Molecular characterization of alpha-lactalbumin folding variants that induce apoptosis in tumor cells

This study characterized a protein complex in human milk that induces apoptosis in tumor cells but spares healthy cells. The active fraction was purified from casein by anion exchange chromatography. Unlike other casein components the active fraction was retained by the ion exchanger and eluted after a high salt gradient. The active fraction showed N-terminal amino acid sequence identity with human milk alpha-lactalbumin and mass spectrometry ruled out post-translational modifications. Size exclusion chromatography resolved monomers and oligomers of alpha-lactalbumin that were characterized using UV absorbance, fluorescence, and circular dichroism spectroscopy. The high molecular weight oligomers were kinetically stable against dissociation into monomers and were found to have an essentially retained secondary structure but a less well organized tertiary structure. Comparison with native monomeric and molten globule alpha-lactalbumin showed that the active fraction contains oligomers of alpha-lactalbumin that have undergone a conformational switch toward a molten globule-like state. Oligomerization appears to conserve alpha-lactalbumin in a state with molten globule-like properties at physiological conditions. The results suggest differences in biological properties between folding variants of alpha-lactalbumin.     

Micellization of casein-graft-dextran copolymer prepared through Maillard reaction

Casein is almost insoluble at around pH 4.6, which is its isoelectric point (pI). Grafting copolymer, casein-g-dextran, was prepared through the Amadori rearrangement of the Maillard reaction. The copolymer has a reversible pH sensitive property: micellization at the pI of casein forming a casein core and dextran shell structure and dissociation when pH differs from the pI. The micelles produced at pH 4.6 have a spherical shape and their size is dependent on the Maillard reaction: reaction time, molar ratio of casein to dextran, and molecular weight of dextran used. Typically, the hydrodynamic diameter of the micelles is about 100 nm and the critical micelle concentration is about 10 mg/L. The micelles are very stable in aqueous solution and can be stored as lyophiled powder. The micelles are able to encapsulate hydrophobic compounds such as pyrene.     

Biocompatible micro-gel particles from cross-linked casein micelles

The stability of internally cross-linked casein micelles against disruption by urea (which disrupts hydrogen bonds and hydrophobic interactions) and trisodium citrate (which sequesters micellar calcium phosphate) was investigated. Addition of urea (0-6 mol L-1) and/or citrate (0-50 mmol L-1) progressively reduced the turbidity of a suspension of casein micelles cross-linked by transglutaminase and increased particle size (determined by dynamic and static light scattering and small-angle neutron scattering), which was attributed to swelling of the micelles. Furthermore, model calculations, assuming a completely stable casein network, were performed to describe the decreases in turbidity on addition of urea and citrate. Measured and described turbidity values are in agreement, indicating that cross-linking of casein micelles with transglutaminase results in a covalently bound protein network, which is entirely stable to disruption by urea and/or citrate. This may offer potential applications for the use of cross-linked casein micelles as biocompatible protein micro-gel particles.     

Studies on the properties of cow's-milk tributyrinases and their interaction with milk proteins

1. The tributyrinases in milk are mainly associated with casein micelles. Dilution or addition of sodium chloride increases the enzyme activity, probably by dissociating the micelle–tributyrinase complexes. 2. Tributyrinase activities of milks activated by dilution and sodium chloride addition were in the range 0·2–1·7μequiv. of acid liberated/ml. of milk/min. from tributyrin emulsion at pH8·5 and 25°. The enzymes have a bivalent-cation requirement for full activity and are rather unstable when separated from casein. 3. Ultracentrifugation of skim milks containing sodium chloride (0·75m) gave preparations low in casein but containing about 70% of the milk tributyrinases. The tributyrinases in such preparations appear to be bound in complexes of molecular weight about 350000. Dilution may result in dissociation to give the free enzymes. 4. Pancreatic lipase also formed complexes with casein micelles, but wheat-germ esterase, xanthine oxidase, milk alkaline phosphatase and other enzymes did not.     

Casein aggregates built step-by-step on charged polyelectrolyte film surfaces are calcium phosphate-cemented

The possible mechanism of casein aggregation and micelle buildup was studied in a new approach by letting α-casein adsorb from low concentration (0.1 mg·ml(-1)) solutions onto the charged surfaces of polyelectrolyte films. It was found that α-casein could adsorb onto both positively and negatively charged surfaces. However, only when its negative phosphoseryl clusters remained free, i.e. when it adsorbed onto a negative surface, could calcium phosphate (CaP) nanoclusters bind to the casein molecules. Once the CaP clusters were in place, step-by-step building of multilayered casein architectures became possible. The presence of CaP was essential; neither Ca(2+) nor phosphate could alone facilitate casein aggregation. Thus, it seems that CaP is the organizing motive in the casein micelle formation. Atomic force microscopy revealed that even a single adsorbed casein layer was composed of very small (in the range of tens of nanometers) spherical forms. The stiffness of the adsorbed casein layer largely increased in the presence of CaP. On this basis, we can imagine that casein micelles emerge according to the following scheme. The amphipathic casein monomers aggregate into oligomers via hydrophobic interactions even in the absence of CaP. Full scale, CaP-carrying micelles could materialize by interlocking these casein oligomers with CaP nanoclusters. Such a mechanism would not contradict former experimental results and could offer a synthesis between the submicelle and the block copolymer models of casein micelles.     

