Biochimica et Biophysica Acta, 415 (1975) 273-309 © Elsevier Scientific Publishing Company, A m s t e r d a m - Printed in The Netherlands BBA 85150

THE

MILK

FAT

GLOBULE

MEMBRANE

S. P A T T O N and T. W. K E E N A N

Department of Dairy Science, Pennsylvania State University, University Park, Pa. 16802 and Department o f Animal Sciences, Purdue University, West Lafayette, Ind. 47907 (U.S.A.) (Received March 19th, 1975)

CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

273

II.

Origin of the membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

274

A. The alveolus and the lactating cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Formation of milk constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Milk lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Milk proteins and lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

274 277 277 277

C. Milk secretion mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Membrane transformation and flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Secretion of milk fat globules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Emptying of secretory vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Membrane material in skim milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

278 278 278 280 282

IlL Isolation of the membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Composition of the membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V.

283 288

A. Gross composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

288

B. Lipid composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

290

C. Protein composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

292

D. Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

296

Critique of the membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

299

A. Origin of skim milk membrane

299

..............................................

B. Membrane sidedness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary

....................................................................

300 300 301 303

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

304

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

304

I. I N T R O D U C T I O N S i n c e t h e 1 7 t h c e n t u r y o b s e r v a t i o n s o f v a n L e e u w e n h o e k it h a s b e e n k n o w n t h a t the fat in milk exists as minute globules, a few #meters in diameter.

During the 19th

c e n t u r y , s c i e n t i s t s b e g a n t o i n t e r e s t t h e m s e l v e s in w h a t s t a b i l i z e s t h i s f i n e e m u l s i o n .

274 In time, such investigations were pressed forward for very practical reasons. The separation of milk into cream and skim milk, the whipping of cream, the churning of butter and many considerations in the flavor and texture of milk products were subsequently shown to be related to properties of the milk fat globule surface. A key fact emerged quite haltingly from these efforts; namely, that the milk fat globule is secreted from the lactating cell by envelopment in plasma membrane. As recently as 1955, King [1] espoused a popular misconception that the milk fat globule membrane derived from a physico-chemical ordering of milk constituents on the surface of the triglyceride core, with the less polar molecules grading toward the more polar ones at the surface. It is evident from King's review (see ref. 1, p. 41) that the truth of the matter was not far off, since a number of workers had been speculating on the biological origin of the membrane. Moreover Jeffers [2], using light microscopy in 1935, quite accurately described the milk fat globule secretion process as involving an enmeshing of the globule in the limiting membrane of the cell. This was shown more precisely in the late 1950's by the electron microscopy studies of Bargmann et al [3,4]. That the milk fat globule is wrapped in plasma membrane of a cell, which under other circumstances is involved in mammary malignancies makes this membrane of special interest. This is rendered the more intriguing in that both lactation and mammary cancer, at least certain forms of it, are mediated by the hormone prolactin. This review covers the origin, isolation and composition of the milk fat globule membrane. It attempts to analyze some controversies about the membrane and it supplements earlier and related reviews, [1,5-9]. The role of the milk fat globule membrane in industrial dairy processing (milk, cream, butte1, etc.) is not covered since it has recently received thorough treatment by Mulder and Walstra 19]. Such knowledge as we have, indicates that ultrastructural appearances of the lactating tissue, its secretory mechanisms and its product, milk, are similar from one species to another*. All milks studied to date contain fat globules averaging several #meter in diameter. However, globules of only one species, the bovine, have been subjected to extensive investigations.

II. ORIGIN OF THE MEMBRANE IIA. The alveolus and the lactating cell The nature of the milk fat globule membrane is best understood with some knowledge of the globule's origin. A fundamental unit of structure in lactating mammary tissue is the alveolus (Fig. 1, left), a sphere-shaped arrangement of lactating ceils around a lumen. Each lumen is a receptacle for the milk secreted by the ceils adjoining it and all the lumens are connected by a duct system draining to the outlet(s) at the skin surface. Each alveolus has an arterio-venous capillary system which * As revealed by electron photomicrographs of lactating tissue from the human, cow, goat, horse, pig, deer, cat, rabbit, guinea pig, rat and mouse.

275

D DROPLE~ HONDRION OPLASM~ ~CULUM

Fig. 1. Appearance of the principal microstructures involved in lactation. At left is a single alveolus showing arrangement of lactating cells and (hollow) lumen area into which milk is secreted. At right is enlarged version of a single lactating cell illustrating the mechanism of protein and fat secretion and showing the principal membrane systems of the cell, i.e., endoplasmic reticulum, mitochondrial, Golgi apparatus and plasma membrane (cell envelope). (From Milk, by S. Patton, Copyright C 1969 by Scientific American, Inc. All rights reserved).

supplies it with raw materials for making milk. The actual synthesis of the milk fat globule takes place in the lactating cell (Fig. 1, right) as does synthesis of lactose, the principal and distinctive carbohydrate of milk, and most of the milk proteins. As portrayed in the figure, fat droplets* form in the cell and ultimately move to the apical region where they become enveloped in plasma membrane and are expelled from the cell into the lumen. Impressive electron photomicrographs of milk fat globules at the ultimate point of secretion from the cell have been presented by Bargmann and K n o o p [4] and Wellings [10] among others. In those presentations the fat droplet is seen to be connected to the cell by only a narrow neck of cytoplasm as in Fig. 1. F r o m the nature of this secretion mechanism it is evident that the secreted milk fat globule has several surfaces. There is the surface that the globule had when it existed in the cell prior to secretion and there are the inner and outer surfaces of the plasma membrane, which are super-imposed on the droplet surface at secretion. It is important to remember that the outside of the cell's plasma membrane becomes the exposed surface on the milk fat globule. This point is relevant to subsequent discussion of membrane "sidedness". Another consideration is that the lactating cell

* For the sake of distinction throughout this review, the formative milk fat globule within the cell is called a droplet whereas after secretion from the cell it is designated a globule.

