Structural,histochemical and biochemical observations on horse milk-fat-globule membranes and casein micelles

Summary Horse milk fat glubules (MFGs) and casein micelles were studied using freeze fracturing, freeze etching and thin-section electron microscopy, as well as lectin histochemistry, gel electrophoresis, and Western blotting. Horse MFGs were found to be relatively small, their average volume-surface diameter being about 2.75 m. The MFG membrane is composed of three layers: an inner proteinaceous coat occasionally having a paracrystalline substructure, a unit membrane, and a prominent filamentous glycocalyx. The last is rich in glycoconjugates, as revealted by its binding of various lectins. In addition, the glycocalyx binds cationized ferritin, which indicates the presence of negative electric charges. Gel electrophoresis revealed the presence of high-molecular-weight glycoproteins in the MFG membrane of horse milk. Such glycoproteins are also present in human MFG membranes but are absent in the bovine MFGs. The casein micelles in horse milk are relatively large, their average volume-surface diameter being about 200 nm.Dedicated to Professor Dr. T. H. Schiebler on the occasion of his 65th birthdayS. Patton gratefully acknowledges the generous support of the Alexander von Humboldt Foundation for his research in scientific institutions in the Federal Republic of Germany     

Location of glycoproteins on milk fat globule membrane by scanning and transmission electron microscopy, using lectin-labelled gold granules

Membrane glycoproteins of bovine and human milk fat globules (MFG) were located by scanning electron microscopy using lectin-labelled gold granules (50 nm diameter) as specific markers. Receptors for wheat germ agglutinin (WGA) and soybean lectin (SBA) were localized in clusters over the whole MFG surface. When MFG were treated with neuraminidase, the density of marking with SBA increased. Marking of MFG with Concanavalin A (ConA) was weak. No marking was obtained with lectins specific for -fucose, -galactose and α- -galactose. When thin sections of MFG marked with WGA (18 nm diameter gold granules) were examined by transmission electron microscopy, the membrane was uniformly marked. Using markers of different sizes (5 and 18 nm diam.) prefixed milk fat globule membranes (MFGM) were simultaneously marked with WGA and SBA. The lectin receptors appeared to belong to different glycoproteins which were clustered. Thin sections of this material showed that the receptors were located on one side of the membrane. No difference was observed between bovine MFG and human MFG from donors having blood group O and A. All results indicated that MFGM is a true biological membrane.     

Relationships between milks differentiated on native milk fat globule characteristics and fat,protein and calcium compositions

Many studies have shown that milk fat globule (MFG) diameter varies in dairy cows in relation to diet and/or breed. However, the mechanisms governing the size of the fat globules remain hypothetical. Our objective was to determine the variable biochemical characteristics (fat, protein, fatty acids (FA), casein and calcium (Ca) contents) between individual milk which differed in both MFG diameter and membrane content, in order to speculate about the links between milk synthesis and MFG secretion. With this aim, we built five databases of individual milk samples from 21 experiments performed between 2003 and 2011. Three of them grouped data from trials dealing with breed/diet effects and included information about: (i) MFG size/membrane, fat and protein contents (n=982), (ii) previous parameters plus FA profile (n=529) and (iii) previous parameters plus true protein composition and calcium contents (n=101). A hierarchical clustering analysis performed on these three databases yielded four groups differing in the MFG characteristics. We observed significant differences among groups for the following parameters: (i) fat content and fat : protein ratio; (ii) de novo and polyunsaturated FA contents; (iii) Ca contents. These relationships could result from potential process regulating the synthesis and secretion of MFG: (i) the apical membrane turnover for MFG secretion and (ii) cytoplasmic lipid droplet formation in the lactocyte during its migration from the basal to the apical pole. The two other databases grouped data from trials dealing with milking frequency (n=211), milking kinetics and milk type (residual v. cisternal) (n=224). They were used to study the relationships between the size of the MFG and milk composition for high native fat contents (from 60 up to 100 g/kg in residual milks). We observed curvilinear relationships between the size of the MFG and fat content, as well as with the fat : protein ratio. This result suggests that MFG diameter reaches a threshold but mechanisms are still unknown.     

Rat casein: isolation and characterization of two major fractions.

