much α‐helical organization of the molecule, as previously shown for ϰ‐casein [4]. Both hydrophobic and charged amino acid residues are distributed non‐uniformly along the chain. Two regions, 1–24 and 80–105, are hydrophilic: the very hydrophilic former, with NH2‐terminal pyroglutamic acid, contains a cysteinyl residue located inside a cluster of eleven ionizable residues including six out of the seven total dicarboxylic amino acids; the 80–105 region, which contains the second cysteinyl residue in position 88, is rather hydrophilic, except at the COOH‐terminal end which is hydrophobic in spite of the presence of a cluster of four basic residues. These two hydrophilic regions are very likely to be on the outisde of the molecule and this may favor the formation of intermolecular S‐S bonds. The very hydro‐phobic central part of the chain, viz., 25–79, where most of the aromatic residues are located, has a para‐ϰ‐casein‐like behaviour in aqueous solution, and it may be responsible for the aggregation ability of para‐ϰ‐casein. According to the sequence data of both ϰB1‐caseinomacropeptide [1] and para‐ϰ‐casein, it is concluded that bovine ϰB‐casein is made up of a single polypeptide chain containing 169 amino acid residues: Asp4, Asn7, Thr14, Ser12, SerP1, PyroGlu1, Glu12, Gln14, P...

No abstract

Much is known about bovine lactoperoxidase but no data are available on its primary structure. In this work its main active fraction was isolated from cow's milk and sequenced using a conventional strategy. A clear similarity was found with human myeloperoxidase, eosinophil peroxidase and thyroperoxidase, the sequences of which were recently elucidated from those of their cDNAs and/or genes. The single peptide chain of bovine lactoperoxidase contains 61 2 amino acid residues, including 15 half-cystines and 4 or 5 potential N-glycosylation sites. The corresponding peptide segments of human myeloperoxidase, eosinophil peroxidase and thyroperoxidase display 55%, 54% and 45% identity with bovine lactoperoxidase, respectively, with 14 out of the 15 half-cystines present in each of the four enzymes being located in identical positions. The occurrence of an odd number of halfcystines in bovine lactoperoxidase supports the recent finding of a heme thiol released from this enzyme by a reducing agent, suggesting that the heme is bound to the peptide chain via a disulfide linkage, since the absence of free thiol in the enzyme was reported long ago Closely related heme peroxidases have been detected in most mammalian exocrine secretions. They are believed to protect mucosal surfaces from microorganisms by catalyzing the production of toxic oxidizing agents from HzOz, and halides or SCN-, a pseudohalide. Two of them, bovine lactoperoxidase and human salivary peroxidase, have been especially studied (Tenovuo, 1985). Bovine lactoperoxidase was highly purified and characterized in the early forties (Theorell and Akeson, 1943). Although lactoperoxidase is able to oxidize Br-, I-and SCN-, Reiter and coworkers (1964) demonstrated that oxidation of SCN-was involved in the mechanism of the lactoperoxidase-catalyzed antibacterial effect in bovine milk. Aune and Thomas (1977) and Hoogendoorn et al. (1977) independently concluded that hypothiocyanite (OSCN-) was the main antibacterial species produced from SCN-and H 2 0 z .

This paper describes the elucidation of the primary structure of the three genetic variants of goat asl-casein, asl-Cn D, E and F, which have been found to be associated with reduced amounts of asl-casein in milk. Variant E has the same electrophoretic mobility as variant B, but differs from the latter by the substitutions of Arg for Lys and of Thr for Ala at positions 100 and 195. A genetically controlled event which does not affect the amino acid sequence of this variant might be responsible for its lower rate of synthesis compared to that of asl-casein B. The deletion of 11 amino acids at positions 59 -69 and of 37 amino acids at positions 59 -95 in variant B leads to variants D and F. In both cases the deletions, which start at the same position of the polypeptide chain, include the major phosphorylation site of the protein. On the basis of sequence data for casein genes and cDNAs, it was concluded that the deletions occurring in the D and F variants are due to the exclusion of one and several exons, respectively. The observed deletions in the proteins could thus be the consequence of splice site mutations which would induce altered RNA processing and hence reduce the rate of synthesis of the casein.Early work on the casein family, which includes three phosphoproteins, asl-, as2-, and fl-casein, and one glycophosphoprotein, k--casein, was performed on proteins isolated from cow's milk. The primary structures of these four polypeptides, which contain 169 -209 amino acid residues, have already been established [l]. Whereas genetic polymorphism is widespread for asl-, fl-, and k--casein, it is more restricted and is breed-dependent for as2-casein [2, 31. Segregation studies led to the conclusion that the four proteins are controlled by a cluster of closely linked genes. In this cluster no recombination was observed in a total of 448 informative families [4]. The unit of inheritance is thus the haplotype which is a combination of individual alleles of each of the four clustered loci.Although the same classes of caseins have been identified in goat's milk [5], so far, in this species, only asl and as2-casein are known to exhibit genetic polymorphism [6]. Segregation studies have confirmed that asl-and as2-casein genes are closely linked suggesting that, as in the cow, casein expression in the goat is controlled by a cluster of genes.Electrophoretic patterns of goat asl-casein are unusually complex due to differences in the mobilities of different variants and to varying intensities of electrophoretic bands which reflect different amounts of protein [6]. It is now established [6, 71 that polymorphism of asl-casein is under the control of at least seven alleles denoted as: asl-CnA, asl-CnB, asl-Cnc, asl-CnD, asl-Cn", asl-CnF and asl-Cno. Alleles asl-CnA, asl-