Isolation and Physicochemical Properties of a Soluble Glycoprotein Fraction of Milk Fat Globule Membrane

A soluble apoprotein fraction was prepared from milk fat globule membrane lipoproteins by delipidation with a chloroform-methanol mixture and was fractionated into three fractions by gel filtration on Bio-Gel A–5m.

The major fraction, Fraction II, contained about 30% of carbohydrate, i.e. 13.9% of hexoses, 8.1% of hexosamines, 8.0% of sialic acid and 0.8% of fucose, and was therefore designated a soluble glycoprotein fraction. The fraction was apparently homogeneous on sedimentation velocity analysis and DEAE-Sephadex chromatography, and had 6.1, 3.79, 0.719, f/f⁰ 2.16 and molecular weight 139,000 daltons. However, the diffused pattern on disc electrophoresis and the occurrence of plural N-terminal amino acid residues suggest that the protein of this fraction is likely to be formed by intermolecular association of heterogeneous polypeptide chains.     

Milk Fat Content and DGAT1 Genotype Determine Lipid Composition of the Milk Fat Globule Membrane

During secretion of milk fat globules, triacylglycerol (TAG) droplets are enveloped by a phospholipid (PL) trilayer. Globule size has been found to be related to polar lipid composition and fat content, and milk fat content and fatty acid composition have been associated with the diacylglycerol acyltransferase 1 (DGAT1) K232A polymorphism; however, the association between the DGAT1 polymorphism and fat globule size and polar lipid composition has not been studied. The ratio between polar and neutral lipids as well as the composition of the polar lipids in milk has industrial as well as nutritional and health implications. Understanding phenotypic and genotypic factors influencing these parameters could contribute to improving milk lipid composition for dairy products. The focus of the present study was to determine the effect of both fat content and DGAT1 polymorphism on PL/TAG ratio, as a marker for milk fat globule size, and detailed PL composition. Milk samples were selected from 200 cows such that there were equal numbers of samples for the different fat contents as well as per DGAT1 genotype. Samples were analyzed for neutral and polar lipid concentration and composition. PL/TAG ratio was significantly associated with both fat content and DGAT1 genotype. Phosphatidylinositol and phosphatidylserine concentrations were associated with fat content*DGAT1 genotype with a stronger association for the AA than the KK genotype. Sphingomyelin concentration tended to interact with fat content*DGAT1 genotype. Phosphatidylethanolamine (PE) concentration showed a biphasic response to fat content, suggesting that multiple biological processes influence its concentration. These results provide a new direction for controlling polar lipid concentration and composition in milk through selective breeding of cows.     

Milk Fat Globule Membrane Proteins in Aseptically Packed Ultra-heat-treated Milk: Changes during Storage

Five genes for tryptophan biosynthesis, trpE, trpD, trpC, trpB, and trpA of Brevibacterium lactofermentum, a coryne form glutamic acid-producing bacterium, were cloned as a 9.6 kb BamHl DNA fragment by colony hybridization. A previously cloned 1.2 kb Pst I DNA fragment containing a major part of the trpE gene was used as a probe. By complementation tests using the subclones of this 9.6 kb BamHl fragment and various tryptophan auxotrophs of B. lactofermentum and Escherichia coli, this fragment was found to contain a gene cluster composed of trpE, trpD, trpC, trpB, and trpA in this order. It suggests that genes for tryptophan biosynthesis in B. lactofermentum may be an operon.     