1. In rat milk, casein exists as particles of 77 nm mean diameter, similar in appearance to the casein micelles in the milk of other species. 2. The heterogeneity of rat casein was investigated by polyacrylamide gel electrophoresis and by chromatography on DEAE-cellulose, both in the presence of 8 M-urea. 3. The chromatography yielded two fractions, D2 and D4. 4. In chemical composition, D2 resembles the kappa-caseins of other species, while D4 resembles the alpha 5-caseins of other species. 5. D2 is soluble in 80 mM-CaCl2 at 37 degrees C, but D4 is insoluble under these conditions. 6. In 80 mM-CaCl2 at 37 degrees C, D2 prevents the precipitation of D4 by the formation of particles similar to those found in rat milk.     

Composition and ultrastructure of calcium phosphate-citrate complexes in bovine milk systems

The present studies show that the colloidal calcium phosphate of cow's milk has a (Ca + Mg)/P_i ratio of 1.67 (± 0.10; n = 22) and contains citrate, Mg and Zn at molar ratios to Ca averaging 0.05, 0.03 and 0.003, respectively. The composition of the natural colloidal phosphate of milk is similar to the precipitates formed by neutralization of ultrafiltrates obtained from acidified milks, and to that of the calcium phosphate-enriched fraction produced by extensive enzymic hydrolysis of the casein micelles in milk. Examination by electron microscopy of these artificial preparations of milk calcium phosphate revealed in both a very fine and uniform substructure which consisted of granules having an average, true diameter of approx. 2.5 nm. The size and shape of these tiny granules closely resemble the morphologies reported for the colloidal phosphate particles in native casein micelles, as well as for the subunits of amorphous calcium phosphate observed during calcification in other biological systems such as mitochondria and bone.     

Interaction of lactoferrin and lysozyme with casein micelles

On addition of lactoferrin (LF) to skim milk, the turbidity decreases. The basic protein binds to the caseins in the casein micelles, which is then followed by a (partial) disintegration of the casein micelles. The amount of LF initially binding to casein micelles follows a Langmuir adsorption isotherm. The kinetics of the binding of LF could be described by first-order kinetics and similarly the disintegration kinetics. The disintegration was, however, about 10 times slower than the initial adsorption, which allowed investigating both phenomena. Kinetic data were also obtained from turbidity measurements, and all data could be described with one equation. The disintegration of the casein micelles was further characterized by an activation energy of 52 kJ/mol. The initial increase in hydrodynamic size of the casein micelles could be accounted for by assuming that it would go as the cube root of the mass using the adsorption and disintegration kinetics as determined from gel electrophoresis. The results show that LF binds to casein micelles and that subsequently the casein micelles partly disintegrate. All micelles behave in a similar manner as average particle size decreases. Lysozyme also bound to the casein micelles, and this binding followed a Langmuir adsorption isotherm. However, lysozyme did not cause the disintegration of the casein micelles.     

Differences between individuals in high-molecular weight glycoproteins from mammary epithelia of several species

Milk fat globules are secreted by envelopment in plasma membrane of the lactating cell. SDS-gel electrophoresis of proteins from this membrane has revealed differences between milk donors in two mucin-like glycoproteins. One of these glycoproteins resolves in 3% acrylamide stacking gel and the other in 4% running gel. The proteins vary in number of bands (one or two) and band mobilities. This polymorphism arises, at least in part, from expression of hypervariable genes. In this study, gel electrophoretic evidence of similar polymorphism in glycoproteins from cow, chimpanzee, horse and human milks is presented. In distinction to the other species, the cow expressed only one of these proteins which was detected in the running gel at Mr 180,000 to 200,000. The electrophoresis pattern for this protein from six cows was highly varied with respect to number (one or two) and position of bands. Peanut agglutinin, wheat germ agglutinin and concanavalin A all were bound specifically by bands of the bovine glycoprotein. Binding of concanavalin A distinguishes the bovine protein from the two human glycoproteins. Further studies of species differences should help shed light on the evolution of these unique glycoproteins and their possible functions in mother and young.     

