Enzymic characteristics of fat globule membranes from bovine colostrum and bovine milk.

Abstract

Fat globule membranes have been isolated from bovine colostrum and bovine milk by the dispersion of the fat in sucrose solutions at 4~ and fractionation by centrifugation through discontinuous sucrose gradients. The morphology and enzymic characteristics of the separated fractions were examined.Fractions comprising a large proportion of the total extracted membrane were thus obtained having high levels of the Golgi marker enzymes UDP-galactose Nacetylglucosamine fl-4-galactosyltransferase and thiamine pyrophosphatase. A membrane-derived form of the galactosyltransferase has been solubilized from fat and purified to homogeneity. This enzyme is larger in molecular weight than previously studied soluble galactosyltransferases, but resembles in size the galactosyltransferase of lactating mammary Golgi membranes.In contrast, when fat globule membranes were prepared by traditional procedures, which involved washing the fat at higher temperatures, before extraction, galactosyltransferase was not present in the membranes, having been released into supernatant fractions. When the enzyme released by this procedure was partially purified and examined by gel filtration, it was found to be of a degraded form resembling in size the soluble galactosyltransferase of milk. The release is therefore attributed to the action of proteolytic enzymes.Our observations contrast with previous biochemical studies which suggested that Golgi membranes do not contribute to the milk fat globule membrane. They are, however, consistent with electron microscope studies of the fat secretion process, which indicate that secretory vesicle membranes, derived from the Golgi apparatus, may provide a large proportion of the fat globule membrane.While the composition and origin of the membranes surrounding the fat globules of milk have been the subject of many studies (1,8,16,23,24), there are serious differences between the conclusions reached on the basis of electron microscope and biochemical analyses.The elegant microscope studies of Wooding (23, 24) suggest that, within the lactating mammary epithelial cell, fat droplets originate near the basal portion with no surrounding membrane, but that during passage to the apex of the cell before secretion they become associated with membranebounded vesicles derived from the Golgi apparatus. Under some conditions, fat droplets are observed within the cell included in vesicles which also contain aqueous constituents and casein micelles (25). Fat globule membranes can therefore originate without any contribution from the plasmalemma. Normally the acquisition of the membrane occurs at the plasmalemma during secre-

Freeze-fracture observations of the lactating rat mammary gland. Membrane events during milk fat secretion.

“…Morphological and biochemical studies of secretion in different cells, including mammary epithelial cells during lactation, provide evidence for a continuous turnover between endomembranes and the plasma membrane segments related to the secretory processes (20). These events could explain the presence of Golgi (24,25,34) and endoplasmic reticulum enzymes (17,25) in MFGM preparations and their phospholipid composition intermediate between that of the Golgi and the plasma membranes (34), even if contami-2 Although clearing of membrane particles was more frequently observed, it may eventually not correspond to the prevalent mechanism if the extrusion without particle segregation is accomplished faster. a The segregation of actin during extrusion is, in view of the particle rearrangements which we observed, a preferable alternative to account for the absence of actin in MFGM preparations.…”

“…However, it has been recently assumed that the overall composition of the MFGM relative to the plasma membrane of the alveolar epithelial cells is not affected (19). Unfortunately, analysis of the apical plasmalemma of the lactating mammary cell is not accessible (34).…”

Physiological role of indigenous milk enzymes: An overview of an evolving picture

“…This phenomenon probably reflects the cellular sources of these membranes, i.e., Golgi membranes (Powell, Jarlfors, & Brew, 1977), the rough endoplasmic reticulum (RER) (Jarasch, Bruder, Keenan, & Franke, 1977) and plasma membranes (Dowben, Brunner, & Philpott, 1967). A prominent marker of Golgi membranes from bovine mammary gland epithelial cells is galactosyltransferase (Keenan, Morre, & Huang, 1972), which also has high specific activity in the MFGM (Powell et al, 1977). A b-type cytochrome which is specifically associated with the RER in cows has also been identified in MFGM and milk microsomes (Jarasch et al, 1977).…”

“…Thus, if blood serum is a source of a given milk enzyme, it is probably through dedicated transcellular transport systems, such as that responsible for the transfer of immunoglobulins (McManaman & Neville, 2003) The largest group of indigenous milk enzymes is that associated with the MFGM and vesicle membranes (Shahani et al, 1973). This phenomenon probably reflects the cellular sources of these membranes, i.e., Golgi membranes (Powell, Jarlfors, & Brew, 1977), the rough endoplasmic reticulum (RER) (Jarasch, Bruder, Keenan, & Franke, 1977) and plasma membranes (Dowben, Brunner, & Philpott, 1967). A prominent marker of Golgi membranes from bovine mammary gland epithelial cells is galactosyltransferase (Keenan, Morre, & Huang, 1972), which also has high specific activity in the MFGM (Powell et al, 1977).…”

Antibodies to the major insoluble milk fat globule membrane-associated protein: specific location in apical regions of lactating epithelial cells.

“…It has been suggested or implied by other workers that MFGM originates, at least to a considerable degree, directly from secretory vesicles or Golgi apparatus without first being integrated into apical plasma membrane (24,39,44,46) . Although our results do not exclude the possibility of some contribution from other cell membranes to the formation of MFGM, they appear to strongly favor the concept that apical plasma membrane is the major source of the membrane complex enveloping the milk lipid globules .…”