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In the lactating mammary gland, epithelial cells secrete triacylglycerols in the form of droplets enveloped by an apical surface membrane. This membrane is known as the milk fat globule membrane (MFGM; Mather & Keenan, 1983). MFGM-associated proteins have been studied and employed in developing antibodies against surface antigens of breast epithelial cells, which are used in breast cancer immunodiagnosis and histopathology (Salinas et al. 1987; Larocca et al. 1991; Peterson et al. 1995). So far, only a small number of proteins have been analysed and their sequence identified in bovine, murine and human MFGM; among them are butyrophilin (Jack & Mather, 1990; Ishii et al. 1995; Taylor et al. 1996), MFG-E8 (Stubbs et al. 1990), PAS 6/7 (Hvarregaard et al. 1996) and lactadherin or breast antigen BA46 (Couto et al. 1996; Taylor et al. 1997). Several minor proteins have yet to be characterized, since it is not easy to isolate them in large quantities from the membrane. SDS gel patterns give useful information about MFGM proteins, such as concentration, relative molecular mass and presence of carbohydrate. Over forty membrane components have been separated by electrophoretic techniques from bovine MFGM (Mather et al. 1980).The research reported here combined SDS-PAGE with sequencing analysis and describes the composition of human MFGM, with the exception of high molecular mass mucin, which only penetrates an acrylamide gel of 40 g/l. Mucins have been extensively studied and the sequence predicted from cDNA (Gendler et al. 1990). Surprisingly, identification of the protein bands in the present study revealed that three proteins alone constituted the major components of human MFGM: xanthine oxidase (EC 1.1.3.22), butyrophilin and lactadherin. Lactadherin belongs to a family of proteins possessing epidermal growth factor-like and factor V/VIII C1/C2-like domains, including bovine PAS 6/7, guinea pig GP55 (Hvarregaard et al. 1996) and murine MFG-E8 (Stubbs et al. 1990). In a previous investigation, we characterized lactadherin (formerly breast antigen BA46) and its truncated 30 kDa form as components of healthy human MFGM (Giuffrida et al. 1998). Human butyrophilin has recently been cloned and sequenced (Taylor et al. 1996); the presence of two extracellular immunoglobulin superfamily domains suggested a potential cell surface receptor function. This study was aimed at identifying and characterizing the multiple forms of the major proteins of MFGM.