Comparative Proteomics of Milk Fat Globule Membrane Proteins from Transgenic Cloned Cattle

The use of transgenic livestock is providing new methods for obtaining pharmaceutically useful proteins. However, the protein expression profiles of the transgenic animals, including expression of milk fat globule membrane (MFGM) proteins, have not been well characterized. In this study, we compared the MFGM protein expression profile of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle, i.e., expressing recombinant human α-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland, with those from cloned non-transgenic (C) and conventionally bred normal animals (N). We identified 1, 225 proteins in milk MFGM, 166 of which were specifically expressed only in the TC-LA group, 265 only in the TC-LF group, and 184 only in the TC-LZ group. There were 43 proteins expressed only in the transgenic cloned animals, but the concentrations of these proteins were below the detection limit of silver staining. Functional analysis also showed that the 43 proteins had no obvious influence on the bovine mammary gland. Quantitative comparison revealed that MFGM proteins were up- or down-regulated more than twofold in the TC and C groups compared to N group: 126 in colostrum and 77 in mature milk of the TC-LA group; 157 in colostrum and 222 in mature milk of the TC-LF group; 49 in colostrum and 98 in mature milk of the TC-LZ group; 98 in colostrum and 132 in mature milk in the C group. These up- and down-regulated proteins in the transgenic animals were not associated with a particular biological function or pathway, which appears that expression of certain exogenous proteins has no general deleterious effects on the cattle mammary gland.     

Formation,Stability and In Vitro Digestion of β-carotene in Oil-in-Water Milk Fat Globule Membrane Protein Emulsions

The formation, stability and in vitro digestion of milk fat globule membrane (MFGM) proteins stabilized emulsions with 0.2 wt% β-carotene were investigated. The average particle size of β-carotene emulsions stabilized with various MFGM proteins levels (1%, 2%, 3%, 4%, 5% wt%) decreased with the increase of MFGM proteins levels. When MFGM proteins concentration in emulsions is above 2%, the average particle size of β-carotene emulsions is below 1.0 μm. A quite stable emulsion was formed at pH 6.0 and 7.0, but particle size increased with decrease in acidity of the β-carotene emulsion. β-carotene emulsions stabilized with MFGM proteins were stable with a certain salt concentrations (0–500 mMNaCl). β-carotene emulsions were quite stable to aggregation of the particles at elevated temperature and time (85 °C for 90 min). At the same time, β-carotene emulsions were stable against degradation under heat treatment conditions. In vitro digestion of β-carotene emulsion showed the mean particle size of β-carotene emulsions stabilized with MFGM proteins in the simulated stomach conditions and intestinal conditions is larger than that of initial emulsions and simulated mouth conditions. Confocal laser scanning microscopy of β-carotene MFGM proteins emulsions also showed the corresponding results to different vitro digestion model. There was a rapid release of free fatty acid (FFA) during the first 10 min and after this period, an almost constant 70% digestion extent was reached. Approximately 80% of β-carotene was released within 2 h of incubation under the simulated intestinal fluid. These results showed that MFGM protein can be used as a good emulsifier in emulsion stabilization, β-carotene rapid release as well as lipophilic bioactive compounds delivery.     

Glycoprotein Properties of the Solubilized Atrial Muscarinic Acetylcholine Receptor

Abstract: The muscarinic acetylcholine receptor from porcine atria exhibits sialoglycoprotein characteristics based on its sensitivity to neuraminidase digestion and its ability to interact specifically with lectin affinity resins when solubilized with a digitonin/cholate mixed detergent system. Differential lectin binding properties of the neuraminidase-treated and untreated receptor suggest that high-affinity binding to immobilized wheat germ agglutinin is accomplished through the presence of both terminal sialic acid and internal N -acetylglucosamine or its β(1→4)-linked oligomers.     

RETRACTED ARTICLE: High Fat Feeding of Lactating Mice Causing a Drastic Reduction in Fat and Energy Content in Milk without Affecting the Apparent Growth of Their Pups and the Production of Major Milk Fat Globule Membrane Components MFG-E8 and Butyrophilin

Lactating mice were fed either a low fat or a high fat diet. Milk samples were collected and the composition was examined. Triglyceride and free fatty acid contents were greatly reduced in the milks of high fat diet group, while protein and lactose contents were almost the same between both diet groups. Although the energy content of each component was also lower in milk of high fat diet group, there was apparently no significant difference in the growth of the pups raised by either diet group. This discrepancy might be in part explained by a hypothesis that the pups might monitor calorie content in milk and keep suckling until the energy intake reaches their satisfaction. Moreover, nearly the same amounts of major milk fat globule membrane proteins MFG-E8 and butyrophilin were shown to be present in the milks from both diet groups and gene expression of both proteins in the mammary glands were also indistinguishable, suggesting that production of major MFGM components is not simply related to fat production and secretion.     

