Preparation and comparison of cytotoxic complexes formed between oleic acid and either bovine or human α-lactalbumin.

α-Lactalbumin is a ubiquitous calcium-binding milk protein with a well-characterized function in regulating the synthesis of lactose. An entirely different activity has been shown to occur when a complex is formed between calcium-free α-lactalbumin and oleic acid. This complex shows strong cytotoxic action against several cancer cells, and several mechanisms have been suggested to account for this cell-killing activity. Most studies have been performed using the human protein, but bovine α-lactalbumin shows similar activity. A new and simple 2-step method for purification of calcium-free α-lactalbumin has been developed, and the resulting highly purified preparation was used to generate a complex with oleic acid. Using 3 different cell lines and 2 types of cell viability assays, the bovine and human α-lactalbumin showed comparable cytotoxic activity. The effect was apparent after 15 min of incubation and was inhibited by the presence of fetal bovine serum or bovine serum albumin. The bovine protein might be a useful alternative to the human protein, but also raises the question whether cytotoxic activity could be generated in different kinds of food containing α-lactalbumin.

Heat-treatment method for producing fatty acid-bound alpha-lactalbumin that induces tumor cell death.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), which was identified in human breast milk as an alpha-lactalbumin (LA)-oleic acid complex, kills tumor cells, selectively. Although it may have potential as a therapeutic agent against various tumor cells, only low-volume methods for its production exist. In this study, heat treatment was used to produce complexes from LAs and oleic acid using a simple method. In the case of human LA and oleic acid, heat-treated samples apparently showed much stronger activities than those treated at room temperature, with cytotoxicities equal to that of HAMLET. Furthermore, circular dichroism spectroscopy revealed that heat-treated samples lost their tertiary structure, suggesting a molten globule as oleic acid-bound LA. BLA samples also showed strong activities by heat treatment. Batch production with heat treatment can efficiently convert LAs into tumoricidal complexes.

A comparative study of peptide models of the alpha-domain of alpha-lactalbumin, lysozyme, and alpha-lactalbumin/lysozyme chimeras allows the elucidation of critical factors that contribute to the ability to form stable partially folded states.

alpha-Lactalbumin (alpha LA) forms a well-populated equilibrium molten globule state, while the homologous protein hen lysozyme does not. alpha LA is a two-domain protein and the alpha-domain is more structured in the molten globule state than is the beta-domain. Peptide models derived from the alpha-subdomain that contain the A, B, D, and 3(10) helices of alpha LA are capable of forming a molten globule state in the absence of the remainder of the protein. Here we report comparative studies of a peptide model derived from the same region of hen lysozyme and a set of chimeric alpha-lactalbumin--lysozyme constructs. Circular dichroism, dynamic light scattering, sedimentation equilibrium, and fluorescence experiments indicate that the lysozyme construct does not fold. Chimeric constructs were prepared to probe the origins of the difference in the ability of the two isolated subdomains to fold. The first consists of the A and B helices of alpha LA cross-linked to the D and C-terminal 3(10) helices of lysozyme. This construct is highly helical, while a second construct that contains the A and B helices of lysozyme cross-linked to the D and 3(10) helices of alpha LA does not fold. Furthermore, the disulfide cross-linked homodimer of the alpha LA AB peptide is helical, while the homodimer of the lysozyme AB peptide is unstructured. Thus, the AB helix region of alpha LA appears to have an intrinsic ability to form structure as long as some relatively nonspecific interactions can be made with other regions of the protein. Our studies show that the A and B helices plays a key role in the ability of the respective alpha-subdomains to fold.

Folate reception by vegetative Dictyostelium discoideum amoebae: distribution of receptors and trafficking of ligand.

We report the first explicit demonstration of post-reception processing of a Dictyostelium chemoattractant. Folic acid stimulates reorganization of the cytoskeleton of vegetative amoebae of D. discoideum. In particular, folic acid is a potent chemoattractant and it causes enlargement of the filopodial array. The distribution of folic acid receptors and the fate of bound folate were investigated by presenting an agonist consisting of the conjugate, folic acid-lactalbumin-FITC (Folate*), to these amoebae. This novel probe was specifically bound to folic acid receptors of these amoebae and it stimulated chemotaxis and enlargement of their filopodial array. Hence, Folate* is a physiologically competent probe. The probe sans-folate moiety was not bound anywhere to living or fixed amoebae. Since Folate* did not bind to amoebae after incubation with equimolar folic acid, this probe is a receptor-specific agonist. We report here the first description, by confocal visualization of a competent agonist, of the distribution of folate receptors of D. discoideum vegetative amoebae and of the fate of this ligand. Examination of fixed amoebae revealed that bound Folate* was distributed generally over their entire surface including their filopodia. However, in living amoebae, Folate* was bound only at the cell body and this bound Folate* was almost completely internalized as concentrated packets into vacuoles. This endocytosis of the probe and the clustering of endocytosed Folate* is consistent with receptor-mediated internalization of a ligand. Possible routes for internalization of the folate probe and the implications of this endocytosis for signal molecule processing and temporal sensing are discussed.