Ernst-Dieter Jarasch

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.

Characteristics of membrane-bound and soluble forms of xanthine oxidase from milk and endothelial cells of capillaries

Xanthine oxidase (xanthine:O2 oxidoreductase, EC 1.2.3.2) was purified from bovine milk lipid globules to electrophoretic homogeneity (Mr 155,000) and antibodies were raised against it in rabbits. By immunolocalization techniques, the xanthine oxidase antigen was detected in milk lipid globules and mammary gland epithelium, but also in capillary endothelium from various tissues, including liver, lung and intestine. These findings were paralleled by measurements of xanthine oxidase activities in the tissues, both in a membrane-associated and a soluble form. Addition of hypoxanthine to fractions containing native xanthine oxidase did not promote lipid peroxidation, in contrast to the widely used in vitro system for lipid peroxidation which involves addition of xanthine oxidase preparations. Extraction with buffers of high ionic strength and with nonionic detergents removed only part of the enzyme from the membranes. Immunoprecipitates from the soluble supernatant fractions, using anti-xanthine oxidase IgG, were enriched in the Mr 155,000 polypeptide. Patterns of proteolytic cleavage products of the xanthine oxidase monomer from capillaries and milk lipid globules were similar but not identical. Immunoprecipitates from soluble fractions of milk lipid globules and tissues were enriched in both xanthine oxidase and NADH-cytochrome c reductase activities. Electrophoretic separation of proteins from milk lipid globule membranes under non-denaturing conditions revealed a close correlation of xanthine oxidase and part of the NADH-cytochrome c reductase activity, but showed different activity profiles of NADH-ferricyanide reductase and xanthine oxidase.

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.

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.

Absence of cytochrome oxidase in nuclear membranes from hepatocytes

Polarographic and spectrophotometric determinations show that highly purified nuclear membrane fractions from rat and bovine liver do not contain significant amounts of cytochrome oxidase activity and cytochrome aa3.