A Novel Method for Separation of Caseins from Milk by Phosphates Precipitation

Milk protein of farm animals is difficult to isolate because of the presence of casein micelles, which are hard to separate from whey by using centrifugation or filtration. Insoluble casein micelles also create an obstacle for purification instruments to operate efficiently. The conventional method, to precipitate caseins by lowering pH to 4.6 and then recover the whey fraction for further purification using chromatography techniques, is not applicable to proteins having an isoelectric point similar to caseins. In addition, the acid condition used for casein removal usually leads to significantly poor yields and reduced biological activities. In this study, a novel method of precipitating caseins under neutral or weak acidic conditions is presented. The method employs a phosphate salt and a freeze–thaw procedure to obtain a casein-free whey protein fraction. This fraction contains more than 90% yield with little loss of bioactivity of the target protein, and is readily available for further chromatographic purification. This method was successfully applied to purify recombinant human factor IX and recombinant hirudin from the milk of transgenic pigs in the presented study. It is an efficient pretreatment approach prior to chromatographic purification of milk protein from farm animals and particularly of great value to collect those recombinants secreted from transgenic livestock.     

Characterization of a secretory vesicle-rich fraction from lactating bovine mammary gland.

Fractions enriched in secretory vesicles were obtained from lactating bovine mammary tissue by a straightforward procedure involving gentle homogenization and centrifugation in isotonic milk salt solution containing Ficoll. Secretory vesicle-rich fractions could also be obtained from lactating rat mammary gland by this procedure. With rats, yields of vesicles were substantially increased by administration of colchicine or thioglucose to animals several hours before sacrifice. Isolated fractions were enriched in lactose and consisted predominantly of 0.2–1.2 μm diameter vesicles, many of which contained casein micelles. Enzymatic, compositional and morphological examination revealed vesicle preparations to be largely free of contamination by rough endoplasmic reticulum, mitochondria, nuclei, peroxisomes and lysosomes. Specific activity of several marker enzymes of the secretory vesicle fraction were similar to, or intermediate between, Golgi apparatus and milk lipid globule membranes. Amounts of cholesterol and gangliosides in vesicle fractions approached levels found in plasma membranes. In distribution of major phospholipids, secretory vesicles were intermediate between Golgi apparatus and milk lipid globule membranes. The pattern of polypeptides of secretory vesicle membrane was qualitatively similar to that of Golgi apparatus membranes. While there were similarities between these polypeptide patterns and that of lipid globule membranes, the latter contained relatively more of certain polypeptides, particularly the internal coat-associated polypeptides of the globule membrane. These observations are discussed in relation to the endomembrane hypothesis and the origin of the membrane of milk lipid globules.     

Enrichment of n-Alkane Assimilation-Deficient Mutants of Candida Yeast by Synergistic Effect of Nystatin and Pyrrolnitrin

In order to clarify further the relationship between the heat stability of casein micelles and the formation of soluble casein upon heating concentrated milk, the effect of formaldehyde was examined. The addition of formaldehyde up to 20 mM markedly increased the heat stability of both concentrated skim milk and concentrated whey protein-free (WPF) milk. The stabilizing effect of formaldehyde was greater for concentrated skim milk than for concentrated WPF milk. The addition of formaldehyde depressed the formation of soluble casein upon heating concentrated milk. No soluble casein was formed on the addition of 20 mM formaldehyde. It was confirmed by Sephadex G-200 gel filtration in the presence of 6.6 M urea that cross-links among the casein components were formed in heated concentrated WPF milk containing formaldehyde. These facts suggest that formaldehyde may introduce cross-links among the casein components and prevent the formation of soluble casein accompanying the release of K-casein from micelles, thus stabilizing the casein micelles.     

