Four rabbit antibodies have been prepared, which are specifically directed against a-lactalbumins from different sources; namely human, cow, goat and sheep milk. Each of these antibodies was tested for its ability to react with, separately, each of the four proteins.The immunological reactions were assessed by means of different techniques : double immunodiffusion in agar gel as well as affinity chromatography of antibodies, using antigens covalently bound to an insoluble matrix. In each case, the strongest reaction was observed between homologous antibody and (matrix-bound) antigen ; heterologous antigens were, however, also capable of cross-reaction.Whereas no cross-reaction between human cc-lactalbumin and antibodies against the bovine protein could be evidenced by immunodiffusion, the occurrence of soluble complexes has been disclosed by means of a gel filtration technique.It has been reported that antisera to bovine a-lactalbumin react with a-lactalbumins from other ruminants, but fail to do so with the parent proteins from non-ruminants [l, 21. However, when the amino acid sequences of human, of bovine and of guinea-pig a-lactalbumins became available [3 -51, their comparison afforded evidence for a strikingly high degree of homology at the structural level. This was difficult to reconcile with the view that the antigenic sites of bovine and human a-lactalbumins, or guineapig, should all be different.Many studies indeed have already demonstrated immunological cross-reactions of iso-functional enzymes from different species. Such is for example the case with lysozymes from duck and from hen egg white [6], or with bovine and porcine trypsins [7]. In this respect, the pattern of cross-reactions between different a-lactalbumins would appear to be anomalous or, possibly, it might result from the experimental procedure that was used.In earlier work, the immunological comparison of a-lactalbumins was in fact performed by means of quantitative, single immunodiffusion, a method that precludes the disclosure of eventual soluble antigen-antibody complexes.Abbreviation. Mops, morpholino-propane sulfonic acid. Eur. J. Biochem. 50 (1975)The present investigation was therefore undertaken in order to find out if antigenic sites of human, of bovine, of goat and of sheep a-lactalbumins are not able to cross-react, to some detectable extent, with heterologous antibodies. EXPERIMENTAL PROCEDURE MaterialsSepharose 4B, Sephadex G75 and DEAE-Sephadex A25 were purchased frorn Pharmacia Fine Chemicals (Uppsala, Sweden). Cyanogen bromide was from Fluka. Freund's complete adjuvant was a Difco product. All other reagents were of analytical grade.Gradients for chromatography were set up with an LKB Ultrograd 11 300 unit, monitored by an LKB Uvicord 11. Purification of a-LactalbuminsThe purification of human a-lactalbumin was carried out according to the procedure already described by Findlay and Brew [3].Bovine, goat and sheep a-lactalbumins were purified by the procedure of Schmidt and Ebner [8], up to the ammonium sulphate precipita...
Through the use of affinity chromatography, a homogeneous preparation of human /?( 1 -4)-D-galactosyltransferase (the A protein of lactose synthase) was obtained. The specificity of this protein for glycoconjugates was studied in the presence and absence of human r-lactalbumin.A kinetic analysis of the transfer of D-galactose to N-acetyl-D-glucosamine and to /j( 1 + 4)-linked N-acetylglucosamine oligomers, suggested that the active site region of the enzyme contains more than one binding site for acceptor molecules. Furthermore, experiments with N-acetylglucosamine-/3( 1 + 4)-N-acetylmuramic-pentapeptide isolated from Micrococcus luteus indicated that the presence of a peptide chain does not enhance enzymic activity, as compared with the corresponding free disaccharide. Similar results were obtained using ovalbumin and the ovalbumin glycopeptide (which have similar apparent K,,, values for A protein) as galactose acceptors. In contrast to its ability to inhibit N-acetyllactosamine production, a-lactalbumin did not inhibit the transfer of D-galactose to the N-acetylglucosamine oligomers or the glycopeptides.Although a-lactalbumin can switch the specificity of A protein from N-acetyl-D-glucosamine to D-ghKOSe resulting in the production of lactose, no transfer of galactose was observed to P(1+4)-linked glucose oligomers or to a collagen glycopeptide, D-glucopyranosyl-a( 1 +2)-~-galactopyranosyloxy-P( 1 --f 5)-lysine. It therefore appears that z-lactalbumin can only modify human A protein for monosaccharide acceptors. ability of the human , / I ( 1 -+4)-~-galactosyltransferase. Furthermore, attempts were made to ascertain whether human a-lactalbumin could function in its specifier role [l] with A protein in the presence of a glycopeptide isolated from chick collagen containing a glucosyl residue at the non-reducing end and also with /?( 1 -4)-linked D-glucose oligomers.On the basis of our results, we have been able to arrive at some conclusions concerning the number of sugar binding subsites of human A protein. EXPERIMENTAL PROCEDURE MuteriulsOvalbumin (type V), peptidase, protease, trypsin, chymotrypsin, UDP-D-galactose and GlcNAc were purchased from Sigma Chemical Co. UDP-D-['4C]-galactose (467 counts min-' nmol-') was from the Radiochemical Centre, Amersham. The cell wall glyco-