276

Fig. 2. An electron photomicrograph of a lactating cell (rat) with parts of several other adjoining cells. Organelles and membranes are as follows: nucleus, N; with small fat droplet, d; mitochondria, m above it; extensive parallel arrays of rough endoplasmic reticulum, er; Golgi vesicles (containing dark staining casein micelles) above Golgi region, G; heavily stained tight junction, j, with protruding array of microvilli, mv. Note evidence of membrane (dark line) around Golgi vesicles, limits of the cells and secreted milk fat globules, g, in lumen, L, but not around fat droplets, d, within cells. Horizontal diameter of nucleus is 3.1 #m. (Micrograph courtesy of B. H. Stemberger).

has three kinds of plasma membrane: that in the basal region of the cell concerned primarily with uptake of substrates from the circulation, that in the lateral region involving contact (communication) with adjoining mammary epithelial cells and that in the apical region concerned particularly with secretion. Only this latter plasma membrane is represented on milk fat globules. The lactating mammary cell contains

277 the classic structures of typical mammalian ceils, Figs 1 and 2. Those interested in further information on structure and function of mammary tissue may consult a threevolume treatise on lactation [11] and recent monographs [8,12]. IIB. Formation of milk constituents From the literature it is evident that membranes accomplish both the synthesis and secretion of milk and that the membranes themselves as well as their products are constituents of milk. In order to document and clarify this matter it is appropriate to start with findings on synthesis of milk constituents. IIB-1 Milk lipids. Greater than 95 % of the lipids in bovine and other milks is triglyceride contained in milk fat globules. Fatty acids for the synthesis of the milk glycerides are derived from the blood lipids, particularly from triglycerides of chylomicrons and very low density liproproteins. A second important source of such fatty acids is de novo synthesis in the mammary tissue. In the bovine it appears that each of these pathways supply roughly one half of the milk triglyceride fatty acids. Milk fatty acid synthesis has been the subject of several extensive reviews [8,13,14]. Milk triglycerides are synthesized by a particulate (microsomal) fraction of mammary tissue [15,16] and electron microscopy - - autoradiography studies of Stein and Stein [17] using the lactating mouse have shown that the precise site is the rough endoplasmic reticulum. Forming milk fat droplets are consistently seen enmeshed in endoplasmic reticulum in the basal region of the cell. These droplets appear to increase in size as they move toward the secreting (apical) end of the cell [4,18]. The limited knowledge of the biophysical processes by which these fat droplets grow in size has been discussed elsewhere [6,8]. One approach to revealing the surface composition of intracellular milk fat droplets is to isolate and analyze the droplets. One such study [19] has indicated that the surface contains phospholipid, cholesterol and protein. Confirming earlier observation [20,21], the phospholipid fraction was found to be substantially enriched in phosphatidylcholine as compared to the total phospholipids of either the tissue or milk. The pattern of phospholipids associated with the droplets was similar to that of endoplasmic reticulum, which is consistent with the electron photomicrographic evidence relating this membrane to the droplet (Section III). Recent reviews have dealt with synthesis, secretion and composition of the milk lipids [5,6,8,9,13,14,22-27]. IIB-2 Milk protein and lactose. Exclusive of many organic compounds at trace levels ( > 0.1%), the other principal milk constituents made and secreted by the cell are the milk proteins (caseins, fl-lactoglobulin and a-lactalbumin) and lactose. The syntheses of these substances are initiated through the action of the pituitary hormone, prolactin, at parturition. In fact one of the milk proteins, a-lactalbumin, together with UDPgalactosyl transferase, forms the enzyme, lactose synthetase [28,29] so that milk protein and lactose synthesis are necessarily coupled. Lactose synthetase activity has been shown to be concentrated in the Golgi apparatus [30]. Casein occurs in milks of various species, including cow, goat, rat, mouse and human, in the form of micelles. These stain densely with heavy metals as viewed in

278 the electron microscope. They exhibit a wide size range and in the case of the bovine and human, average 1000 A in diameter. Casein micelles are identifiable histochemically in intracellular secretory vesicles (Fig. 2). It is established that the peptide chains of this family of proteins are made on ribosomes of the rough endoplasmic reticulum [31] and that phosphorylation of the peptides and their calcium-mediated assembly into micelles occur in the Golgi apparatus and secretory vesicles [32-34]. The concentrations of lactose, casein, fl-lactoglobulin and a-lactalbumin in bovine milk average approximately 5.0, 3.0, 0.4 and 0.l ~ , respectively. Several recent reviews of the milk proteins are available [35-39].

IIC. Milk secretion mechanisms IIC-I Membrane transformation andflow. While there is much remaining to be learned about structure and function of membranes in any cell or tissue, the secretion of milk by the lactating mammary cell is known to remove the membrane from the cell and the process must necessarily involve replacement. In the late 1960's Morr6 and his colleagues [40,41] began documenting earlier proposals that the Golgi apparatus is the site in the cell where membranes are transformed from endoplasmic reticulum-like to plasma membrane-like. The necessity for invoking membrane transformation and flow was also noted by Patton et al. [20,21] in connection with milk secretion. More precisely, they suggested that plasma membrane removed from the cell in the process of milk fat globule secretion is replaced by secretory vesicle membranes, which fuse with the plasma membrane at the time these vesicles empty their contents (the non-fat globule phase of milk) into the lumen. The flow of membrane from Golgi apparatus to plasma membrane requires replacement of Golgi apparatus membrane, especially in that the Golgi apparatus of itself has little or no protein and lipid synthetic capability. A scheme relating the flow of membrane and the synthesis and secretion of milk components is presented in Fig. 3. One problem with the concept of membrane flow is that the amount of surface involved in Golgi apparatus-derived vesicles in the lactating cell greatly exceeds that removed by milk fat globule secretion. It seems necessary to embrace the idea of partial recycling, resorbtion or retention of Golgi vesicle membrane in the lactating cell. A more detailed consideration of this matter and the concept of aging in relation to membrane transformation are presented elsewhere [8]. IIC-2 Secretion of milk fat globules. The envelopment of milk fat droplets in plasma membrane at secretion produces one of the most unique dispositions of a biological membrane in all of nature (Fig. 4). Yet very little is known of how this happens. There is first of all, the unexplained movement of the forming fat droplet from the interior of the cell to a location for secretion in the apical region. Generally, this latter region contains fewer organelles and displacement into unoccupied cytoplasm may be one factor in the movement of fat droplets and secretory vesicles to this location. Pressure effects must also be important since the shape of cells in the alveolus is clearly influenced by the presence or absence of milk in the lumen. It has been noted since the studies of Jeffers [2] that the cells are flattened when the lumen is

279

Q

5

•

t ~

":. @;"

4 @

3

Fig. 3. A conception of the relationship between membrane flow and product flow in the synthesis and secretion of milk. Lower left: protein components of milk and enzymes involved in the synthesis of milk are vectored from the ribosomes, where they are made, into the cisterna of the endoplasmic reticulum. The product and membrane flow in the direction of the arrows from the region of endoplasmic reticulum, (1); to the Golgi apparatus, (2); where the skim milk phase is completed and packaged into secretory vesicles, (3); which fuse with and empty through the plasma membrane, (4); this membrane also accomplishes secretion of milk fat globules (dark spheres) by a packaging-type of envelopment with release into the alveolar lumen, (5).