Further Investigation on Naturally Occurring Tocopherols and Tocotrienols in Bovine Milk Fat

The polar lipid material which contains most of unsaponiriable matter of milk fat was collected by means of neutral alumina column chromatography. After saponification of the polar lipid material, the unsaponiriable matter was purified by repeated Florisil and neutral alumina column chromatography and the total tocopherol fraction was obtained. It was found that the total tocopherol fraction isolated from milk fat contained 6 of the known naturally occurring tocopherols, that is, α-, β-, γ-, and δ-tocopherols and α- and γ-tocotrienols. These were identified by two-dimensional thin-layer and gas-liquid chromatography before and after hydrogenation.     

Isolation and Biological Properties of Osteopontin from Bovine Milk

A procedure for the isolation of osteopontin (OPN) from bovine milk using ion-exchange and hydrophobic chromatography is described. A DEAE-Sephacel column followed by dual phenyl-Sepharose columns yielded ∼8 mg of purified protein per liter of milk. SDS–PAGE analysis revealed that the protein migrated atM_r60,000. NH₂-terminal sequence analysis of the first seven amino acids revealed the protein to be identical to that previously reported for bovine OPN. Also, our preparation demonstrated expected biological properties of OPN including adhesion of both endothelial and vascular smooth muscle cells to OPN in a dose- and Arg-Gly-Asp-dependent manner. Furthermore, OPN coupled to Sepharose was capable of binding the α_vβ₃integrin from a detergent extract of endothelial cells. Thus, our procedure yielded biologically active OPN from an abundant and natural source.     

Glycoprotein Properties of Muscarinic Acetylcholine Receptors from Bovine Cerebral Cortex

Muscarinic acetylcholine receptors from bovine cerebral cortex were solubilized in digitonin for the subsequent determination of several biochemical properties. The digitonin-solubilized receptors were representative of the entire membrane-bound population of muscarinic receptors with respect to carbohydrate content, isoelectric point, and molecular weight. The glycoprotein nature of the solubilized receptors was demonstrated by their quantitative binding to wheat germ agglutinin-agarose. The presence of a bound antagonist did not decrease the extent of receptor binding to this lectin. Treatment of receptors with neuraminidase to remove N-acetylneuraminic acid residues reduced binding to wheat germ agglutinin-agarose by 40%; further treatment with endoglycosidases D and H, to remove all N-linked carbohydrate, decreased binding by a total of 67%. Removal of N-acetylneuraminic acid residues had no effect on agonist binding properties of the membrane-bound receptors. The carbohydrate-specific enzymes were further used to assess the contribution of carbohydrate to the isoelectric point and molecular weight of the receptor. Muscarinic receptors solubilized in either digitonin or Triton X-100 focused as one major species with a pI of 4.3. Neuraminidase treatment resulted in an increase of 0.17 units in the pI of the receptor. Muscarinic receptors labeled with the covalent muscarinic antagonist propylbenzilylcholine mustard migrated as a single major polypeptide with a molecular weight of 73,000 on sodium dodecyl sulfate-urea-polyacrylamide gels. The exclusion of urea from these gels severely retarded receptor mobility, indicating a strong tendency for aggregation of receptors in SDS. Removal of N-linked carbohydrate by endoglycosidase treatment reduced the molecular weight of the antagonist binding polypeptide by no more than 5%. These results demonstrate the glycoprotein nature of muscarinic receptors from mammalian cerebral cortex and provide evidence for their heterogeneity with respect to carbohydrate content.     