Electrosorption of pectin onto casein micelles

Pectin, a polysaccharide derived from plant cells of fruit, is commonly used as stabilizer in acidified milk drinks. To gain a better understanding of the way that pectin stabilizes these drinks, we studied the adsorption and layer thickness of pectin on casein micelles in skim milk dispersions. Dynamic light scattering was used to measure the layer thickness of adsorbed pectin onto casein micelles in situ during acidification. The results indicate that the adsorption of pectin onto casein micelles is multilayered and takes place at and below pH 5.0. Renneting, i.e., cleaving-off kappa-casein from the casein micelles, did not alter the adsorption pH. It did, however, show that pectin arrests the rennet-induced flocculation of casein micelles below pH 5.0. From the findings we concluded the attachment of pectin onto casein micelles is driven by electrosorption. Adsorption measurements confirmed the multilayered nature of the adsorption of pectin onto casein micelles. Both the adsorbed amount and the layer thickness increased with decreasing pH in the relevant range 3.5-5.0. The phase behavior of a casein micelles/pectin mixture was determined and could be explained in terms of thermodynamic incompatibility being relevant above pH 5.0 and adsorption, leading to either stabilization and bridging, being relevant below pH 5.0. The results confirm that electrosorption is the driving force for the adsorption of pectin onto casein micelles.     

Alpha casein micelles show not only molecular chaperone-like aggregation inhibition properties but also protein refolding activity from the denatured state

Casein micelles are a major component of milk proteins. It is well known that casein micelles show chaperone-like activity such as inhibition of protein aggregation and stabilization of proteins. In this study, it was revealed that casein micelles also possess a high refolding activity for denatured proteins. A buffer containing caseins exhibited higher refolding activity for denatured bovine carbonic anhydrase than buffers including other proteins. In particular, a buffer containing α-casein showed about a twofold higher refolding activity compared with absence of α-casein. Casein properties of surface hydrophobicity, a flexible structure and assembly formation are thought to contribute to this high refolding activity. Our results indicate that casein micelles stabilize milk proteins by both chaperone-like activity and refolding properties.     

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.     

High pressure-induced changes in bovine milk proteins: a review

High pressure (HP)-induced changes in the proteins of bovine milk have become an area of considerable research interest in recent years; as a result, there is now a detailed understanding of the effects of HP on casein micelles and whey proteins. HP treatment at pressures >400 or >100 MPa denatures the two most abundant whey proteins, alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg), respectively. The majority of denatured beta-lg in HP-treated milk associates with the casein micelles, although some denatured beta-lg remains in the serum phase or is attached to the milk fat globule membrane; HP-denatured alpha-la is also associated with the milk fat globules. Casein micelles are disrupted on treatment at pressures >200 MPa; the rate and extent of micellar disruption increases with pressure and is probably due to the increased solubility of calcium phosphate with increasing pressure. On prolonged treatment at 250-300 MPa, reassociation of micellar fragments occurs through hydrophobic bonding; this process does not occur at a pressure >300 MPa, leading to considerably smaller micelles in such milk. As a result of HP-induced changes, the size, number, hydration, composition and light-scattering properties of casein micelles in HP-treated milk differ considerably from those in untreated milk.     

Genetic diversity in German draught horse breeds compared with a group of primitive, riding and wild horses by means of microsatellite DNA markers

We compared the genetic diversity and distance among six German draught horse breeds to wild (Przewalski's Horse), primitive (Icelandic Horse, Sorraia Horse, Exmoor Pony) or riding horse breeds (Hanoverian Warmblood, Arabian) by means of genotypic information from 30 microsatellite loci. The draught horse breeds included the South German Coldblood, Rhenish German Draught Horse, Mecklenburg Coldblood, Saxon Thuringa Coldblood, Black Forest Horse and Schleswig Draught Horse. Despite large differences in population sizes, the average observed heterozygosity (H(o)) differed little among the heavy horse breeds (0.64-0.71), but was considerably lower than in the Hanoverian Warmblood or Icelandic Horse population. The mean number of alleles (N(A)) decreased more markedly with declining population sizes of German draught horse breeds (5.2-6.3) but did not reach the values of Hanoverian Warmblood (N(A) = 6.7). The coefficient of differentiation among the heavy horse breeds showed 11.6% of the diversity between the heavy horse breeds, as opposed to 21.2% between the other horse populations. The differentiation test revealed highly significant genetic differences among all draught horse breeds except the Mecklenburg and Saxon Thuringa Coldbloods. The Schleswig Draught Horse was the most distinct draught horse breed. In conclusion, the study demonstrated a clear distinction among the German draught horse breeds and even among breeds with a very short history of divergence like Rhenish German Draught Horse and its East German subpopulations Mecklenburg and Saxon Thuringa Coldblood.     