full and that they are elongated into an empty lumen. Jeffers suggested that this projecting of the cell into the lumen tends to draw the fat droplet into a protruded position in the apical plasma membrane. The readily observed rise in the fat content of milk toward the end of a milking [42] suggests that fat droplet secretion may be favored by change in cell shape (pressure) as the lumen empties. In addition to pressure, or lack of it, established by the relative fullness of the lumen, there are pressures transmitted at the base of the, cell by tension in capillaries and by contraction of myoepithelial cells under influence of the "milk ejection" hormone, oxytocin. This latter action is stimulated by suckling or manipulation of the m a m mary gland. It is a further question as to what attracts the formed fat droplet to the plasma membrane. Affinity due to the substantial lipid content of both seems likely. On this basis Patton and Fowkes [20] presented evidence to show that London-van der Waals forces would achieve an attraction of one atmosphere when the distance between membrane and droplet is reduced to 20 A. Since this attraction is inversely related to distance it predicts that once the two surfaces are brought within a critical distance envelopment of the droplet would automatically progress from a point of close approach to extend ovel the entire surface. By this means one can visualize a relatively rapid snapping of the droplet across the membrane. From his observations with the electron microscope Wooding [18] disagrees with this explanation. He finds the distance between membrane and droplets too large (100-200 A) in secreted milk fat globules to accommodate the data of Patton and Fowkes. Of course the precision with which electron microscopy practices may reproduce distances in the living cell, as well as the accuracy with which attraction forces can be imputed in such cells are at question here. But it does seem plausible that lipophilic elements of the plasma membrane and of the fat droplet generate hydrophobic forces, which assist in bringing the two surfaces together. It is evident that secretion of milk fat globules as well as

280

Fig. 4. Milk fat globule (rat) in process of being secreted from cell. Large mitochondrion lies under globule inside cell. Two casein micelles are evident in alveolar lumen (upper left by arrow). Note plasma membrane (diameter 74 ~) extending out from cell surface, upper left, around protruding globule. Well-resolved unit membrane can be seen at various sites on globule (arrow and lower right opposite arrow). Globule is 1.60 x 1.27 #m in long and short diameters. (Electron micrograph courtesy of P. S. Stewart). e m p t y i n g o f secretory vesicles, discussed following, are p o o r l y u n d e r s t o o d biophysical p h e n o m e n a . 11C-3 Emptying of secretory vesicles. As discussed r e g a r d i n g milk fat d r o p l e t s there m a y be general physical forces, bearing on the cell, which tend to displace secretory vesicles into the apical region o f the cell. However, the p r o b a b i l i t y o f m o r e discrete r e g u l a t i o n o f intracellular m o v e m e n t o f secretory vesicles is suggested by recent re-

281 search on microtubules. Drugs such as colchicine and vincristine which disassemble microtubules have been observed to interfere with secretory processes in the liver[43, 44] and pancreas [45]. Recently these two alkaloids have been shown to suppress lactation in the goat [46]. Both fat globules and aqueous phase secretions were inhibited. Microtubules are known to exist in the apical web of the lactating cell [47]. The possibility that they are involved in milk secretion opens up an interesting new research area. Milk fat globules have been photographically recorded rather convincingly in the midst of their secretion from the cell (e.g. Fig. 4). To see secretory vesicles in such a circumstance is problematical. What must occur is an interaction between the vesicle and the plasma membrane such as to allow the vesicle contents to be emptied out of the cell. Photomicrographs of the fusion and opening, involving two membranes each roughly 100 A in diameter, could readily be criticized as artifactual because of membrane fragility. Beery et al. [32] have recorded an instance which appears to be at the very moment before emptying of the vesicle (see Fig. 13 in ref. 8). Carollet al. [48] show by photomicrograph, the actual opening of the vesicle through the plasma membrane (see their figure in ref. 34). Whereas the milk fat globule has a core of more or less pure triglyceride, the content of the secretory vesicle is presumed to be the skim milk phase of milk, i.e., casein micelles, other milk proteins, lactose, salts, water, etc. That casein micelles, dark staining granules of about 1000 A diameter, are observable within secretory vesicles and in the alveolar lumen shows that these particles by some means get across the plasma membrane. There is no evidence that this occurs by a mechanism other than that depicted in the photomicrograph of Carollet al. and in the scheme, Fig. 3. Since secretory vesicle contents and milk fat droplets are secreted by the same membrane and approach this membrane from the same side, it is evident that there must be important differences in the surface composition and properties of the vesicle and the droplet, or they would be secreted by the same type of mechanism. One seldom, if ever, sees an intact secretory vesicle in milk, the casein micelles being free-floating in the continuous aqueous phase. If it is assumed that fusion of at least some vesicle membrane with plasma membrane replaces membrane removed in secretion of milk fat globules, it can be reasoned that the interior side of plasma membrane must be similar to the exterior of secretory vesicle membrane. This results from a consideration of the geometry of the event (see Fig. 3). It has been shown by Papahadjopolous et al. [49] that artificial vesicles composed of phosphatidylcholine, phosphatidylserine and cholesterol, exhibiting a classical bilayer membrane structure, will fuse with plasma membranes of mammalian cells. These lipids are principal components of the plasma membrane and presumably also of secretory vesicle membranes of the lactating cell (see Section IV, Composition). From considerations of "sidedness", discussed in Section V, the membrane surfaces in question may contain most of the phosphatidylserine in the membranes. Membrane fusion is implicit at many points in the functioning of the Golgi apparatus including the initial blebbing of vesicles from the apparatus, the merging of these relatively small vesicles to form

