Primary structure of a kappa B I bovine casein macropeptide

Abstract

This paper deals with the amino-acid sequence of χB1-caseinomacropeptidc, the soluble COOH-terminal fragment split from xB,-casein by rennin. χB1-casein, the major component of χB1-casein, is devoid of carbohydrate and was used as starting material. This component, which contains 2 methionyl residues [1-3], was cleaved with cyanogen bromide. The 3 expected peptides, designated as CN peptides, were separated first on Sephadex G-50 and further purified on Dowex 50-X2. Peptide χB1CN2, which is 63 residues long, is the COOH-terminal part of χB1-casein chain according to the following observations : it is devoid of homoserine-homoserine lactone; the 11 amino acids released by carboxypeptidase A are those of the COOH-terminal end of χ-casein [4]. Peptide χB1CN2 represents the χB1-caseinomacropeptide devoid of its NH2-terminal methionyl residue [5-7]. Its complete amino-acid sequence was determined by classical methods, using thermolysin to produce smaller fragments, then exopeptidases and chemical procedures, such as subtractive Edman degradation and hydrazinolysis, for establishing the structure of these fragments. Overlapping of the 11 “thermolysin” peptides was obtained by studying peptic fragments from χB1CN2. The sequence of the first 18 amino acids at the NH2-terminal end is identical with that reported earlier by Jolles' group [8,9] The COOH-terminal sequence is identical to those respectively reported by Jolles and Alais [1,10] and Malpress et al. [7], but it is only in partial agreement with those suggested by De Koning et al. [6] and Pujolle et al. [4]. From sequence data of peptide χB1CN2, is has been concluded that χB1-caseinomacropeptide is a single polypeptide chain containing 64 amino acid residues: Asp1, Asn3, Thr11, Ser5, SerP1, Glu8, Gln2, Pro8, Gly1, Aha6, Val6, Met1, Ile7, Leu1, Lys3, and its molecular weight has been calculated to be 6755. There is a higher number of acidic than basic residues and the negative net charge of the molecule has been estimated to be 7.5 at pH 6.8. Since Bigelow's parameter for the average hydrophobicity [11] is 1083, χB1-caseinomacropeptide can be considered to be rather hydrophobic. The sequence obtained emphasizes the uneven charge distribution along the polypeptide chain: the 3 lysyl residues are located at the NH2-terminal end, and the 32-53 region contains 7 of the total 10 acidic residues. Another remarkable feature of the chain is the great number of hydroxy-amino acid residues : one in four of the total 64 residues is serine or threonine, and this relative proportion rises to one in three in the 12-64 region. According to Hill and Wake [12], this distribution might be intimately associated with the micelle-stabilizing properties of χ-casein. As expected from the high amount of some amino acids, and thc lack of cysteine, arginine, histidine, phenylalanine, tyrosine and tryptophane, many regions in the caseinomacropeptide have identical or very similar structures. Therefore, some difficulties were encountered when ovcrlapping the “thermolysin” peptides. The high amount (12.50%) and rather uniform distribution of prolyl residues indicate, as previously shown for χ-casein [13], that the caseinomacrogeptide has limited structural-coiling possibilities. In addition, the COOH-terminal sequence of para-χB1-casein has been investigated by study- ing peptide χB1CN3 which represents, without taking its additional COOK-terminal methionyl residue into account, the COOH-terminal end of para-χB1-casein. The peptide χB1CN3 contains 3 histidyl residues instead of 2 as expected from Jolles' results [9,14]. In connection with recent works dealing with synthetic substrates of rennin, the sequence of the first 11 amino acids a t the COOH-terminal end of para-χB1-casein reported here is of interest since the amino acids in the vicinity of the rennin-sensitive linkage seem to contribute to its lability [15–21]. Thus, we report the sequence of the first 75 amino acids at the COOH-terminal end of χB1- casein. The localization of the two amino acid substitutions which differentiate the known variants A and B will be reported in a forthcoming paper [22].