I n previous reports [l-51, we have described some of the features of the primary structure of bovine oL,l-casein. I n the present work, the complete amino acid sequence has been established and the salient features of this phosphoprotein have been discussed.I n the polypeptide chain, the region containing the phosphopeptides Tml (Ti -T2), TmlT2 and TmlTl [1,4,5], had only been shghtly studied as yet. Because of the difficulties encountered in the breakdown of these phosphopeptides, hydrolysis with endopeptidases and exopeptidases were performed on both native and dephosphorylated peptides.It was confirmed that peptide TmlT2 contains three hydroxy-amino acids ( 2 Ser, 1 Thr), but, instead of three [l], two phosphorus atoms were found with purified preparations. Partial acid hydrolysis and dephosphorylation using an orthophosphoric-monoester phosphohydrolase indicate that the two seryl residues in this peptide are 0-phosphorylated. The remaining gap in the sequence of the central part of peptide TmlT2 was bridged by studying two fragments obtained by papain digestion of the dephosphorylated preparation.Instead of four serines, postulated earlier from the results of the amino-acid composition of peptide TmlTl [I], five were shown to be present after detailed analysis of the fragments. Since the five phosphate groups could be readily removed by an orthophosphoric-monoester phosphohydrolase, the five seryl residues could be 0-phosphorylated. The fragments obtained after hydrolysis with thermolysin of both native and dephosphorylated peptide TmlTl were further degraded using classical methods for the determination of the amino acid sequence.I n the light of all the results obtained on this phosphoprotein (a,l-casein B), the total number of amino acid residues has been corrected to 199, instead of 198, as reported previously [l-51, and the molecular weight has been calculated to be 23616. The following amino-acid composition; Asp,, Asn,, Thr,, Ser,, SerP,, Glu,,, Gln,,, Pro,,, Gly9, Ma,, Val,,, Met,, Ile,,, Leu,,, Tyr,,, Phe,, LysI4, His,, Trp,, Arg,, indicates that there is a higher number of acidic than basic residues in this protein. On the basis of the intrinsic dissociation constants of titratable groups in proteins [6], the negative net charge of the molecule was estimated to be 22 at pH 6.5 and 28 at pH 8.6. Since Bigelow's parameter for the average hydrophobicity [7] of this protein is 1170, it could be considered to be a hydrophobic protein. The high amount (8.50/,) and uniform distribution of prolyl residues indicate that this protein has limited structuralcoiling possibilities.The polypeptide chain contains three hydrophobic regions, viz. 1-44,90-113 and 132-199. The fist two are characterized by the fact that basic residues predominate over acidic residues. The third region, where most of the aromatic residues are located, contains very few basic residues and is therefore of more acidic character.Two regions, 45-89 and 114-131, are hydrophilic. The former contains more than half of the total acidic residue...

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ABSTRACTa-Lactalbumin is an abundant milk-specific calcium metalloprotein which has an evolutionary relationship to lysozyme. It modifies the substrate specificity of a Golgi galactosyltransferase by forming the lactose synthetase binary complex. Lactose, together with other sugars and diffusible ions, is responsible for the osmotic pressure of milk. To assess the involvement of a-lactalbumin in lactogenesis, a-lactalbumin-deficient mice were created by disrupting the gene by homologous recombination in embryonic stem cells. Homozygous mutant mice are viable and fertile but females cannot feed their offspring. They produce a highly viscous milk that pups appear to be unable to remove from the mammary gland. This milk is rich in fat and protein and is devoid of a-lactalbumin and lactose.

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...

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