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Previous reports [1-5], we have described some of the features of the primary structure of bovine αs1-casein. In the present work, the complete amino acid sequence has been established and the salient features of this phosphoprotein have been discussed. In the polyeptide chain, the region containing the phosphopeptides Tm1 (T1-T2), Tm1T2 and Tm1T1 [1,4,5], had only been slightly 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 Tm1T2 contains three hydroxy-amino acids (2 Ser, 1 Thr), but, instead of three [1], two phosphorus atoms were found with purified preparations. Partial acid hydrolysis and dephosphorylation using an orthophosphoric-monoester phosphohydrolase indicate that the two seryl residues m this peptide are O-phosphorylated. The remaining gap in the sequence of the central part of peptide Tm1T2 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 Tm1T1 [1], five were shown to be present after detailed analysis of the fragments. Since the five phosphate groups could be ready removed by an orthophosphoric-monoester phosphohydrolase, the five seryl residues could be O-phosphorylated. The fragments obtained after hydrolysis with thermolysin of both native and dephosphorylated peptide Tra1T1 were further degraded using classical methods for the determination of the amino acid sequence. In the light of all the results obtained on this phosphoprotein (αs1-casein B), the total number of amino acid residues has been corrected to 199, instead of 198, as reported previously [1-5], and the molecular weight has been calculated to be 23616. The following amino-acid composition; Asp7, Asn8, Thr5, Ser8, SerP8, Glu25, Gln14, Pro17, Gly9, Ala9, Val11, Met5, Ile11, Leu17, Tyr10, Phe8, Lys14, His5, Trp2, Arg6, 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 thc average hydrophobicity [7] of this protein is 1170, it could be considered to be a hydrophobic protein The high amount (8.5%) 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 first 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 residues, in particular seven of the total eight phosphoseryl residues. Another remarkable feature is the concentration of amino acids with carboxylic side chains, particularly glutamic acid, in the vicinity of two clusters of phosphoseryl residues. It may be noticed that the phosphoseryl residues m αs1-casein are arranged m a manner similar to those in β-casein [8]. In particular, in αs1-casein, the 62-70 region containing four phosphoseryl residues, is similar to the 13-21 region in β-casein [8]. It is interesting to point out that ail but one of the phosphoseryl residues always occur m position n with relation to a glutamyl or a phosphoseryl residue, which is in position n + 2. From this, it would appear that there is an enzymatic phosphorylation of the polypeptide chum (carried out by a phosphoryl kinase which may require a negative charge in the phosphorylation site) rather than a direct incorporation of phosphoserine into the polypeptide chain during protein synthesis. The location of the amino-acid substitutions that characterize the αs1-Casein D and the β-casein C variants has provided evidence for an enzymatic phosphorylation of casein [9]. The problem of the phosphorylated sites in αs1- and β-caseins will be discussed in a forthcoming paper. The repeating amino-acid sequences located in the two regions of αs1-casein, viz. 70-84 and 110-125, are also of interest. The primary structure of αs1-casein given here is that of the genetic variant B. The three other variants A, C and D have also been studied. The C and D variants differ from the B variant by the substitutions of Gly/Glu in position 192 [10] and ThrP/Ala in position 53 [9], respectively. The A variant is characterized by a deletion of thirteen amino-acid residues from position 14 to position 26 in the hydrophobic NH2-terminal part of the polypeptide chain [11]. Because of this deletion, there m an important change in the physico-chemical properties of αs1-casein [12, 13]. Some important features of the amino-acid-sequence determination should be noted. Firstly, the great value of maleic anhydride as a reversible-blocking reagent for amino groups of proteins [14], and the usefulness of thermolysin as a specific enzyme for degrading polypeptides [15]. Secondly, the difficulties encountered during the study of the phosphopeptides: these peptides were difficult to purify since they have similar anionic characters and only react slightly with ninhydrin during paper revelation; other difficulties have been the high destruction of phosphoserine during hydrolysis with HCl 5.7 N, the poor yield in the substractive Edman degradation when the NH2-terminal residue is a phosphoserine, and the failure of endopeptidases and exopeptidases to split off peptide bonds that are close to phosphate groups. Fortunately, alkaline phosphatase has proved to be a useful tool for removing phosphate groups, thus avoiding most of these difficulties. Since the amino-acid sequence of αs1-casein is now known, the degradation of this protein by coagulating and other proteolytic enzymes during cheese ripening may be studied. In particular, bitter peptides originating from αs1-casein may be located. As we have already briefly noted [4], it is obvious that one of the three bitter peptides isolated by Matoba et al. [16] from a tryptic hydrolysate of whole casein, was the segment 23-34 of the polypeptide chain of αs1-casein. In addition, the knowledge of the primary structure will be of help in understanding many aspects of the relations between structure and physical properties in this phosphoprotein. [ABSTRACT FROM AUTHOR]