282 larger ones, as well as the emptying of vesicles through fusion with the plasma membrane. Bovine milk is the best known dietary source of calcium, containing an average 120 mg/100 ml. Very little attention has been given at the cellular level to the role of calcium ions in synthesis and secretion of milk but the potential importance of these ions in regulating (and inhibiting) both of these processes is intriguing. Calcium ions appear to facilitate membrane fusion [50], protein secretion [51], microtubule disassembly [52,53] and casein micelle formation [34,54]. HC-4 Membrane material in skim milk. It is common experience in commercial dairying that no matter how efficiently milk is separated into cream and skim milk a small content of lipid, about 2 ~ of the total milk lipid, remains in the skim milk. The common explanation given for this is the existence of very small fat globules, which cannot be removed from skim milk with complete efficiency. In 1964 evidence was presented that the lipids in skim milk are not fat globules but are contained in membranes which actually sediment in a strong centrifugal field [55]. Stewalt et al [56] characterized these membranes in an ultrastructural study. By ultracentrifugation of skim milk, membrane material could be recovered as a fluffy layer on the surface of the casein pellet. Spherical vesicles ranging up to 300 or 400 nm in diameter were observed. A substantial portion of the smaller sac-like vesicles were identified as microvilli by their shape and dimensions, as viewed in both embedded and negatively stained preparations under the electron microscope. No doubt these were sloughed from the surface of mammary cells (see Fig. 2). Microvilli have been prepared from human placentas by gentle agitation in ice cold 0.9 ~o NaCI solution for 30 min [57]. Of course, milk of cows and goats is retained in the udder for hours between milkings and thus milk may be viewed similarly, as a physiological salt solution washing cell surfaces. Consistent with this point, less membrane material (phospholipid and cholesterol) is recovered in goat milk after milking the animal several times, at hourly intervals [58]. This suggests that the amount of microvilli in milk is a function of contact time between extracellular milk and tissue. These observations may assist cell scientists looking for sources of microvilli. Biochemical studies of membrane material in skim milk [59,60] have shed light on the general question of membrane origin. One conception of the biosynthesis of membranes is that they might be built from lipoprotein subunits. Since membrane fragments exist in milk along with cell debris and even intact epithelial cells, milk would seem to be an appropriate site in which to search for such subunits. By passing bovine skim milk through a column of Sepharose 4B, it was shown that all of the lipid phosphorus and 5'-nucleotidase activity were excluded into the void volume, indicating the membrane material exceeded a particle weight of 20 × 106 and the absence of subunits [59]. These membrane vesicles have the lipid composition and certain marker enzymes (5'-nucleotidase, glycosyl hydrolase) characteristic of plasma membrane [60]. The origin and nature of this membrane material is discussed further in Section V. The tendency of membranes to form vesicles is well known. Such membranous

283 vesicles, as occur in skim milk and as may be released from milk fat globules under various conditions, seem to be identifiable with the "milk microsomes" or "lipoprotein particles" described and characterized earlier (1954-1958) by Morton and Bailie [61,62].

ItI. ISOLATION OF THE MEMBRANE In a sense isolation is a misnomer since the actual separation of the milk fat globule membrane from the secretory cell is accomplished during the process of cellular discharge of the fat globule. To obtain fat globule membranes, all that need be done is to separate fat globules from the milk serum, remove and collect the membrane. Palmer and Samuelsson [63] were apparently the first to describe a method for dissociating the membrane from the globule and recovering this membrane for further study. Many of the steps used in the procedure developed by Palmer and Samuelsson are similar or identical to those in use today. The first step in recovery of the globule membrane is to separate fat globules from milk serum. Since they have a density less than that of water by virtue of their high lipid content, fat globules float to the top of columns of milk. Globules are thus readily isolated by centrifugation in laboratory centrifuges or in mechanical cream separators; the latter devices being particularly useful for large scale isolations. Speed of centrifugation is not critical as all but the smallest fat globules can be nearly quantitatively separated by centrifugation at a few thousand g forces for periods as brief as 10 min. After recovery, fat globules are dispersed in water or, better, buffered or unbuffered isotonic solutions and reseparated; this process being repeated a sufficient number of times to remove all entrained milk serum constituents. In our hands, better yields of membrane are obtained with isotonic wash solutions (sucrose or NaCI) than with water or dilute buffers. This appears to be due to greater stability of the globules and consequently, less separation of membrane fragments during washing. For certain studies of membrane constituents (e.g., enzyme activities, electrophoretic mobility, labeling of external membrane constituents), washed globules can be used as such. For other studies, it is necessary to dissociate the membrane from the globule by physical or chemical methods. Direct extraction of membrane constituents from the globule is a common chemical procedure. For example, extraction with sodium dodecyl sulfate has been used to recover solubilized membrane proteins for electrophoretic analysis [64,65] and membrane phospholipids have been recovered by direct extraction of globules with such solvents as chloroform/methanol [66,67]. These procedures are valid, since the floating lipids recovered after removal of the membrane are devoid of phospholipid and protein [19,68-70]. While to date there have been no reports of stabilization of large membrane fragments from fat globules by chemical means, approaches based on methods used during dissociation of plasma membranes from cultured cells may be feasible [71,72]. Physical methods which have been used to dissociate the membrane from the globule include

284 churning, freezing - - thawing and sonication. Slow freezing and thawing is quite effective for dissociation of the membrane from the globule; rapid freezing and thawing is much less effective [66,73]. Churning can be accomplished by agitation, with a hand mixer or blender, or with homogenizers or butter churns. Sonication, while useful, is time consuming with large volumes. On lysis, the lipid in water emulsion is destabilized and the core lipids coalesce or, at higher temperatures, rise as an oil. The membrane material is concentrated in the aqueous phase and can be obtained as a pellet by centrifugation. There has been no systematic evaluation of the centrifugal forces necessary to quantitatively sediment membranes released by different procedures. In our hands, centrifugation at 100 000 x g for 60-90 min has been sufficient to pellet all membrane material released either by churning or freezethawing (as judged by the absence of phospholipid and 5'-nucleotidase activity in the aqueous supernatants). If lysis and centrifugation are conducted at temperatures above the melting range of the globule core, the resultant oil is free of phospholipid [66-69]. However, if these operations are performed below the melting range, considerable quantities of membrane become entrained in the lipid phase. This membrane material can be recovered by melting the solidified fat, washing with some aqueous medium and centrifugation [5]. As an alternative to high speed centrifufugation, membrane material can be precipitated from the aqueous phase by lowering the pH or by addition of ammonium sulfate and the precipitated membrane can then be collected by filtration or low speed centrifugation [5,74,75]. From the above, it will be obvious to the experienced tissue fractionator, that fat globule membranes can be isolated much more rapidly than can plasma membranes from homogenates. It is also obvious that there is considerable latitude in choice of conditions for isolation of this membrane from milk. The conditions selected are dictated largely by the constituents of interest. In some cases washing of the fat globules may be unnecessary, whereas for some studies it is critically important to remove all milk serum constituents. Washing procedures will necessarily remove loosely associated membrane constituents as well as entrained or adsorbed milk serum constituents. While this is a disadvantage, nearly all fractionations of tissue or cell homogenates suffer the same disadvantage. Similarly, all other steps in recovery of fat globule membranes are subject to certain criticisms. Nevertheless, recovery of constituents of interest is dictated largely by the isolation method chosen. For details of representative isolation methods the reader is referred to refs [5,66, 73-76]. Yield of milk fat globule membranes is dictated by many factors. The lipid content of the milk determines in large measure the amount of fat globule membrane material which is present. The milks of all species which have been examined contain fat globules with diameters in the range of about 1-10 tzm [37, 77]. The lipid content of milks varies from as low as 0.2 9/o or less in species such as the rhinoceros, to as high as 50 9/00in certain marine pinnipeds [37,77]. By far, globule membranes obtained from the milk of domestic cows have been the most widely studied. The lipid content of cows' milk varies with breed, diet and stage of lactation, but averages 2.5-6.0~

