Lactalbumin amino acid sequence

The human a-lactalbumin amino acid sequence was used in the refinement since the baboon sequence has not been determined, although it was known from the unpublished work by R. Greenberg to be close to the human sequence. However, it became evident in the course of the X-ray work that there were eight sequence changes in baboon a-lactalbumins (see Section VII,B). 

Bovine a-lactalbumin is one of the two enzymes in lactose synthetase, and its amino acid sequence shows striking similarities to that of lysozyme.118 A model based on the lysozyme model has been built, and the side-chain interactions found are convincing, showing that the model is essentially correct. The active cleft in the crystal is, however, shorter than that in the model, and is consistent with a mono- or di-saccharide as the substrate. Thus, the lysozyme structure may serve as a model for some enzymes that synthesize and hydrolyze carbohydrates. 

Brew, K., Castellino, F. J., Vanaman, T. C, and Hill, R. L. 1970. The complete amino-acid sequence of bovine a-lactalbumin. J. Biol. Chem. 245, 4570-4582. 

Schewale, J. G., Sinha, S. K. and Brew, K. 1984. Evolution of a-lactalbumins. The complete amino acid sequence of the a-lactalbumin from a marsupial (Macropus rufogri-seus) and corrections to regions of sequence in bovine and goat a-lactalbumins. J. BioL Chem. 259, 4947-4956. 

About 20% of milk protein is soluble in the aqueous phase of milk. These serum proteins are primarily a mixture of /3-lactoglobulin, a-lactalbumin, bovine serum albumin, and immunoglobulins. Each of these globular proteins has a unique set of characteristics as a result of its amino acid sequence (Swaisgood 1982). As a group, they are more heat sensitive and less calcium sensitive than caseins (Kinsella 1984). Some of these characteristics (Table 11.1) cause large differences in susceptibility to denaturation (de Wit and Klarenbeek 1984). 

The application of the primary databases and structural analytical tools will be introduced using a protein from a future experiment. In Experiment 4, you will extract, purify, and characterize a-lactalbumin from bovine milk. To prepare for this activity, here you will learn about the structure of a related protein, a-lactalbumin from humans. We will search databases to find and view its primary and secondary structure and also determine if there are other proteins with a similar amino acid sequence and structure. After completion of these exercises, you will be able to apply these computer tools to proteins of your own choice. 

Another useful structure tool is RasMol (or RasMac). This will allow you to view the detailed structure of a protein and rotate it on coordinates so you can see it from all perspectives. A hyperlink to RasMol is present under the View Structure function just above Chime. You may need to study RasMol instructions provided under Help, or you may use a Ra.s Mol tutorial listed in Table El.2. Another useful protein viewer is tin-Swiss-Protein Pdv Viewer (Table El.2). BLAST is an advanced sequence similarity tool available at NCBI. To access this, go to the NCBI home page (www.ncbi.nlm.nih.gov) and click on BLAST. Then click on Basic BLAST search to obtain a dialogue box into which you may type the amino acid sequence of human a-lactalbumin. This process may be stream lined by downloading the amino acid sequence in FASTA format into a file and transferring the fde into the BLAST dialogue box. BLAST will provide a list of proteins with sequences similar to the one entered. 

Use the techniques outlined in the experimental procedure to explore two enzymes you will study in later experiments. Study the two enzymes malate dehydrogenase (Experiment 10) and tyrosinase (Experiment 5). View structures and look at amino acid sequences as you did for human a-lactalbumin. 

Use the BLAST tool to compare the amino acid sequences for human a-lactalbumin and lysozyme. Repeat the process using BLAST to compare the nucleotide sequences for the genes coding for human a-lactalbumin and lysozyme. 

In 1958 Yasunobu and Wilcox drew attention to certain similarities between a-lactalbumin and lysozyme (see Gordon, 1971). A few years later Brew and Campbell (1967) also drew attention to their marked similarity in molecular weights, amino acid composition, and the amino-and carboxy-terminal amino acid residues. They stated, To the extent that the properties mentioned reflect similar primary structures, the a-lactalbumins may have evolved by gradual modification from lysozyme, which is found in the milk of many species (p. 263). This proposal prompted Brew etal. (1967, 1970) to determine the amino acid sequence of bovine a-lactalbumin, which proved to have a high level of sequence identity with domestic hen egg-white lysozyme. Thus, these studies were in accordance with the proposal that the two proteins had diverged from a common ancestor (see also Hill etal., 1969, 1974). They stated that although lysozyme does not participate in lactose synthesis and a-lactalbu-... 

Because of the high level of identity in amino acid sequence between lysozyme and a-lactalbumin (see Fig. 10), it was inevitable that interest turned to the three-dimensional structure of a-lactalbumin when the structure of lysozyme was determined in 1965 by the group at the Royal Institution. However, there were unforeseen difficulties in the direct experimental determination, as discussed below. Hence, attention was directed to models for the a-lactalbumin structure based on the coordinates for lysozyme and on energy minimization programs. 

