T.W. Keenan

Localization of xanthine oxidase in mammary-gland epithelium and capillary endothelium

Xanthine oxidase, an iron-sulfur molybdenum flavoprotein known to generate superoxide radical, was demonstrated in several bovine tissues. The enzyme (155 kd polypeptide) was purified from bovine milk lipid globules and antibodies were raised that allowed precipitation of the enzyme without inactivation of enzymatic activity. By immunolocalization techniques at light and electron microscope levels, the antigen was found in milk-secreting epithelial cells but not in epithelial cells of several other tissues. In a number of tissues, including mammary gland, liver, heart, lung and intestine, antibodies to xanthine oxidase stained only endothelial cells of capillaries, including sinusoids, but not endothelia of larger blood vessels and endocard. In both milk-secreting epithelial and capillary endothelial cells, xanthine oxidase was distributed throughout the cytoplasm. Results from biochemical and immunological studies suggest that xanthine oxidase is similar in the various tissues examined and may serve similar redox functions.

Origin and secretion of milk lipids.

The cream fraction of milk comprises droplets oftriacylglycerol coated with cellular membranes. In thisreview, we discuss how these droplets are formed andsecreted from mammary epithelial cells during lactation. This secretory system is especiallyinteresting because the assembled lipid droplets aresecreted from the cytoplasm enveloped by cellularmembranes. In other cells, such as hepatocytes andenterocytes, lipid is secreted by exocytosis frommembrane-bounded compartments of the secretory pathway.Milk lipids originate as small droplets oftriacylglycerol, synthesized in or on the surfaces ofrough endoplasmic reticulum (ER)4 membranes. Thesedroplets are released into the cytoplasm as microlipiddroplets (MLDs) with a surface coat of protein and polarlipid. MLDs may fuse with each other to form largercytoplasmic lipid droplets (CLDs). Droplets of varyingsize, are transported to the apical cytoplasm by unknownmechanisms and are secreted from the cell coated with anouter bilayer membrane. CLDs may increase in size in all regions of the cell, especially atthe plasma membrane during secretion. Two possiblemechanisms for lipid secretion have been proposed: anapical mechanism, in which lipid droplets are enveloped with apical plasma membrane, and asecretory-vesicle mechanism, in which fat droplets aresurrounded by secretory vesicles in the cytoplasm andare released from the surface by exocytosis fromintracytoplasmic vacuoles. A combination of both mechanisms maybe possible. Following secretion, a fraction of themembrane surrounding the globules may be shed from thedroplets and give rise to membrane fragments in the skimmilk phase.

Tightly bound cardiolipin in cytochrome oxidase.

Tightly bound cardiolipin has been found in cytochrome oxidase. The molar ratio of bound cardiolipin to cytochrome oxidase approaches 1:1. The tightly bound cardiolipin cannot be removed by many solvents which extract loosely bound lipids, but can be extracted with alkaline chloroform-methanol. Phospholipase A does not hydrolyze the bound lipid. After partial removal by repeated cholate-(NH4)2SO4 treatment maximum activity can be restored in the oxidase by cardiolipin but not by detergents. The bound cardiolipin has a fatty acid composition similar to the bulk mitochondrial cardiolipin but small amounts of three other phospholipid fractions with a high level of saturated fatty acid are also found to be closely associated with the oxidase.

Studies on the structure of milk fat globule membrane

SummaryMilk fat globule membrane was solubilized with sodium dodecyl sulfate and mercaptoethanol and the membrane proteins were separated by SDS-polyacrylamide gel electrophoresis. The membrane preparations contained three major size classes of polypeptide of 155,000, 62,500 and 43,500 daltons. At least five glycopeptides were separated of which two stained intensely with periodic acid-Schiff reagent, but poorly with coomassie blue. Trypsin hydrolysis of whole cream and isolated milk fat globule membrane revealed major differences in the rates of protein hydrolysis. Many of the membrane proteins of whole cream resisted proteolysis compared with the same proteins in the isolated membrane. Two glycopeptides were resistant to trypsin digestion in either preparation. Treatment of whole cream with neuraminidase led to the release of at least 70% of the protein-bound sialic acid. Whole cream and isolated membrane samples were iodinated with125I in the presence of lactoperoxidase and hydrogen peroxide. The membrane proteins were significantly more accessible to lactoperoxidase-125I in isolated membrane compared with the proteins of whole cream. Polypeptides of molecular weight 43,500 and approximately 48,000 daltons were predominantly labelled in whole cream and could be eluted from the fat globules with magnesium chloride (1.5m). The results strongly suggest that the proteins of milk fat globule membrane are asymmetrically arranged in the membrane and that most of the protein-bound sialic acid is present on the external surface of milk fat globules.

Membranes of mammary gland. X. Adenosine triphosphate dependent calcium accumulation by Golgi apparatus rich fractions from bovine mammary gland.

Abstract Golgi apparatus rich fractions from lactating bovine mammary gland had an Mg 2+ -dependent, Ca 2+ -stimulated adenosine triphosphatase. These Golgi apparatus fractions also accumulated Ca 2+ in vitro. Accumulation of Ca 2+ required ATP and could be abolished by treatment either with low concentrations of deoxycholate followed by ultrasound, or by heating at 100 °C for 10 min. The adenosine triphosphatase activity of Golgi apparatus was strongly stimulated by low concentrations of Ca 2+ and moderately stimulated by high concentrations of K + . This activity was unaffected by Na + and was not inhibited by ouabain. The pH optimum for the Mg 2+ -dependent hydrolysis of ATP was 7.5, the K m was 5 × 10 −5 M and the activation energy was 6 000 calories/mole. This Mg 2+ -dependent adenosine triphosphatase activity was also found in rough endoplasmic reticulum, smooth microsomes and milk fat globule membrane, the latter membrane being derived directly from the apical plasma membrane. All of these membrane fractions had the ability to specifically accumulate Ca 2+ . Specific accumulation was highest with smooth microsomes and lowest with milk fat globule membrane with Golgi apparatus and rough endoplasmic reticulum being intermediate. These observations provide one plausible explanation for intracellular Ca 2+ accumulation and secretion into milk. Further, these results help explain the ultrastructural observations of casein micelle formation in secretory vesicles elaborated by Golgi apparatus.