Commercial lima bean inhibitor was fractionated into four apparently homogeneous components as previously described by Jones, Moore and Stein in 1963. Reduced and alkylated component IV was hydrolyzed with trypsin and the resulting peptides were separated by ion exchange chromatography on Dowex 50 X2 or gel filtration on Bio-Gel P-6. Where necessary, further purification of the peptides was carried out by paper chromatography, paper electrophoresis or a combination of both. Seven peptides were obtained in pure form and their compositions are reported here. The amino acid sequence of six of these peptides was determined, using classical methods. When allowance is made for the occurrence of two homologous peptides, presumably resulting from heterogeneity m the original protein preparation, the tryptic peptides isolated, account for the complete composition of the protein. In an attempt to obtain overlapping sequences the tryptic digest was also performed on the reduced, alkylated and guanidinated protein. Five tryptic peptides, including one containing homoarginine as the carboxy-terminal residue, were isolated in pure form from the digest; their amino acid compositions are reported. [ABSTRACT FROM AUTHOR]

A purified glycoprotein with blood-group-A specificity, was digested with insoluble pronase to give a serologically active product enriched in peptide-bonded serine and threonine residues; this was degraded with 0.25 M sulphuric in glacial acetic acid for 24 h. The resultant N-acetylgalactosaminyl-peptide were hydrolysed with 0.2 M hydrocholoric acid for 24 h at 110 °C. fractionation of the products on Sephadex G-10, followed by preparative separation on amino-acid analyzer and paper electrophoresis at pH 1.9 yielded the following homogeneous oligopeptides; one tripeptide with the N-terminal sequence Thr-Thr-Ser, three tetrapeptides with the sequences, Thr-Ser-Thr-Ser, Thr-Pro-Thr-Ser and Pro-Thr-Thr-Ser, one pentapeptide with the sequence Thr-Pro-Thr-Ser, one hexapeptide with the sequence Pro-Thr-Thr-Thr-Pro-Ser and one heptapeptide with the sequence Ala-Pro-Thr-Thr-Ser-Gly-Ser. Galactosamine was present in several of these fragments and the hexapeptide and heptapeptide both contained sufficient of this sugar for all the serine and threonine residues to be glycosylated. The following five tetrapeptides sequences were deduced from mixtures of two peptides which differed by the amino-acid substituent at only one position in the chain; Pro-Thr-Ala-ser, Thr-Thr Pro-Ser, thr-Thr-Ala-Ser, Ser-Pro-Thr-Ser, and Ser-Ala-Thr-Ser. A tripeptide mixture which differed only in the nature of the third amino acid yielded the following three tripeptide sequences: Pro-Thr-Ser, Pro-thr-Ala. These peptides do not indicate a clearly defined amino-acid sequence surrounding the glycosylated hydroxy amino acids but establish that in the human blood-group-specific glycoproteins there is close packing of the threonine in the region of the peptide moiety carrying the carbohydrate chains. [ABSTRACT FROM AUTHOR]

The isolated α-chain of sea anemone collagen was cleaved at methionyl residues with cyanogen bromide. Eleven unique peptides were isolated by ion-exchange and molecular-sieve chromatography, eight of them in approximately equimolar amounts. Characterization of the eleven peptides with regard to amino-acid composition and molecular weight demonstrated that the isolated peptides account for all of the amino acids and for the molecular weight of the α-chain within experimental error. Since the molecule of sea anemone collagen contains ten methionyl residues, the data of the present paper confirm earlier analytical and preparative results that this invertebrate collagen is made up of three identical α-chains. In comparison with known collagens, on the basis of quantitative and qualitative features of amino-acid compositions, the cyanogen bromide peptides of sea anemone collagen indicate genetical relationship of sea anemone collagen to basement membrane collagen. [ABSTRACT FROM AUTHOR]

Peptide B, the N-terminal fragment of thioredoxin obtained by cyanogens bromide treatment was aminoethylated and digested with pepsin. Eight peptides were separated by high voltage paper electrophoresis and chromatography and characterized with respect to amino acid sequence. The results account for all 37 residues peptide B. The N-terminal sequence of peptide B was determined as: Ser-Asp-Lys-Ile-Ile-His-Leu-Thr-Asp-Asp-Ser-Phe. [ABSTRACT FROM AUTHOR]