Isolation and properties of human kappa-casein

Human kappa-casein was isolated from human whole casein by gel filtration with Sephadex G-200 and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate (SDS). The kappa-casein was calcium-insensitive and did stabilize human beta-casein and bovine alpha s1-casein against precipitation by calcium ions. Formation of micelles from human beta- and kappa-caseins, and calcium ions was confirmed by electron microscopic observation. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a single band was obtained. The formation of para-kappa-caseins by chymosin was confirmed by SDS-PAGE. Two para-kappa-caseins with apparent molecular weights of 13,000 and 11,000 appeared. The molecular weight of intact human kappa-casein was estimated to be approximately 33,000. The human kappa-casein contained about 40% carbohydrate (15% galactose, 3% fucose, 15% hexosamines, and 5% sialic acid) and 0.10% (1 mol/mol) phosphorus. Its amino acid composition was similar to that of bovine kappa-casein except for serine, glutamic acid, and lysine contents.     

Reconstitution of Human Casein Micelle and Its Properties

Human casein micelles were reconstituted from isolated κ- and β-caseins and calcium ions. Micelle formation was recognized in the presence of calcium chloride even at the low concentration of 5mM. At pH levels ranging from 5.5 to 8.0, the re-formed micelles were quite stable so that precipitation of β-casein was not observed. The large micelles were constituted by a higher ratio of β-casein to κ-casein (16:1 by weight) than the small micelles (3: 1). The κ-casein in the small micelles contained carbohydrates to about 43% (w/w) in the molecule, whereas that in the large micelles was only about 25%. When the casein micelles were re-formed from κ-easein and fractionated β-casein components, the extent of phosphorylation of the β-casein component was found to influence the micelle formation; i.e., the β-casein component with no phosphate (the 0-P form) was disadvantageous to form micelles, but the component with 5 phosphates (the 5-P form) formed micelles most easily.     

Scattering and turbidity study of the dissociation of casein by calcium chelation

The dissociation of casein was studied after addition of polyphosphate that leads to calcium chelation, using light and X-ray scattering and turbidimetry. It is shown that the dissociation is a cooperative process; that is, a casein complex is either completely dissociated or remains largely intact. A systematic study was done of the dependence of the rate and extent of dissociation on the polyphosphate concentration and was found to be determined by the ratio between casein and polyphosphate. The structures of the casein complex and the small micellar particles formed after dissociation were compared. Additional experiments with a different chelatant (EDTA) gave similar results.     

Self-association of calcium and magnesium complexes of dentin phosphophoryn

Self-association of rat dentin phosphophoryn in the presence of calcium and magnesium ions was examined by chemical cross-linking and electron microscopy. Highly phosphorylated phosphophoryn (HP) binds a maximum of 1.33 calcium ions or 1.07 magnesium ions per organic phosphate residue at pH 7.4-8.0. The Ca-HP complexes are predominantly linear when the calcium content of the complex is less than about 65% of the saturation level. At higher calcium levels, the protein has a folded conformation, and transient protein-protein interactions occur. The equilibrium mixture of monomers and oligomers is predominantly monomeric unless the protein is saturated with calcium. The saturated Ca-HP complex forms discrete high molecular weight particles about 25 nm in diameter. The particles are electrically neutral and generally occur in clusters. Mg-HP complexes appear predominantly linear by electron microscopy at all concentrations of bound magnesium up to about 99% of the saturation level; however, protein-protein interaction is measurable when the magnesium content is as little as 65% of the saturation level. At saturation, Mg-HP complexes form high molecular weight particles which are negatively charged. Because of the negative charge, these particles form a stable colloidal suspension and have a rather stellate configuration.     

