Lactose synthase activity

A necessary, but insufficient, property for a protein to be an a-lactalbumin is its ability to act as a specifier in the lactose synthase system. There is, at present, controversy as to whether a true a-lactalbumin occurs in the milk of monotremes. Thus, it is essential to have good methods for the determination of galactosyltransferase and lactose synthase activities. They must enable the detection of low levels of activity. This is... 

Hopper and McKenzie (1974), in their study of the possible ability of echidna lysozyme to act as specifier protein for the production of weak lactose synthase activity, found that the conditions of the determination needed modification for these purposes. A preliminary study was made by H. A. McKenzie and V. Muller (unpublished observations) of optimum conditions for such determinations. However, it was evident that much further work was necessary. The effect of lipids on gtdactosyl-transferase activity has been studied by Mitranic and Moscarello (1980). 

D. Structural Changes on Cation Binding by a-Lactalbumin and Their Implications in Lactose Synthase Activity... 

At the same time the precise delineation of the calcium binding site, discussed in Section VII (see also Table IX and Fig. 8), naturally led to the consideration of the minimum number of these residues that must be present and the relevant conformation of the peptide chain to enable calcium binding to occur. Questions that arise immediately are Can any lysozyme bind Ca(II) in a comparable manner To what extent can lysozymes exhibit weak lactose synthase activity and a-lactalbumins exhibit weak lytic activity ... 

Purification of the membrane-bound lactose carrier protein is a very different problem from the purification of the soluble OMP synthase. Both the approach to purification and the assays for the protein during purification are quite novel. The assay involves reconstituting a transport system with membranes that are free of lactose carrier protein, then adding the partially purified carrier protein and radioactively labeled lactose. The activity in this assay system is proportional to the transport of radioactive lactose across the membrane in the cell-free reconstituted system. 

Recently, the molten globule state of a-lactalbumin has been shown to possess antitumor activity when complexed with a fatty acid [36,37], and hence the protein may possess secondary biological activity in addition to the primary activity of native a-lactalbumin, i.e., substrate specificity modifier activity in a lactose synthase system [38,39]. The molten globule of a-lactalbumin thus provides an example of the folding intermediate of a protein exhibiting a secondary biological activity. 

We open with a brief report of the early discovery of the occurrence and isolation of these proteins and the elucidation of their function and homology, followed by a brief discussion of some problems in their isolation and the determination of their activity. We then consider various aspects of their three-dimensional structures and their significance. We summarize studies on the implications of their sequence similarities, and also on the binding of metal ions, especially calcium(H), and consider their implications. Then follows a brief discussion of lactose synthase, an enzyme of which a-lactalbumin and galactosyltransferase are essential components. We then examine the evidence concerning the evolution of the two proteins, about which there are conflicting views (see Section X,B). Some conclusions and predictions of future directions are made. 

The substrate specificity of the lactose synthase system was studied further by Brew et al. (1968). They confirmed that neither A protein nor B protein alone was active for the synthesis of lactose, but the A protein catalyzed the following reaction (see also Eig. 2) ... 

Fig. 2. Reactions catalyzed by galactosyltransferase (GT). (a) The incorporation of galactose (Gal) into a (1—>4) linkage with JV-acelylglucosamine (NAG) to form N-acetyl-lactosamine (NAL). UDP, Uridine diphosphate, (b) Modification of the activity of GT by a-lactalbumin (a-LA) to convert it to a lactose synthase catalyzing the formation of lactose from UDP-Gal and glucose.

While known sequencing methods may enable the sequence or partial sequence to be established for an impure protein [e.g., cow milk lysozyme (White et al., 1988)], protein of high purity is required for other purposes (e.g., determination of trace lytic activity in an isolated a-lactalbumin or trace lactose synthase specifier activity in a lysozyme). Otherwise, erroneous conclusions may be drawn with respect to structure and function and their evolutionary relationships. 

Musci and Berliner (1985b) concluded that apo-a-lactalbumin is more efficient as the modifier protein in the lactose synthase system than is the Ca(II)-bound form. They found that Vniax for the apo form shows a 3.5-fold increase over that for the Ca(II)-bound form, but there is no difference in (app.) between the two forms. They also confirmed that calcium stabilizes the protein against thermal denaturation (see Section IX,E), but that zinc is crucial in shifting the protein toward the apo-like form that is optimally active in lactose synthase. Their model is summarized schematically in Fig. 9. 

Many years ago Hopper and McKenzie (1974) noted structural similarities between equine and echidna lysozymes. They also obtained some evidence, albeit controversial, of a weak ability of echidna lysozyme to act as a modifier in the lactose synthase system. More recently, McKenzie and White (1987) noted very weak lytic activity in a variety of a-lactalbumin preparations. Also, Teahan et al. (1986, 1990) confirmed certain essential structural features for Ca(II) binding in echidna lysozymes I and II and noted the potential binding of Ca(II) by equine and pigeon lysozymes. D. C. Shaw and R. Tellam (quoted by Godovac-Zimmermann et al., 1987) made preliminary fluorometric observations that indicated binding of Ca(II) by echidna and equine lysozymes. 

Much has been accomplished, especially in recent years, toward the goal of elucidating the active sites of galactosyltransferase and a-lactal-bumin. To this end, alteration of specific residues with observations of consequent effects on structure and activity is enlightening, as are metal ion effects. Where the substrate is concerned, we now have some detailed structural information for both galactosyltransferase and the lactose synthase system. 

It has been conventional wisdom that lysozyme is not active in the lactose synthase system and that a-lactalbumin does not have lytic activity. The essential residues for interaction of specifier protein with ga-lactosyltransferase have not yet been unequivocably defined, nor has the role of Ca(II) in this system. Thus, it is not, at present, possible to rule out weak specifier activity for lysozyme in the lactose synthase system. 

UDP-A-acetyl-5-thio-D-galactosamine (UDP-5.SGalNAc) was active as a donor substrate of lactose synthase, the complex of galactosyltransferase (EC 2.4.1.38) and lactalbumin. By tliis method the disaccharide /9-5SGalNAc/9(1 4)GlcNAc was prepared. UDP-5.S GalNAc was synthesized from an A -acetyIgalactosaininc... 

This modified enzyme is lactose synthase. Synthesis of ot-lactalbumin is activated hormonally in mothers shortly after giving birth. The ot-lactalbumin protein combines with preexisting galactosyltransferase, changes its specificity, and activates the large amount of lactose synthesis needed for milk production. 

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