Crossed immunoelectrophoresis of bovine milk fat globule membrane protein solubilized with non-ionic detergent

Nielsen, C. SCHäFER; Bjerrum, Ole J.

doi:10.1016/0005-2736(77)90342-x

Cited by 36 publications

(18 citation statements)

References 44 publications

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“…This occurred in the absence of any significant penetration of the lipid bilayer by the proteinase, since xanthine oxidase which is localized on the cytoplasmic face (14,45,46) remained intact during trypsin digestion (Fig. 7b).…”

Section: Fig 2 Analysis Of Btn By Rt-pcrmentioning

confidence: 97%

Butyrophilin Is Expressed in Mammary Epithelial Cells from a Single-sized Messenger RNA as a Type I Membrane Glycoprotein

Banghart¹,

Chamberlain²,

Velarde³

et al. 1998

Journal of Biological Chemistry

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We investigated the expression of butyrophilin in eukaryotic cells with a view to determining the number of mRNA species, the incorporation of the peptide chain into microsomes, and the topology of the processed protein in biological membranes. Butyrophilin is synthesized from a single sized mRNA in both bovine and murine lactating mammary tissue and associates with microsomal membranes with a type I topology (N exo ⅐C cyto ) via a single hydrophobic anchor in the middle of the sequence. Several isoelectric variants of the protein were detected in cellular membranes from lactating bovine mammary tissue and in the milk-fat-globule membrane. We found no evidence for soluble forms of butyrophilin in postmicrosomal supernatants. The 66-kDa protein appears to be subjected to limited proteolysis, giving rise to a 62-kDa fragment lacking the C terminus and to other more minor fragments of lower M r in the milk-fat-globule membrane. Antipeptide antibodies to epitopes within the N-and C-terminal domains were used to show that butyrophilin retains a type I topology in plasma membranes when expressed in insect cells from a baculovirus vector, and in secreted milk-fat globules. These data do not agree with previous suggestions that butyrophilin may exist in cytoplasmic soluble forms, or be reorganized in the plane of the lipid bilayer during secretion in lipid droplets from mammary cells. The results are discussed with reference to the role butyrophilin may play as the principal scaffold for the assembly of a complex with xanthine oxidase and other proteins that functions in the budding and release of milk-fat globules from the apical surface during lactation.Several years ago we cloned a cDNA encoding the major milk-fat-globule membrane (MFGM) 1 protein, butyrophilin (BTN) (1) with a view to understanding the function of this mammary-specific protein in milk-fat secretion (for reviews of this process, see Refs. 2-4). From the derived amino acid sequence we predicted that BTN is an integral protein with a single membrane anchor and a glycosylated exoplasmic N terminus (type I orientation) (1). Subsequent comparisons with more recently cloned cDNAs have established that BTN is a member of the immunoglobulin superfamily (5) with two Nterminal immunoglobulin domains, one of the intermediate type (IgI) (6) toward the N terminus and one of the constant C1 type (7) toward the membrane bilayer (8). A large proportion of the C-terminal region comprises the RFP or B30.2 domain, a sequence which is present in a subfamily of zinc-finger proteins (9, 10), and a group of recently identified BTN-like genes (11, 12). Several workers have proposed that BTN may function as an integral receptor for cytoplasmic fat droplets and that budding of the droplets at the cell surface is initiated by interactions between the cytoplasmic tail of BTN and other proteins, notably the redox enzyme xanthine oxidase (1, 13-16). However, the assumption that BTN behaves as an integral protein in mammary epithelial cells has been confounded by several observati...