On the basis of monomer molecular weights (from sedimentation-equilibrium and sedimentation—diffusion studies, amino acid sequences and compositions), the a-lactalbumins, with one exception (rat a-lactalbumin, 140 residues, see Section VII,B), have a single chain of — 123 residues and values of —14,000. The mammalian lysozymes have 128-130 residues and values of —14,400, except echidna lysozyme, which has —125 residues. The c-type hen egg-white lysozymes have -127—131 residues, in contrast to the g type, which has —185 residues. 

The X-ray crystallographic determination of the structure of a-lactalbumin has been made on baboon (Papio cynocephalus) a-lactalbu-min. Its amino acid sequence has not been determined. However, on the basis of preliminary work by R. Greenberg (personal communication), it is evident, as would be expected, that its sequence is similar to that of human a-lactalbumin. On the basis of their X-ray studies, Acharya et al. (1989) concluded that the following are possible sequence changes from that of the human protein ... 

The assignment for residue 57 was discussed by Teahan (1986). She pointed out that KDII and PDl lysozymes have identical amino acid sequences, except for residue 57, which is given as Glu by Hermann et al. (1971) for KDIII and as Gin by Kondo et al. (1982) for PDl. Prager and Wilson (1972) have shown that both proteins have identical electrophoretic mobilities. Thus, it is likely that KDII has Gin in position 57. This is in accord with the later view of Rodriguez et al. (1987). Further, we note that all other c-type lysozymes and all a-lactalbumins have Gin in this position. Hence, we conclude that KDII and KDIII have Gin in position 57, as shown in Fig. 10. This, then, means that KDII and PDl are identical. 

In Section VII,B and Fig. 10 we compared the sequences of 13 a-lactalbumins (if the bovine A variant, equine B and C variants, and ovine variant are included), 23 mammalian c-type lysozymes (if donkey, mouse M, bovine stomach 1 and 3, caprine 1 and 2, ovine 1-3, camel 1, deer 2, echidna II, and porcine 1 and 2 are included), and 13 avian c-type lysozymes (if KDIII and PD2 and PD3 are included). Analysis of the sequence differences indicates that, with the recent considerable increase in the number of lysozymes sequenced, there has been an appreciable decrease in the numbers of residues that are invariant in lysozymes as well as for both proteins. Nevertheless, there is still significant overall homology (—35%) between a-lactalbumin and c-type lysozyme. From the similarities in amino acid sequences, three-dimensional structures, intron—exon patterns, etc., there can be little doubt that the concept of divergence is still valid for these proteins. What is controversial are the rate of evolution and the details of the way in which ct-lactalbumin arose, although it is conceded generally that the mechanism involves gene duplication. 

Figure 4.25 Amino acid sequence of a-lactalbumin showing intramolecular disulphide bonds ( ) and amino acid substitutions in genetic polymorphs (from Brew and Grobler, 1992).

Brodbeck and Ebner found that the soluble lactose synthetase from milk can be separated into two protein components, A and B, which individually do not exhibit any catalytic activity however, their recombination restores full lactose synthetase activity. The B fraction has been crystallized from bovine skim milk and bovine mammary tissue, and was identified as a-lactalbumin. It was thus found that a-lactalbumin can be substituted for the B protein of lactose synthetase. Lactose synthetases from the milk of sheep, goats, pigs, and humans were also resolved into A and B proteins, and the fractions from these species were shown to be qualitatively interchangeable in the rate assay of lactose synthesis. Determination of the amino acid sequence of a-lactalbumin (B fraction) has shown a distinct homology in the sequence of amino acids of a-lactalbumin and hen s egg-white lysozyme, suggesting that lysozyme and a-lactalbumin have evolved from a common ancestral gene. 

K. Brew, T. C.Vanaman and R.L. Hill, Comparison of the Amino Acid Sequence of Bovine (x-Lactalbumin and Hens Egg White Lysozyme, J. biol. Chem. 242, 3747-3749 (1967). 

For example, the respective values at pH 10.6 are 0.262, 0.494, and 1.04 mole per cent (ratio of about 1 2 4) at pH 11.2 the values are 0.420, 0.780, and 1.32 mole per cent and at pH 12.5 (pH of 1% protein solution in 0.IN NaOH), the respective values are 0.762, 0.780, and 2.62 mole per cent. (Note that the value of casein approaches that of gluten at this pH). The observed differences in lysinoalanine content of the three proteins at different pH values are not surprising since the amino acid composition, sequence, protein conformation, molecular weights of protein chains, initial formation of intra- versus intermolecular crosslinks may all influence the chemical reactivity of a particular protein with alkali. Therefore, it is not surprising to find differences in lysinoalanine content in different proteins treated under similar conditions. These observations could have practical benefits since, for example, the lower lysinoalanine content of casein compared to lactalbumin treated under the same conditions suggests that casein is preferable to lactalbumin in foods requiring alkali-treatment. 

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