The Effect of Manipulating Fat Globule Size on the Stability and Rheological Properties of Dairy Creams

Recombined cream (RC, 23 % fat w/w) and standardised commercial cream (CC, 28 % fat w/w) were studied to understand the effects of manipulating fat globule size at the micron-/nano-scale on the stability and rheological properties of cream. All samples were adjusted to a fat: protein ratio of 5:1 and a fat: emulsifier (Tween 80) ratio of 30:1 to stabilize emulsion. For both CC and RC, different emulsions with droplet sizes covering micron- (3.9 μm), sub-micron (0.5 – 0.6 μm) and nano-metric scales (0.13 – 0.29 μm) were obtained using either the homogeniser (7/3 MPa) or the microfluidiser (85 MPa and 42 MPa). Fat globules from both RC and CC had high zeta potential values (-28 to -43 mV) and maintained their reduced size after 1 month of storage at 4 °C, providing evidence of emulsion stability. Droplet size had a significant effect on rheological characteristics of all creams produced. Nano-sized RC tended to have a rigid structure (solid/gel-like form) as compared to micron-sized RC (liquid-like form) as reflected by higher phase angle. Surprisingly, the rheological properties of CC exhibited an opposite tendency to that of RC. This implies that the observed rheological properties of CC and RC could not be fully explained by the discrepancy in droplet size. Differences in interfacial properties between RC and CC might also play a role in the rheological behaviour of the creams. Results indicated the stable high milk fat emulsions could be successfully created by reducing the globule size. These findings would be useful in understanding how micron-/nano-sized emulsions can be utilised in further application or processing of creams.     

Selective Extraction of Alkaline Phosphatase and 51 -Nucleotidase from Milk Fat Globule Membranes by a Single Phase n-Butanol Procedure

ABSTRACT

A single phase extraction procedure employing 8% (v/v) n-butanol at room temperature extracted over 90% of alkaline phosphatase activity and over 60% of 5'-nucleotidase activity from bovine milk fat globule membranes (MFGM). For 5'-nucleotidase, higher n-butanol concentrations lead to loss of activity, while lower concentrations were ineffective in extracting the enzyme. When extractions were performed at 0°C, similar yields were obtained for alkaline phosphatase extraction with 8% (v/v) n-butanol, but 5¹- nucleotidase extraction required 10% (v/v) n-butanol for similar yields. However, 5'-nucleotidase was less susceptible to denaturation during extraction at 0°C. The Km values and substrate specificities for both alkaline phosphatase and 5'-nucleotidase were unchanged by extraction with 8% (v/v) n-butanol. The 8% (v/v) n-butanol extraction procedure provides a 3-fold purification step, and an enzyme preparation suitable for further purification.     

Effects of Soy Proteins and Hydrolysates on Fat Globule Coalescence and Whipping Properties of Recombined Low-Fat Whipped Cream

The interaction and synergetic effect of soy protein isolate (SPI) and its hydrolysates with different concentrations of monoglycerides were explored at the air-water/oil interfaces in recombined low-fat whipped cream (20%). The creams were made with 20% palm oil, 18% carbohydrate, 0.22% stabilizers, and 0.25–1.00% monoglycerides. The proteins used were native soy protein isolate (NSPI), commercial soy protein isolate (CSPI), soy protein hydrolysates by pepsin (SPHPe), soy protein hydrolysates by papain (SPHPa), and SC (sodium caseinate). Overrun, stability, rheological behavior, and texture of recombined low-fat whipped cream were studied. Results indicated that increasing concentration of monoglycerides was effective in improving the textural, whipping properties, and stability of recombined low-fat whipped cream. Increasing concentration of monoglycerides in the mix prompted the displacement of adsorbed protein from fat globules, built up a firmer structure of fat aggregates, and stabilized the trapped air bubbles in the structure of recombined low-fat whipped cream. At the same level of monoglycerides, SPHPa whipped cream produced a similar overrun, stability, and texture as SC. Due to the high proportion of β-conglycinin in SPHPe, a low degree of fat globule partial coalescence occurred and led to low overrun and weakened structure in recombined low-fat whipped cream.