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Section: Fig 2 Analysis Of Btn By Rt-pcrmentioning

confidence: 97%

Butyrophilin Is Expressed in Mammary Epithelial Cells from a Single-sized Messenger RNA as a Type I Membrane Glycoprotein

Banghart¹,

Chamberlain²,

Velarde³

et al. 1998

Journal of Biological Chemistry

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“…First, cream can be separated from milk by a laboratory centrifuge or in a large scale, bench-top cream separator. Next, the separated cream is washed two [Ye et al, 2002], three [Fong et al, 2007;Kanno & Kim, 1990] or more number of times [Asker, 1974;Mangino & Brunner, 1975] in 3-15 fold volumes of distilled or deionized water [Kanno & Kim, 1990;Newman & Harrison, 1973], sucrosesaline solution with [Erickson et al, 1964;Mather et al, 1977;Nejjar et al, 1986;Snow et al, 1977] or without [Dowben et al, 1967] pH buffering, pH buffered sucrose solution [Khodaparast-Sharifi & Snow, 1989], isotonic phosphate buffer solution [Nielsen & Bjerrum, 1977], phosphate-saline buffer [Innocente et al, 1997], or simulated milk ultrafiltrate [Ye et al, 2004]. In some cases, detergents [Diaz-Maurino & Nieto, 1976;Mather et al, 1977] or dissociating agents [Ye et al, 2002] are added to facilitate the washing.…”

Section: Isolation Of Mfgm From Milkmentioning

confidence: 99%

“…After washing the cream, the MFGM is released from the triglyceride fat core into the aqueous phase by churning [Diaz-Maurino & Nieto, 1976;Dowben et al, 1967;Fong et al, 2007;Mather et al, 1977;Nielsen & Bjerrum, 1977], agitation [Harrison et al, 1975] at reduced temperatures or applying cycles of freezing-thawing [Dowben et al, 1967;Khodaparast-Sharifi & Snow, 1989;Snow et al, 1977Snow et al, , 1980. Alternatively, MFGM can directly be released from washed cream by the use of polar aprotic solvents [Bingham & Malin, 1992;Dapper et al, 1987], bile salts [Erickson et al, 1964;Snow et al, 1980], or nonionic detergents [Patton, 1982]; in addition these authors reported that direct extraction normally results in a lower yield, and a certain difference in composition depending on the concentration of the applied chemicals, the time and temperatures of extraction.…”

Section: Isolation Of Mfgm From Milkmentioning

confidence: 99%

“…Two fractions, the soluble supernatant and the MFGM pellet, are obtained by ultracentrifugation. Precipitation of MFGM fragments at low pH [Fong et al, 2007;Kanno & Kim, 1990] or by 'salting out' with ammonium sulfate [Kanno & Kim, 1990;Nielsen & Bjerrum, 1977] may be applied to MFGM suspensions, after which the MFGM material is separated by centrifugation. All the above reviewed methods are laboratory applications for the isolation of MFGM material from untreated milk.…”

Section: Isolation Of Mfgm From Milkmentioning

confidence: 99%

“…Prior to measuring the activity of MFGM enzymes, extra washing steps of the pellet are often applied to remove most of the contaminating whey protein [Diaz-Maurino & Nieto, 1976;Khodaparast-Sharifi & Snow, 1989;Nielsen & Bjerrum, 1977;Snow et al, 1977Snow et al, , 1980. Solutes used in the washing solution can affect the membrane enzyme activity [Diaz-Maurino & Nieto, 1976;McPherson et al, 1984;Snow et al, 1980] and dialysis does not always remove the solutes completely [Bash et al, 1976].…”

Section: Isolation Of Mfgm From Milkmentioning

confidence: 99%

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Composition, Properties and Nutritional Aspects of Milk Fat Globule Membrane – A Review

El-Loly¹

2011

Pol. J. Food Nutr. Sci.

