Gerda Bruder

Butyrophilin, an apical plasma membrane-associated glycoprotein characteristic of lactating mammary glands of diverse species

Lipid globule membranes were isolated from human and bovine milk and from the milk of sheep, goat, pig, rat and guinea pig, and their polypeptide compositions were analyzed. The major polypeptides with molecular weights similar to that of bovine butyrophilin were separated by gel electrophoresis, isolated and characterized with respect to isoelectric point, molecular weight, immunological cross-reactivity and peptide composition after proteolytic cleavage. We show that in all species examined these proteins are similar to bovine butyrophilin in (i) their relative insolubility in buffers of low and high ionic strength and in non-denaturing detergents, (ii) the occurrence of several isoelectric variants, and (iii) patterns of peptides obtained by protease digestion. It is concluded that closely related proteins are major constituents of the cytoplasmic coat structures associated with milk lipid globule membranes of many species, and we propose the name butyrophilins for this group of proteins. Bovine and human butyrophilins are glycosylated with relatively large amounts of glucosamine, mannose, glucose and galactose but little fucose, sialic acids or galactosamine. Most if not all of the sugar residues are associated with an acetone-soluble peptide fragment of Mr 12000-16000 focusing at about pH 4.0. We suggest that this fragment contains a membrane-spanning peptide sequence and is involved in the attachment of the cytoplasmic coat to the membrane of the milk lipid globule.

The B-type cytochrome in endoplasmic reticulum of mammary gland epithelium and milk fat globule membranes consists of two components, cytochrome b5 and cytochrome P-420

Abstract The cytochrome contents of rough endoplasmic reticulum (ER) of lactating bovine and rat mammary epithelial cells and of the membranes surrounding the fat globules in bovine and human milk (MFGM) were analyzed with spectrophotometric (at +20 °C and −196 °C) and immunological methods. Two cytochrome components were found. One was identified as cytochrome b 5 by the characteristic split of the α-band in reduced versus oxidized difference spectra at low temperature, by the reduction with NADH, which was insensitive against rotenone and antimycin, and by the solubility upon trypsin treatment. This component showed cross-reaction with the microsomal cytochrome b 5 from rat hepatocytes using rabbit antibodies against the purified cytochrome b 5 fragment released from rat liver microsomes by trypsin treatment. The in situ localization of cytochrome b 5 in mammary epithelial cells was demonstrated by indirect immunofluorescence microscopy in both frozen sections of tissue and cultured cells. The second cytochrome component was identified as cytochrome P-420 by the characteristic spectral bands in the CO-difference spectrum and the dithionite-difference spectrum, by the reaction with cyanide, and by the insolubility upon trypsin treatment of the membranes. In addition, we found evidence for the existence of a form of cytochrome P-420 in these membranes which does not bind CO. The presence of cytochrome P-420 in mammary gland ER and MFGM fractions was not due to preparative artifacts. No cytochrome P-450 was observed in these membranes. The significance of the occurrence of these redox components in ER and surface membrane of mammary gland epithelium and other cells is discussed.

Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420

The presence of cytochromes b5, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c redutases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b5. In addition, cytochrome b5 fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with Mr values in the range 10 000-13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.

Protein synthesis in lactating guinea-pig mammary tissue perfused in vitro: II. Biogenesis of milk-fat-globule membrane proteins

Guinea-pig mammary tissue was perfused in vitro, radiolabelled with [35S]methionine and intracellular protein precursors of the milk-fat-globule membrane (FGM) recovered by immunoabsorption techniques. Labelled xanthine oxidase was solely detected in post-microsomal supernatants and butyrophilin in carbonate-washed membranes. A major glycoprotein (Gp 55), was initially present in a membrane-bound form, but after longer perfusion times a fraction of this protein was recovered in the post-microsomal supernatant. These results are discussed with reference to formation of the apically-derived FGM.

Protein synthesis in lactating guinea-pig mammary tissue perfused in vitro. I. Radiolabelling of membrane and secretory proteins.

A method for the in vitro perfusion of isolated guinea-pig mammary tissue is described that allows the radiolabelling of secretory and membrane proteins. Glands were depleted of methionine, labelled with [35S]methionine for 5 min and perfused with medium containing an excess of unlabelled methionine for varying times. The structural integrity of the alveoli in the perfused glands appeared well maintained. Epithelial polarity was preserved and junctional complexes were evident. About 20% of the methionine provided in the medium was extracted by glands of 10 g wet weight under the labelling conditions employed. With chase periods from 15 to 40 min, 50-70% of the methionine was incorporated into trichloroacetic-acid (TCA)-precipitable material. The principal radiolabelled proteins recovered from the tissue fractions had Mrs and isoelectric points similar to the major secretory proteins (i.e. caseins and alpha-lactalbumin) of guinea-pig milk. Autoradiography of tissue sections at the resolution of the light microscope showed that secretory proteins were transported from sites of synthesis within secretory cells to the alveolar lumina after 45 min. These highly labelled secretory proteins could be almost completely removed from microsomal fractions by treatment with sodium carbonate solutions. Proteins with Mrs from 30 000 to 200 000 were detected in the washed membranes by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. These labelled membrane-associated proteins persisted in the microsomal membrane fraction after chase periods from 7.5 to 40 min.