285 for the common domesticated breeds [37,77]. In cows' milk fat, the globules range in diameter from 1 to 10/~ and the size distribution profile has been shown to vary with stage of lactation [78-80]. Given a constant fat percentage, milks with a greater abundance of smaller globules contain more globule membrane material. In addition to the surface area to volume ratio of the lipid globules, membrane yield can be affected by the age, the degree of agitation or temperature manipulation of the milk. Extended washing will eventually cause destabilization and consequent loss of membrane material from the lipid globules. There appear to be breed and seasonal variations in stability of lipid globules [5]. In his extensive survey of the literature, Brunner [5] found that reported yields of membrane from milks of cows ranged from 0.5 to 1.5 g dry weight per 100 g milk. The possible loss of membrane material from globules before or after withdrawal of milk from the animal is considered herein. To what extent the milk fat globule membrane fraction finally isolated consists of membrane fragments derived from cellular membranes, other than the apical plasma membrane, is an important consideration. Evidence that the milk fat globule membrane is similar to plasma membranes in composition will be presented in a following section. When isolated by the general scheme outlined above, any membrane material associated with fat globules could be present in the final membrane fraction. One potential source of such contaminating membranes is entrainment of intracytoplasmic organelles or membrane fragments between the apical plasma membrane and the lipid globule during cellular discharge of the globule [18,22,81-83]. It has been repeatedly observed that a proportion of extracellular milk fat globules of several species include crescents of cytoplasm entrained in this manner [18,22, 81-83]. These cytoplasmic crescents contain membranes identifiable as originating from mitochondria, rough endoplasmic reticulum and smooth endomembranes (Golgi apparatus or smooth endoplasmic reticulum). While quantitative data have not been forthcoming, it has been claimed that there is species variation in the proportion of globules containing cytoplasmic crescents [83]. Since constituents or enzymatic activities characteristic of mitochondria [61,62,84,85], Golgi apparatus [86,87] or endoplasmic reticulum [74,85] are absent or present in only very low levels in globules or globule membranes from bovine milk, it is apparent that crescents contribute at most only a small amount of membrane material to fat globules in at least certain breeds of domestic cows. The presence of constituents characteristic of intracellular membranes in globule membrane fractions from human [88] and rat [89] milks, for example, indicate that entrained material may make significant contributions to fat globule membrane fractions from certain species. Where such contamination becomes a significant problem, the possibility of purification of the milk fat globule membrane by centrifugation in density gradients can be explored. Some caution must be exercised in this approach, since the milk fat globule membrane is not homogenous with respect to buoyant density [64,90]. By centrifugation of milk fat globule membrane on sucrose step-gradients, seven fractions ranging in density from less than 1.13 g/cm 3 to greater than 1.19 g/cm 3, were collected. While all fractions had identical 5'-nucleotidase and xanthine oxidase specific activities on a protein

286 basis, the total lipid and phospholipid contents of these fractions varied inversely with density (K. Weber, I. H. Mather and T. W. Keenan, unpublished). Kobylka and Carraway [64] separated three distinct fractions of milk fat globule membrane by sucrose density gradient centrifugation and observed that the electrophoretic pattern of polypeptides in all fractions was essentially identical to that of the initial milk fat globule membrane. Swope and Brunner [90] obtained three separate fractions by differential centrifugation of milk fat globule membrane suspensions. These fractions were found to have the same amino acid composition but widely different amounts of total lipid, phospholipid and cholesterol; the lipid content varied inversely with sedimentation velocity. These observations lead to the conclusion that milk fat globule membrane can be subdivided into several fractions with homogenous protein composition but with different lipid contents. Whether this variation in lipid content reflects true differences in plasma membrane composition or simply manipulation-induced differences or varying entrainment of globule triglyceride remains to be determined. That some of this difference may be due to isolation procedure is suggested by observations that a high melting triglyceride fraction is tightly adsorbed to the interior face of the milk fat globule membrane, when isolation is performed in the cold. Membranes isolated at 37 °C contain much lower amounts of these high melting triglycerides (compare refs 91 and 92). Another potential source of contamination of the milk fat globule membrane is the aquisition of constituents existing on the presecretory globule surface. Within the cell, lipid droplets have some protein, phospholipid and ganglioside, yet the lipid fraction recovered after removal of the membrane from milk fat globules is nearly completely devoid of these constituents [19,66,68-70). It is thus reasonable to assume that these materials add to the fat globule membrane and are recovered in this fraction. Because electron micrographs of fat droplets in situ have failed to reveal the presence of a limiting membrane, e.g. in refs 18, 22, 81, 83 and 93-95, it has been assumed that the proteins and polar lipids associated with intracellular fat droplets are adsorbed at the surface and serve to stabilize the droplet in the aqueous cytoplasm [19,20,69,70,76]. However, recent observations suggest that failure to detect a limiting membrane around intracellular lipid droplets may be due to limitations of the classical glutaraldehyde-osmium tetroxide fixation technique. As shown in Fig. 5, direct osmication of lactating rat mammary tissue reveals the presence of a single thin membrane, which is nearly but not completely continuous around the surface of intracellular lipid droplets (E. D. Jarasch, T. W. Keenan and W . W . Franke, in preparation). This membrane appears to represent one-half of the cisternal bilayer of agranular endoplasmic reticulum. As the droplet grows in size, the cisterna appears to be pulled apart gradually with one membrane enveloping the lipid droplet. That ~h!s membrane does not appear to be completely contiguous around extracellular fat globules makes quantification of its contribution to the total membrane material surrounding milk fat globules difficult. However, analyses of bovine milk fat globule membrane and endoplasmic reticulum fractions has shown the former to contain less than 10~ of the b-type cytochrome of the latter on a protein basis, suggesting

287

Fig. 5. Electron micrograph showing the partial envelopment of an intracellular lipid droplet, LD, by a membrane, which appears to represent one-half of the cisternal membrane of endoplasmic reticulum (arrows). The membrane on the surface of the lipid droplet lacks ribosomes. This membrane is still seen in milk lipid globules, MLG, which are enveloped by plasma membrane (arrowheads in the insert). CV, casein-containing secretory vesicles; AL, alveolar lumen. Lactating rat mammary tissue was fixed by soaking directly in osmium tetroxide and then processed for sectioning by standard methods. Magnification, × 30 000. Insert magnification, × 50 000.