p( 1 -4)-Galactosyltransferase from human milk (the A protein of lactose synthase) has been found to be heterogeneous when fractionated by affinity chromatography against insolubilized a-lactalbumin, using a linear gradient of decreasing N-acetylglucosamine concentration. Three forms were isolated. Molecular weights of the different species, as determined by sodium dodecylsulphate gel electrophoresis, were found to be 38000, 43000 and 50000. The 38000 and 50000 species were studied for their catalytic ability to synthesize either lactose in the presence of a-lactalbumin, or N-acetyllactosamine in the presence and absence of the 'specifier' protein.Appreciable difference was observed between the two enzyme forms with respect to their catalysis of lactose synthesis with a-lactalbumins from various sources. Differences in the rate of production of N-acetyllactosamine in the presence of a-lactalbumin were also observed. For the lowest-molecularweight species it was found that the inhibitory effect of a-lactalbumin upon N-acetyllactosamine synthesis becomes an activating effect at higher a-lactalbumin concentrations, while no such inversion was observed for the other species.The results suggest that the conformation at the site of association of the enzyme with the acceptor saccharide or cr-lactalbumin has been changed, presumably by a partial enzymic hydrolysis.
a-Lactalbumin isolated from human milk was reacted with tetranitromethane in molar excess of 8 -32 mol/mol of tyrosine. After gel filtration on Sephadex G-75, followed by chromatographic fractionation using DEAE-Sephadex A-25, three main components were separated, which differed from one another in the extent of nitration.These protein fractions were found to contain, respectively, one and two nitrotyrosine residues, or two nitrotyrosine residues together with one nitrotryptophan. The lactose synthase specifier activity of each of these components was measured and compared with that of unsubstituted cr-lactalbumin.Comparison of kinetic parameters showed the chemically modified proteins to be only slightly less active when tyrosines were the sole residues modified. In sharp contrast the additional nitration of a single tryptophan residue totally abolished the specifying activity of a-lactalbumin.Circular dichroism spectra of the tryptophan derivative revealed some structural alteration when compared with the other two and with the native protein. The conclusion could also be confirmed by using a double-immunodiffusion technique. After hydrolysis of the derivatives with thermolysin, it was possible to localize the substituted residues in the known sequence of human a-lactalbumin. Tyrosine-103 was found to be more easily nitrated than tyrosine-18. These two residues seem, therefore, to be on the outer surface of the molecule and more exposed than tyrosine-36 and tyrosine-50.Some precautions are indicated in the use of tetranitromethane as a nitrating agent on the basis of complex products observed in the nitration of the free amino acids tyrosine and tryptophan and their derivatives. Since the nitration of tryptophan residues was claimed to take place only when high molar excess of reagent over protein was employed, the use of C(NO;)4 as a chemical modifying agent appeared to retain much of its interest in studying which site of human cr-lactalbumin is interacting with human A protein in the lactose synthase system.Tyrosine side-chains were indeed likely candidates for playing a definite role in either the primary or even the secondary structure of a-lactalbumin. Align- ment of sequences for the three a-lactalbumins which have been completely worked out (bovine, guinea-pig and human; [6-81) indicates that each of the four tyrosines occupies the same position throughout. Furthermore, their immediate neighbours are also unchanged. Such is, however, not the case for kangoroo a-lactalbumin ([9] partial sequence from 1 -42) in which tyrosines-18 and 36 are replaced by, respectively, valine and leucine ; moreover, their neighbours are not conserved.From these results it seemed worthwhile to attempt a specific substitution of tyrosines in order to find out whether these residues are part of some site of association between a-lactalbumin and the A protein.The chemical modification of tyrosyl residues in bovine a-lactalbumin has already been reported. Reaction with C(N02)4 was used [lo-121 leading, however, to contradicto...
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