Water interactions with varying molecular states of bovine casein: 2H NMR relaxation studies

The caseins occur in milk as spherical colloidal complexes of protein and salts with an average diameter of 1200 A, the casein micelles. Removal of Ca2+ is thought to result in their dissociation into smaller protein complexes stabilized by hydrophobic interactions and called submicelles. Whether these submicelles actually occur within the micelles as discrete particles interconnected by calcium phosphate salt bridges has been the subject of much controversy. A variety of physical measurements have shown that casein micelles contain an inordinately high amount of trapped water (2 to 7 g H2O/g protein). With this in mind it was of interest to determine if NMR relaxation measurements could detect the presence of this trapped water within the micelles, and to evaluate whether it is a continuum with picosecond correlation times or is associated in part with discrete submicellar structures with nanosecond motions. For this purpose the variations in 2H NMR longitudinal and transverse relaxation rates of water with protein concentration were determined for bovine casein at various temperatures, under both submicellar and micellar conditions. D2O was used instead of H2O to eliminate cross-relaxation effects. From the protein concentration dependence of the relaxation rates, the second virial coefficient of the protein was obtained by nonlinear regression analysis. Using either an isotropic tumbling or an intermediate asymmetry model, degrees of hydration, v, and correlation times, tau c, were calculated for the caseins; from the latter parameter the Stokes radius, r, was obtained. Next, estimates of molecular weights were obtained from r and the partial specific volume. Values were in the range of those published from other methodologies for the submicelles. Temperature dependences of the hydration and Stokes radius of the casein submicelles were consistent with the hypothesis that hydrophobic interactions represent the predominant forces responsible for the aggregation leading to a submicellar structure. The same temperature dependence of r and v was found for casein under micellar conditions; here, the absolute values of both the Stokes radii and hydrations were significantly greater than those obtained under submicellar conditions, even though tau c values corresponding to the great size of the entire micelle would result in relaxation rates too fast to be observed by these NMR measurements. The existence of a substantial amount of trapped water within the casein micelle is, therefore, corroborated, and the concept that this water is in part associated with submicelles of nanosecond motion is supported by the results of this study.     

Mechanism for Increase in Intracellular Concentration of Free Calcium in Fertilized Sea Urchin Egg : A Method for Estimating Intracellular Concentration of Free Calcium

Intracellular free calcium concentration in the sea urchin egg was calculated to increase from 0.1 mM in an unfertilized egg to 1 mM in a fertilized egg 10 min after fertilization, based on measurement of the dissociation constant between free calcium and sea urchin egg homogenate. The dissociation constant between free calcium (dialyzable calcium) and homogenate of sea urchin eggs was measured by means of dialysis equilibrium. The dissociation constant of the unfertilized egg was about 10^–4 M and that of the fertilized egg was about 10–3 M in three species of sea urchin, Hemicentrotus pulcherrimus, Anthocidaris crassispina, and Pseudocentrotus depressus. An increase in the dissociation constant of the unfertilized egg homogenate was observed after the addition of calcium ion at a concentration above 0.3 mM, the dissociation constant becoming the same as that observed in the fertilized egg homogenate after the administration of CaCl₂ at a concentration above 1 mM. Sodium ion also caused a decrease in the calcium-binding ability of the unfertilized egg homogenate. Therefore, penetration of calcium ion or sodium ion upon fertilization might induce an increase in the dissociation constant and then intracellular concentration of free calcium would increase at fertilization. Almost all calcium-binding ability of the egg homogenate was found in the microsomal fraction, and the substance which bound calcium was thought to be protein in nature, since trypsin could decrease the level of calcium-binding substance in the homogenate of the eggs.     

Fractionation of spectrin by differential precipitation with calcium

The calcium-stimulated ATPase activity of spectrin can be substantially separated from the magnesium-stimulated activity by partial precipitation of spectrin with calcium. The precipitated Ca-ATPase fraction can be dissolved by dialysis against EDTA, and the resulting solution can be polymerized into characteristic spectrin fibrils. The supernatant contains Mg-ATPase and, either before or after dialysis, contains predominantly “torus” forms. The two fractions are essentially identical on gel electrophoresis, being composed primarily of the high molecular weight peptides with a constant level of minor bands. These results imply that the Ca-ATPase is associated firmly with the high molecular weight peptides.     

Properties of Acid Casein and Casein Micelles from Milk Destabilized by Frozen Storage

No difference was found in calcium sensitivity, electrophoretic and optical properties between acid caseins prepared from skimmilk before and after frozen storage (up to 180 days at ?7°C).

Destabilization of casein micelles can not be explained either by the reduction of solvation of the micelles or by the liberation of κ-casein from the micelles. However, when storage period was extended (about six months), splitting of a part of κ- and β-casein from the micelles to soluble form was observed, suggesting a drastic change of structure of the destabilized casein micelles.     

Fractionation by size of casein micelles on controlled-pore glass

Casein micelles have been separated from skim milk by chromatography on CPG-10 3000 glass beads. Fractionation of the micelles according to size has been demonstrated. Polyacrylamide gel electrophoresis of urea treated micelles reveals that different relative amounts of the major casein components occur in the various micelle fractions. No discernible dissociation of the micelles into monomeric caseins has been observed.     

Physicochemical characterization of casein phosphopeptide-amorphous calcium phosphate nanocomplexes

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.     

Caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate

Artificial casein micelles were prepared by adding 30 mM calcium, 22 mM phosphate and 10 mM citrate to sodium caseinate solutions, and the content of the casein aggregates cross-linked by colloidal calcium phosphate was determined by high-performance gel chromatography on a TSK-GEL G4000SW column in the presence of 6 M urea. The content of the casein aggregates cross-linked by colloidal calcium phosphate in artificial whole casein micelles was 48% of total casein, and their relative casein composition determined by high-performance ion-exchange chromatography was 53.1% for alpha s1-casein, 15.8% for alpha s2-casein, 31.1% for beta-casein and 0% for kappa-casein. The order of cross-linking by colloidal calcium phosphate agreed with that of the ester phosphate content of casein constituents. The content of the casein aggregates cross-linked by colloidal calcium phosphate was higher in alpha s1-kappa-casein micelles than in beta-kappa-casein micelles. kappa- and gamma-caseins and dephosphorylated alpha s1-casein were not cross-linked by colloidal calcium phosphate. Although kappa-casein was not cross-linked, chemically phosphorylated kappa-casein, of which the average phosphate content was 8.5 per molecule, was cross-linked. It is concluded that caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate.     

The minerals of milk

The salt of milk constitutes a small part of milk (8-9 g.L(-1)); this fraction contains calcium, magnesium, sodium and potassium for the main cations and inorganic phosphate, citrate and chloride for the main anions. In milk, these ions are more or less associated between themselves and with proteins. Depending on the type of ion, they are diffusible (cases of sodium, potassium and chloride) or partially associated with casein molecules (cases of calcium, magnesium, phosphate and citrate), to form large colloidal particles called casein micelles. Today, our knowledge and understanding concerning this fraction is relatively complete. In this review, the different models explaining (i) the nature and distribution of these minerals (especially calcium phosphate) in both fractions of milk and (ii) their behaviour in different physico-chemical conditions, are discussed.     

Synthesis of α-Adenosine 5′-Monophosphate

The distributions of protein, calcium and inorganic phosphate among casein micelles of skimmilk before and after frozen storage were investigated by gel filtration through Sephadex G-200 with 6.6 m urea solution as eluant.

The results showed that the native casein micelles were fractionated into two fractions. Fast eluting fraction contained large amount of calcium and inorganic phosphorus. Slow eluting fraction contained calcium but was essentially free of inorganic phosphorus. Frozen storage caused the increase of proportion of the fast eluting fraction accompanying the increase of calcium and inorganic phosphorus contents in it. It is suggested that the salt linkage newly formed during frozen storage causes the increase of proportion of the fast eluting fraction.     

Analysis of chromatin reconstitutiion.

The ability of high molecular weight chicken erythrocyte chromatin to spontaneously self-assemble into native-like material, after dissociation by high ionic strength and reassociation by salt gradient dialysis, was critically examined. The native conformational state of the reassembled nucleoprotein complex was regenerated to the extent reflected by circular dichroism spectra and thermally induced helix--coil transition of the nucleoprotein DNA. However, internucleosomal packing of approximately 205 base pairs of DNA per repeating unit, as probed by digestion with micrococcal nuclease, was not regenerated upon reassembly and was replaced by a packing of approximately 160 base pairs per repeating unit. Thus, high molecular weight chromatin containing only lysine-rich histones (H1 and H5) and core histones (H2A, H2B, H3, and H4) is not a true self-assembling system in vitro using the salt gradient dialysis system used herein. Circular dichroism and thermal denaturation studies on core chromatin (lysine-rich histones removed) showed that core histones alone are not capable of reassembling high molecular weight DNA into native-like core particles at low temperature (4 degree C). Reassembly at 21 degree C restored the circular dichroism but not the thermal denaturation properties to those characteristic of undissociated core chromatin. Nonetheless, micrococcal nuclease digestions of both reassembled core chromatin products were identical with undissociated native core chromatin. Ressembly in the presence of the complete complement of histones, followed by removal of the lysine-rich histones, did regenerate the thermal denaturation properties of undissociated native core particles. These results indicated multiple functions of the lysine-rich histones in the in vitro assembly of high molecular weight chromatin.     

Sedimentation Property of Casein Micelles from Milk Destabilized by Frozen Storage

Casein micelles destabilized in milk under frozen storage was compared with stable casein micelles before frozen storage by means of analytical ultracentrifugation.

The stable micelles disaggregated with urea-containing buffer showed a single homogeneous peak of ~0.9S, while a fast sedimenting subpeak, in addition to the major peak of ~0.9 S, appeared in the destabilized micelles. However, any difference was not found between the stable and destabilized caseins when they were analyzed after removal of calcium. It is suggested that a new type of association is formed, possibly through salt linkages, in the casein system destabilized under frozen storage. Sedimentation pattern of calcium paracaseinate phosphate complex in urea-containing buffer suggests that the destabilization of the micelles by rennin does not involve the change of salt linkages.