288 that endoplasmic reticulum contributes no more than 10 ~ of the protein in milk fat globule membrane fractions (E. D. Jarasch et al., in preparation). These observations, considered together with previous studies showing that milk fat droplets form and grow in close proximity to a granular endoplasmic reticulum [17,18,81 ], suggest that this latter membrane may provide the nucleation site for initiation of lipid droplet formation and also serves to stabilize lipid globules in the cytoplasm. Support for this suggestion comes from observations that the enzymatic machinery involved in incorporation of fatty acids into triglycerides and phospholipids are contained in endoplasmic reticulum [15-17] and that intracellular lipid droplets have a phospholipid distribution pattern strikingly similar to that of endoplasmic reticulum (compare refs 19, 76 with 96). A further consideration is that relatively mature intracellular fat droplets in the apical region may have more or different surface materials associated with them, than do growing droplets embedded in endoplasmic reticulum. While there are limitations to the use of milk fat globule membrane as a source of plasma membrane, these are not so severe as to rule out its utility for such studies. Certainly, many of the criticisms of the isolation procedures and the purity of the final fraction are also applicable to plasma membranes isolated from tissue or cultured cell homogenates. As will be documented in a following section, the milk fat globule membrane compares favorably in composition with the cleanest plasma membrane fractions obtained from tissues such as liver. Further purification of the milk fat globule membrane fraction to yield a preparation even more characteristic of apical plasma membrane is certainly feasible. In addition to methods such as density gradient centrifugation, the high negative charge of fat globule membranes [97] suggests that preparative zone electrophoresis [98] may be a useful method for further purification.

IV. COMPOSITION OF THE MEMBRANE IVA. Gross composition While the composition of milks from a great diversity of species has been cataloged [37,77], the only milk fat globule membranes which have been studied extensively are those of the bovine. Investigators have recently begun characterization of fat globule membranes from milks of other species, but the data are still meager and relatively incomplete. This section will be restricted largely to a review of bovine milk fat globule membranes; only general comparisons with globule membranes from other species will be made. Gross composition of bovine milk fat globule membranes is given in Table I. Taken together, proteins and lipids account for over 90 ~ of the dry weight of the membrane [5,64,66,90,99], but the relative proportions of these two constituent classes vary widely. Protein values ranging from 25 to 60 ~o or more of the dry weight have been reported [5,64,66,90,99]. Part of this difference may be attributable to

289 TABLE I GROSS COMPOSITION OF BOVINE MILK FAT GLOBULE MEMBRANES The glycerides include mono-, di- and triglycerides. Constituent

Amount

References

Proteins Total lipids Neutral lipids Hydrocarbons Sterols Sterol esters Glycerides Free fatty acids Cerebrosides Gangliosides Sialic acids Hexoses Hexosamines

25 to 60~o of dry wt 0.5 to 1.1 mg/mg protein 56 to 80~ of total lipids 1.2 ~ of total lipids 0.2 to 5.2~ of total lipids 0.1 to 0.8 ~ of total lipids 53 to 74 ~ of total lipids 0.6 to 6.3 ~ of total lipids 3.5 nmol/mg protein 6 nmol/mg protein 63 nmol/mg protein 0.6/~mol/mg protein 0.3/~mol/mg protein

5, 66, 108 5, 90, 96, 99, 108 66, 90, 96, 99, 108 99 66, 90, 99 66, 99 66, 99 66, 99 128 69 69, 96 74 90

factors such as breed of the animal, season and stage of lactation and thus represent true variation in composition of the membrane. As discussed in Section II, the age and treatment of the milk as well as the method used to isolate the membrane can influence the composition of the final fraction. Since milk fat globules are > 95 ~o triglyceride, the amount of such lipid contaminating the membrane preparation can greatly influence the analytical results for protein content of the membrane. On a protein basis the phospholipid content of the membrane averages about 0.25 mg/mg protein [5,90,96]. In a carefully controlled study, where care was taken to minimize manipulation artifacts and to remove all milk serum constituents, Swope and Brunner [90] found the protein content of milk fat globule membranes to be about 41 to 43 ~o of the dry weight. This appears to be one of the more reliable values available. Fat globule membrane fractions contain both protein- and lipid-bound carbohydrates (Table I). There are about 55 nmol of protein-bound sialic acid and 6 nmol of ganglioside-bound sialic acid per mg protein in the membrane [7,69,96,100]. Hexoses occur at a level of about 0.6 # m o l and hexosamines at about 0.3/~mol per mg protein [74,90]. Which constituents are present in the neutral hexose fractions is unknown but both glucosamine and galactosamine are found in the protein fraction [64,90] and only galactosamine in the ganglioside fraction [69]. Martel et al. [101] found similar levels of carbohydrates in human milk fat globule membranes. R N A has been identified in bovine milk fat globule membranes [102] and D N A has been detected by Giesma staining (E. D. Jarasch, personel communication). While reliable quantitative data for the bovine membrane are absent, Martel et al. [!01] found 15/zg R N A / m g protein and failed to detect D N A in human milk fat globule membranes. Whether this low level of R N A is a true membrane constituent, as suggested

290 for liver plasma membranes (e.g., ref. 103), or simply reflects the presence of low levels of endoplasmic reticulum in the fraction [17,18,81,82] remains to be determined. A number of elements, notably sulfur, copper, iron, molybdenum, manganese, magnesium, calcium, sodium, potassium and zinc, have been identified in globule membrane fractions by Brunner and his colleagues [5,75,104]. Most of these elements are protein-bound. Changes in the milk fat globule membrane as a result of bovine mastitis have been analyzed [105,106] and an interesting comparison of lipid and protein in the membrane and in a mammary tumor virus from milk of infected mice has also been made [107].

IVB. Lipid composition While the total neutral lipid content of globule membranes is highly variable, the same constituents are commonly detected in this fraction. Glycerides are by far the most abundant constituents of the total membrane lipids (Table I) [66,99,108]. Triglycerides normally constitute 90 ~ or more of the glyceride fraction with diglycerides and monoglycerides accounting for the remainder [66,99,108]. These triglycerides contain a higher proportion of longer chain-length fatty acids, than do the bulk milk triglycerides and are referred to as high melting triglycerides [91,92,99]. In milk fat globule membranes prepared at 0 and 4°C from precooled milk, triglycerides (and possibly other neutral lipids) have been shown to form a thick coat along one face of the membrane [92,109]. These triglycerides are tightly bound in that they resist removal by acetone during dehydration for fixation and electron microscopy [92]. Much lower levels of high melting glycerides are found in membranes prepared at 37 °C from uncooled milk [91]. Microelectrophoretic characteristics of milk fat globules subjected to various treatments led Newman and Harrison [97] to conclude that the membrane surface is predominately ionogenic and contains little neutral lipid. This and the above observations imply that the high melting triglycerides are localized along the inner face of the membrane. Whether any of the triglycerides associated with the membrane are actually present in the lipid bilayer is problematic. Free fatty acids and hydrocarbons such as squalene and fl-carotene are also present in milk fat globule membrane lipid fractions [66,99]. Sterols constitute a significant proportion of the membrane lipids (Table I) [64,66,96,99]. About 8 0 ~ of the total sterol is present in the free form and the remainder is accounted for as sterol esters [66,99]. Cholesterol and its esters represent the dominant sterol; the only other sterols found in milk fat are lanosterol and dihydrolanosterol [110,111]. According to Schwartz [110], the latter two sterols occur in unesterified form only and at about one-sixtieth the concentration of cholesterol. The sterol esters of milk as well as the milk fat globule membrane are unique in that they contain higher proportions of odd carbon chain length saturated and unsaturated fatty acids than do other milk lipid classes [76,112]. Milk fat globule membrane phospholipid fractions contain sphingomyelin,