Crosslinking of casein by microbial transglutaminase and its resulting influence on the stability of micelle structure

The influence of enzymatic crosslinking by microbial transglutaminase (mTG) on the stability of casein micelles of ultrahigh temperature (UHT)-treated milk in the presence of EDTA (0-0.45 mM) or ethanol (0-74 vol%) as well as under high hydrostatic pressures up to 400 MPa was investigated. Disintegration of micelles and changes in micelle size were monitored by the measurement of turbidity as well as by dynamic light scattering. The results show that the incubation of UHTtreated milk with mTG resulted in an improved micelle stability toward disintegration on addition of EDTA, ethanol, or pressure treatment. Intramicellar formed isopetides significantly enhanced the stability of casein micelles. It is supposed that net-like crosslinks are formed within the external region of the micelles and they adopt the stabilizing role of colloidal calcium phosphate within the micelles, thus making the micelles less contestable for disrupting influences.     

Topo-optical investigations of the human erythrocyte glycocalyx-age related changes

Summary The conformational state of the glycocalyx of the intact and altered erythrocyte membrane was studied by means of the topo-optical toluidine blue reaction, i.e. induced membrane birefringence. High membrane anisotropy represents the normal glycocalyx structure and its decline represents their perturbation. The results show that the glycocalyx structure is changed during ageing of the erythrocytes in vivo as well as in vitro. During fluid preservation, in vitro ageing and vesiculation of cells in vitro, a subpopulation of cells showed a decline of membrane anisotropy, but other cells demonstrated abnormally high values. In the latter cases, there is usually a correlation to spherocytes. From this point of view, it is to be assumed that spherogenesis during cell ageing is induced by cell vesiculation. This leads to a remodelling of an intact plasmalemma. In contrast, the cell fractions which are probably non-vesiculating seem to be more or less damaged by membrane and/or plasmic hydrolases. This can be mimicked by neuraminidase and protease treatment of erythrocytes in vitro. Membrane lesions caused by freeze preservation of red blood cells are rare. The topo-optical results are interpreted according to the assumptions of the theory of membrane anisotropy, i.e. the formation of dye-stuff micelles at distinct, clustered, sialylated carbohydrate chains of the gluycophorin A.     

A lactational study of the composition and integrity of casein micelles from the milk of the tammar wallaby (Macropus eugenii)

The amount of casein found in the milk of the tammar wallaby increases as lactation progresses. The increase is due to increasing amounts of β-casein; the α-casein remains largely constant. The α-casein is the more highly phosphorylated; the most abundant form is the 10-P, throughout lactation. The level of phosphorylation of β-casein shifts to lower average values in late lactation, possibly indicating the enzymatic reaction is overloaded by the increasing amounts of β-casein. Unlike bovine casein micelles, the wallaby micelles are not completely disrupted at pH 7.0 by sequestration of their calcium content with ethylene diamine tetraacetic acid (EDTA). Complete disruption only follows the addition of sodium dodecyl sulphate, indicating considerably greater importance for hydrophobic bonds in maintaining their integrity. This micellar behaviour indicates that, despite the evolutionary divergence of marsupials millennia ago, the caseins of wallaby milk assemble into micelles in much the same fashion as in bovine milk.     

Filter-aided sample preparation with dimethyl labeling to identify and quantify milk fat globule membrane proteins

Bovine milk is a major nutrient source in many countries and it is produced at an industrial scale. Milk is a complex mixture of proteins, fats, carbohydrates, vitamins and minerals. The composition of the bovine milk samples can vary depending on the genetic makeup of the bovine species as well as environmental factors. It is therefore important to study the qualitative and quantitative differences of bovine milk samples. Proteins in milk can be present in casein micelles, in the serum (the water soluble fraction) or in fat globules. These fat globules have a double membrane layer with proteins being bound to or being incapsulated in the membrane layer. The identification and molecular composition of the milk proteins have gained increased interest in recent years. Proteomic techniques make it now possible to identify up to many thousands of proteins in one sample, however quantification of proteins is as yet not straightforward. We analyzed the proteins of the milk fat globule membrane using dimethyl labeling methods combined with a filter-aided sample preparation protocol. Using these methods, it is now possible to quantitatively study the detailed protein composition of many milk samples in a short period of time.