     

Isolation and Characterization of Full and Truncated Forms of Human Breast Carcinoma Protein BA46 from Human Milk Fat Globule Membranes

We have isolated and characterized two proteins of 50 and 30 kDa from human milk fat globule membranes of healthy donors. N-terminal and internal sequencing revealed that the 50-kDa protein is the full-length human breast carcinoma protein BA46 that is highly expressed in human breast tumors. The 30-kDa protein is a truncated form of protein BA46 which consists of the C-terminal factor V/VIII-like domain of BA46 and which appears to anchor BA46 to the milk fat globule membrane. Defective release of the epidermal growth factor domain containing a surface RGD motif may be related to involvement of BA46 in breast cancer     

On the Mechanism of Sphingomyelin Interaction with Solubilized Membrane Proteins

Studies on the high-affinity receptor for IgE from rat basophilic leukemia cells (RBL-2H3) have shown that the phospholipid sphingomyelin remains attached to the protein complex during washing of the affinity immobilized complex under solubilizing conditions. Here we extended these findings and compared the species distribution patterns in sphingomyelin and phosphatidylcholine of the receptor-bound lipids to those of the plasma membrane lipids. Fc_?-receptor-bound sphingomyelin but not phosphatidylcholine was enriched in long-chain fatty acids. We then examined other membrane proteins with respect to sphingomyelin enrichment. RBL-2H3 cell surface proteins, immobilized on concanavalin A-Sepharose and washed under solubilizing conditions, also showed a two-to six-fold enrichment in the associated sphingomyelin. Similar observations were also derived from other cell types, such as the mouse fibroblast cell line A-9 and the pig kidney epithelial cell line PK-1. Since this has been observed in all the three cell sources, it was suggested that sphingomyelin enrichment in Fc_?-receptor preparations, although reproducible, was not specific for this protein. That this phenomenon was not specific for a particular protein might also be concluded from experiments that have shown nonhomogenous distribution of sphingomyelin in protein-free lipid-detergent mixtures. These results are compatible with a model whereby the interaction between sphingomyelin and soluble membrane proteins results from preference to nonmicellar phases or to structures with extended hydrophobic domains, probably due to the imperfect fitness of the detergent micelles to properly accomodate these lipids. This feature makes long-chain sphin gomyelin a plausible candidate for the lipid responsible for the stabilizing effect that crude lipid preparations exert on the structural and functional properties of some membrane protein, e.g., Fc_? R.     

Electrophoretic and Immunological Properties of Folate-binding Protein Isolated from Bovine Milk

Changes of the folate-binding protein (FBP) concentration in bovine milk after parturition were investigated. The FBP was highly purified from mature milk by affinity chromatography. The purified FBP showed a single protein band in polyacrylamide gel electrophoresis and was immunologically homogenous in double immunodiffusion. However, in two-dimensional gel electrophoresis, the FBP was separated into several spots in isoelectric focusing in the first dimension, and each spot also showed two molecular weights in SDS-gel electrophoresis in the second dimension. But these FBP molecules were immunologically identical with each other. The neuraminidase treatment obviously diminished the number of isoelectric points of the FBP. Thus, the variety of FBP molecules was at least partially due to the variability of the sialic acid content in the carbohydrate moieties. Moreover, the milk FBP showed species-specificity among mammals immunologically as well as physicochemically.     

Comparison of Bovine Milk Protease with Plasmin

Comparative studies of bovine milk protease and bovine plasmin were performed. It was found that milk protease was very similar to plasmin in various properties such as optimum pH, pH-stability, heat-stability, inhibition by various inhibitors and molecular weight. The changes of casein by both enzymes as observed by polyacrylamide gel electrophoresis were also quite similar. From these results, it is suggested that milk protease may be plasmin itself transported from bovine plasma.     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.