291 TABLE II PHOSPHOLIPID DISTRIBUTION IN MILK FAT GLOBULE MEMBRANE AND MEMBRANE FRACTIONS FROM BOVINE MAMMARY GLAND Phospholipid

Sphingomyelin Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidylethanolamine Cardiolipin Lyso derivatives Others

~ of total lipid phosphorus Mito [117] NM [118] RER [96] GA [96]

PM [76,96] MFGM [76,96]

3.0 43.2 0.4 4.9

5.0 54.5 4.7 10.3

5.7 57.1 4.1 5.7

12.7 46.8 6.0 7.1

23.5 32.9 4.4 11.6

21.9 36.2 4.1 10.7

25.1 17.8 2.1 3.6

19.6 3.2 3.9 1.5

23.6 0.2 3.7

27.1

25.3 0.4 1.8

27.5

0.5

2.3

Abbreviations: Mito, mitochondria; NM, nuclear membrane; RER, rough endoplasmic reticulum; GA, Golgi apparatus; PM, plasma membrane; MFGM, milk fat globule membrane.

phosphatidylcholine and phosphatidylethanolamine as the major constituents (Table II). Lesser amounts of inositol and serine phosphatides are also present, as are low but variable amounts of lysophosphatides [7,66,67,76,84,96,99,113]. Choline and ethanolamine plasmalogens are also present in milk phospholipids; they account for about 10 and 3 ~o, respectively, of the parent phosphatide fraction [114]. Phospholid distribution patterns similar to that of the bovine membrane have been observed in fat globule membranes from rat [89] and human [115] milks. In bovine and caprine species, about 60 ~ of the milk phospholipids are associated with fat globule membranes, the remainder being contained in the skim milk membrane fraction [59,66,67]. The distribution patterns of phospholipids in milk fat globule membranes and skim milk are identical [66,67]. Whether this is true in milks of other species, remains to be determined but it is of interest that phospholipid distributions in milks of all species examined are similar to that of bovine milk [116]. The only m a m m a r y cellular membrane with a phospholipid distribution pattern similar to the fat globule membrane is the plasma membrane (Table II). Compared to these two membranes, which have nearly identical phospholipid distribution profiles, intracellular membranes have a lower sphingomyelin and higher phosphatidylcholine content [7,76,89,96,117,118]. Less than 6 ~ of the lipid phosphorus of nuclear membrane, endoplasmic reticulum and mitochondrial fractions is present in sphingomyelin and about l 3 ~o of the Golgi apparatus lipid phosphorus is found in sphingomyelin. In contrast, more than 20 ~ of the lipid phosphorus of plasma membrane and milk fat globule membrane is present in sphingomyelin. The relation of these phospholipid differences to endomembrane differentiation has been discussed elsewhere [7,89,96,119-123]. The phospholipid distribution in fat globule membranes is nearly identical to that of liver plasma membranes; in fact, the phospholipid patterns

292 of intracellular membranes from mammary gland are very similar to the phospholipid distribution in respective membrane fractions from liver (e.g., refs. 119,121). The virtual absence of cardiolipin, a characteristic mitochondrial constituent [124] in milk fat globule membranes [84] implies that mitochondria or inner mitochondrial membrane fragments [125] are absent from this fraction. The fatty acid composition of individual phospholipids from plasma membrane and milk fat globule membrane are nearly identical [76]. Phosphoglycerides in these membranes contain appreciable amounts of the unsaturated acids 18:1 and 18:2 (number of carbons: number of double bonds) but only low levels of longer chainlength unsaturated fatty acids [76,96]. The same is true of phosphoglycerides from intracellular mammary membranes [96]. Sphingomyelins in milk fat globule and intracellular membranes of mammary gland are characterized by low levels ( < 10 weight 700)of unsaturated fatty acids and appreciable quantities of 22:0, 23:0 and 24:0 acids [66,76,96,126]. Morrison and Smith [127] identified mono- and dihexose ceramides in lipid fractions from bovine milk. These were subsequently shown to be concentrated in the milk fat globule membrane [126,128] and to have the structures fl-glucosyl(1 ~ 1)-N-acylsphingosine and fl-galactosyl-(1 ~ 4)-fl-glucosyl-(1 -~ l)-N-acylsphingosine [129]. Glucosyl and lactosyl ceramides occur in nearly equal proportions in milk fat globule membrane [126,128] and together are present in levels of about 3.5 nmol/mg protein [128]. Free ceramide has also been identified in total milk lipids [130] and may well be a constituent of the milk fat globule membrane. Neutral glycolipids with longer carbohydrate chains have not been detected in milk fat globule membranes. This may be due to the presence of a highly active CMP-sialic acid : lactosylceramide sialyltransferase in mammary gland [69,87]. Gangliosides are present in milk fat globule membrane in concentrations of about 6 nmol/mg protein (sialic acid basis) [69,100]. This level is very close to that determined for rat liver plasma membranes [100]. At least six (and possibly as many as nine [131]) chromatographically distinguishable gangliosides are present in the milk fat globule membrane [69,100]. Structures of three of these have been shown to be sialic acid-galactoseglucose-ceramide, N-acetylgalactosamine-(sialic acid)-galactose-glucose-ceramide, and (sialic acid)2-galactose-glucose-ceramide (GM3), GM2, and GDa, respectively, in the nomenclature of Svennerholm [132]). [69,133]. GD 3 is the most abundant ganglioside, accounting for more than 50 ~o of the lipid-bound sialic acid of the membrane [69,100]. Both N-acetyl- and N-glycolylneuraminic acids are present in the membrane ganglioside fraction [69]. Ceramide [130] the cerebrosides [126,128] and gangliosides [69] are strikingly similar to sphingomyelin in their low content of unsaturated fatty acids and their appreciable content of the 22:0, 23:0 and 24:0 acids. This and other evidence [69,87] suggests that these sphingolipids arise from a common pool of ceramide within the mammary gland. IVC. Protein composition As early as the mid 1800's investigators were aware of the protein nature of the