Preparation and characterization of the inner coat material associated with fat globule membranes from bovine and human milk.

Abstract Milk fat globules of many species are characterized by a dense 10–50 nm thick layer sandwiched between the milk fat globule membrane (MFGM) and the outer shell of the fat droplet. This coat material is tightly associated with the membrane and survives isolation and extensive washing of the isolated MFGM. We have prepared these MFGM-associated coat structures from bovine and human milk by removal of membrane and loosely associated material using extractions in low and high salt buffers, non-ionic detergents such as Triton X-100, and/or solutions of lithium diiodosalicylate. Residual fractions obtained after such treatments are devoid of identifiable membrane structures but are enriched in MFGM coat material which appears in the form of densely stained plaques of a finely filamentous texture. MFGM fractions are enriched in some polypeptide bands seen after electrophoresis two of which are especially prominent in both species (band 3, apparent mol. wt 155 000; band 12, apparent mol. wt 67 000). Human and bovine MFGM coat fractions and isolated bovine band 12 polypeptide material separated after dissociation in sodium dodecylsulfate (SDS) by gel filtration, chromatography on hydroxylapatite or preparative electrophoresis in SDS-polyacrylamide gels are intimately associated with small amounts of phospholipids and gangliosides of a pattern different from that of total MFGM, contain carbohydrates (relatively high contents of mannose, glucosamine, galactose, and galactosamine; low levels of fucose and sialic acids) and show similar amino acid compositions. The relationship of band 12 polypeptide to components of MFGM coat preparations from various other species and to components present in other membrane fractions has been examined by immunodiffusion techniques and immunofluorescence microscopy using rabbit, mouse and guinea pig antibodies against purified band 12 polypeptide. Evidence is presented for the occurrence of related polypeptides in MFGM coat preparations from different species. The unusual structure and resistance of the MFGM coat material, especially the occurrence of glycopeptides in association with the cytoplasmic side of a membrane structure, are discussed in relation to the stabilization of the emulsified state of milk fat and the process of milk fat globule budding as well as a general model for local differentiation of membrane character.

Composition and synthesis of gangliosides in mammary gland and milk of the bovine

Gangliosides were identified as constituents of milk and mammary gland of cows. On a sialic acid basis, the average concentrations of gangliosides in mammary gland and milk were 88.4 and 5.6 nmoles/g wet weight. Nearly 90% of the milk gangliosides were contained in milk fat globule membrane. The same six chromatographically distinguishable gangliosides were found in mammary gland and the fat globule membrane. Structures of three of the gangliosides were shown to be ceramide-glucose-galactose-sialic acid, ceramide-glucose-galactose-(sialicacid)-N-acetyl-galactosamine, andceramide-glucose-galactose-sialicacid-sialicacid. The latter was the most abundant ganglioside, accounting for more than 50% of the lipid-bound sialic acid in mammary gland and milk. Both N-acetyl- and -N-glycolylneuraminic acids were found in mammary ganglioside fractions. Fatty acids with even carbon numbers from C14 to C24 were contained in gangliosides. Appreciable amounts of n-tricosanoic acid were also present. Mammary gland had all glycosyltransferases involved in synthesis of N acetylneuraminyl2 -galactosylglucosyl ceramide (GD3) starting with ceramide.

Distribution of gangliosides among subcellular fractions from rat liver and bovine mammary gland

That gangliosides are specifically localized in plasma membranes of extraneural cells has been assumed [ I] or suggested based on their enrichment in isolated plasma membrane fractions [2,3]. However, we recently demonstrated that gangliosides are not specifically localized in the surface membranes of either rat liver or bovine mammary gland; only 10 to 25% of the cellular gangliosides were recovered in these membrane [4]. These sialic acid containing glycolipids are ubiquitous among mammalian tissues (e.g. [S]) and have been implicated as having immunological activity, and as functioning in cellular recognition [6] and in cell-cell adhesion [7]. More recently abnormal ganglioside metabolism has been implicated as a possible causal agent in tumorigenesis [8-lo]. In view of their physiological significance, we examined the subcellular distribution of gangliosides.

Glycosyltransferases: Do they exist on the surface membrane of mammalian cells?

According to the membrane flow or endomembrane hypothesis [ 1,2], membrane biogenesis includes the physical transfer of membranes from one subcellular compartment to another. Endoplasmic reticulum membrane is transferred to the Golgi apparatus where it is transformed into plasma membrane-like membranes. This transformed membrane is utilized in formation of secretory vesicles which arise from cisterna of the Golgi apparatus. The secretory vesicle membranes fuse with the plasma membrane and during this process the vesicle contents are discharged into the extracellular space [3,4]. Secretory vesicle membranes can thus contribute to growth or renewal of membranes of the cell surface. Additionally, constituents of surface membranes, especially glycolipids and glycoproteins, have the potential for being synthesized within the context of this export route; their synthesis need not be restricted to the cell surface [5 -9] . Yet, recent views and hypotheses assume partial or even exclusive localization of the carbohydrate transferases of glycoprotein and glycolipid synthesis in or on the surface membrane [10-25]. In this report we critically evaluate the evidence relevant to localization of these transferases within eucaryotic cells.

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.