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processes and defense mechanisms in the newborn [Cavaletto et al., 2006].The stabilizing membrane acts as reactive sorts on the interface between the barriers of milk serum. As such, it can be globule and rate-controlling for a host of physical and of enzymes chemical interactions, e.g., binding mace elements; controlled release of the polar materials products of lipolysis; transfer of milk serum; maintenance of emulsion into stability by prevention of globule fusion; availability of fatty acids and cholesterol for micellar absorption in the small intestine; and destabilization by creaming, clumping, churning, freeze-thaw, and heating, resulting in loosely bound substances into milk transfer serum. Worthy of notice is that the interactions are dynamic.Moreover, it is assumed that the MFGM proteins also possess specific nutritional properties. As such, due to their origin, composition and structure, MFGM polar lipids and proteins could be used as an emulsifier or stabilizer, combining technological and nutritional functionality [Mulder & Walstra, 1974;Anderson & Cawston, 1975;Patton & Keenan, 1975;Patton & Jensen, 1975, 1976Keenan et al., 1983;Mather & Keenan, 1983; McPherson & Kitchen, 1983; Walstra & Jenness, 1984;Keenan & Dylewski, 1985 Bucbheim, 1986;Mather, 1987; Keenan et al., 1988;Keenan & Dylewski, 1995;Keenan & Patton, 1995;Mather & Keenan, 1998;Danthine et al., 2000; Keenan, 2001; Keenan & Mather, 2002; Ollivier--Bousquet, 2002;Lopez et al., 2006Lopez et al., , 2007Sanchez-Juanes et al., 2009;Zamora et al., 2009]. Composition, Properties and Nutritional Aspects of Milk Fat Globule Membrane -a Review Mohamed Mansour El-Loly Dairy Department, National Research Centre, Dokki, Cairo, EgyptKey words: milk fat globule membrane, mammary gland, isolation, structure, stability, nutritional, nutraceuticalIn the last few years, knowledge on the composition and properties of the milk fat globule membrane (MFGM) increased significantly. It is now recognized that the MFGM is highly complex in structure and composed of different protein and lipid components with specific technological and nutritional properties. As such, MFGM materials have been isolated and characterized as valuable ingredients for incorporation into new food products. However, MFGM are also sensitive to modification during isolation and processing, and care should be taken to standardize the composition and characteristics of the membrane to maintain its unique properties during application in food products.The MFGM is subject to changes in composition and structure from the moment the fat globule leaves the mammary secretory cell. Upon milk harvesting and further milk handling, further changes to the MFGM take place. Depending on the type and degree of treatment, this may involve different physico-chemical interactions between various membrane components, the loss of membrane components and/or adsorption of components from the milk plasma. However, the effects appear to be variable and dependent on physiological (animal) factors, and much remains ...

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Electroimmunochemical analysis of amphiphilic proteins and glycolipids stained with Sudan Black‐containing detergent micelles

Bjerrum¹,

Gerlach²,

Bøg-Hansen³

et al. 1982

Electrophoresis

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Micelles of non-ionic detergents can be colored through incorporation of the hydrophobic dye Sudan Black. This property can be used to identify amphiphilic antigens in complex mixtures. Thus, crossed immunoelectrophoresis with 0.05 % v/v Triton X-100 plus 0.002 % w/v Sudan Black shows selective staining of major amphiphilic proteins from human erythrocytes, thrombocytes and serum, from bovine milk fat globules and from yeast plasma membranes. The method may also give information about glycolipid antigens in detergent-solubilized membrane material, e. g. mannosyl-diinositolphosphoryl-ceramide from yeast. In this case, however, the membrane has to be solubilized with detergent plus Sudan Black and analyzed in detergent-free gels, where a stable micellar structure seems to exist. In a surplus of detergent, the glycolipid antigen may enter new micelles during electrophoresis, reducing the number of epitopes per micelle below two, thus avoiding precipitation. Analysis of purified lipopolysaccharide from Bordetellapertussis followed this pattern, whereas lipopolysaccharides from Pseudomonas aeruginosa and Escherichia coli, although heavily influenced by the presence of Triton X-100, were precipitated in the presence of a surplus of detergent. The described method is simple, fast and easy to perform, It requires neither extra equipment nor expensive chemicals. Sometimes heavily stained, detergent-containing precipitates of a non immunological nature may appear, but these can easily be discerned from immunoprecipitates.

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Crossed immunoelectrophoresis of bovine milk fat globule membrane protein solubilized with non-ionic detergent

Cited by 36 publications

References 44 publications

Butyrophilin Is Expressed in Mammary Epithelial Cells from a Single-sized Messenger RNA as a Type I Membrane Glycoprotein

Butyrophilin Is Expressed in Mammary Epithelial Cells from a Single-sized Messenger RNA as a Type I Membrane Glycoprotein

Composition, Properties and Nutritional Aspects of Milk Fat Globule Membrane – A Review

Electroimmunochemical analysis of amphiphilic proteins and glycolipids stained with Sudan Black‐containing detergent micelles

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