293 material surrounding milk fat globules. Total membrane preparations and soluble or insoluble fractions from the membrane obtained by various extraction procedures have been thoroughly characterized with respect to composition and immunologic, electrophoretic and sedimentation characteristics. These studies, which have been extensively reviewed by Brunner [5], were important to further development of the area. However, results obtained were characteristic, not of individual protein species but at best, were averages for the aggregate of different proteins present in the fraction. To date, no non-enzymatic protein has been purified to a state of electrophoretic homogeneity from the milk fat globule membrane. Early attempts at characterization of milk fat globule membrane proteins by polyacrylamide gel electrophoresis revealed the presence of about 9 polypeptides in this fraction [76]. Since the electrophoretic system used in this study had but limited resolving power and the solubilization procedure brought only a fraction of the total membrane protein into solution [134], 9 different polypeptides was at best a lower limiting number. Nevertheless, these analyses did show that milk fat globule membranes and the plasma membrane from bovine mammary gland had virtually identical electrophoretic patterns [76]. Subsequently, it was found that more than 98 700of the milk fat globule membrane protein can be solubilized by treatment with sodium dodecyl sulfate and 2-mercaptoethanol [65] and electrophoresis in sodium dodecyl sulfate-containing polyacrylamide gels has greatly expanded the number of polypeptides known to occur in the milk fat globule membrane. Using such methods, Anderson et al. [135] and Kobylka and Carraway [64,136] found 6 major coomassie blue-positive polypeptides in the membrane, whereas Kitchen [74] described the presence of 7 major polypeptides and, in a separate study, Anderson et al. [137] reported molecular weights for 10 major polypeptide constituents. There is remarkable agreement among the various authors with regard to the molecular weights of the major polypeptides. These molecular weights range from about 240 000 down to about 15 000 [64,65,135-137]. In the above studies several minor polypeptides were also detected but their number was not enumerated. Within the above molecular weight range, Keenan and Huang [96] detected at least 14 polypeptides in the milk fat globule membrane. More recently, Mather and Keenan [65] found at least 21 polypeptides within the above molecular weight range in this membrane. In this study three major size classes of polypeptides of about 155 000, 63 000 and 44 000 were found. A typical electrophoretic pattern is shown in Fig. 6. When heavier protein loads are applied to gels, even more polypeptides are seen, particularly in the 10 000 to 45 000 dalton range (I. H. Mather, unpublished). These studies have revealed that there are a minimum of at least 30 electrophoretically distinguishable polypeptides in the bovine milk fat globule membrane. The electrophoretic pattern of human milk fat globule membrane proteins is somewhat similar to that obtained with the bovine membrane (compare Fig. 6 with Fig. 6 in ref. 101). No enzymatic activity has yet been ascribed to any of the electrophoretically distinguishable membrane polypeptides and indeed, it is not yet known if any of the electrophoretic bands represent individual protein species or are composed of several different proteins of similar molecular weights.

294

! 0'8 0-7 --'- 0 - 6 E ,t-

O

0-5

1.0 (,0

v

0.4

tO

-,-" 0.3 u E

x

0-2

W

0-1

2i !

0 0

0.25

!

0.50

!

0.75

1.0

Mobility Fig. 6. Electrophoretic pattern of milk fat globule membrane polypeptides. Washed cream was extracted with sodium dodecyl sulfate and 2-mercaptoethanol and the extract was mixed with bromophenol blue and sucrose and applied to a l0 9/0 polyacrylamide gel containing sodium dodecyl sulfate and mercaptoethanol. Staining was with coomassie blue and the gel was scanned at 650 nm. Protein bands routinely seen in gels are numbered from the top of the gel. Positions of periodic acid-Schiff positive constituents are indicated by arrows and designated by Roman numerals. Apparent molecular weights of bands 3, 12, 16 and 21 are 155 000, 62 500, 43 500 and 11 000 respectively. From Mather and Keenan [65].

In addition to the coomassie blue-positive bands, five [65,74], six [64,135,136] and nine (K. Weber, unpublished) periodate-Schiff positive glycoprotein bands have been detected in electrophoretic patterns of milk fat globule membranes. It is the usual observation that some or all of these glycoproteins either do not stain at all or stain, at best, poorly with coomassie blue [64,65,135], a phenomenon also observed with glycoproteins from other membranes [138]. The apparent molecular weights of these globule membrane glycoproteins vary with the acrylamide percentage of the gels [64] but appear to range from about 130 000 to 40 000 (K. Weber, unpublished). While glycoprotein-containing fractions obtained from milk fat globule membranes

295 have been characterized (e.g. ref. 5, 139), no definitive reports of purification of individual glycoproteins from the membrane have appeared. However, Farell and co-workers have begun characterization of an approximately 45 000 mol. wt glycoprotein which they have released from the membrane and purified to near electrophoretic homogeneity (H. W. Farrell, Jr., personal communication). The enzymes 5'-nucleotidase and nucleotide pyrophosphatase, which are present in the globule membrane (see following) may be glycoproteins as are these enzymes from hepatocyte plasma membranes [140, 141]. Protein patterns of intracellular endomembrane fractions from mammary gland have received little study, although Keenan and Huang [96] found that rough endoplasmic reticulum, Golgi apparatus and milk fat globule membrane had eight polypeptide components with nearly identical electrophoretic mobilities. There was also wide ranging similarity in the amino acid composition of rough endoplasmic reticulum, Golgi apparatus and milk fat globule membrane [96]. It was recently shown by Slaby and Brown [142] that there are gestational and lactational differences in the distribution of major membrane polypeptide constituents of rough endoplasmic reticulum from mouse mammary gland. In particular, they found that two major endoplasmic reticulum membrane proteins, of molecular weights 76 000 and 57 000, occurred to a significant extent only during lactation. In his comparative study, Kitchen [74] observed the electrophoretic pattern of polypeptides from milk serum (skim milk) membranes to be qualitatively identical to that of the milk fat globule membrane and to differ, quantitatively, only in the levels of an 85 000 mol. wt polypeptide, which was much more prevalent in the serum membrane fraction. While some variation in amino acid composition of bovine milk fat globule membrane is seen in results from different laboratories, in general, the amino acid distributions reported have been very similar (Table III) [64,74,90,96]. Some difference in amino acid composition due to individual animal or breed differences would be expected. The membrane is characterized by high levels of glutamic and aspartic acids and leucine and is apparently quite low in content of sulfur amino acids [74,90]. Amino acid composition of the skim milk membrane fraction is practically identical to that of the milk fat globule membrane [74]. With the exception of somewhat higher levels of histidine and alanine, the human milk fat globule membrane is similar to that from the bovine in amino acid composition (compare ref. 101 with Table III). Perhaps a more interesting observation was that of Kobylka and Carraway [64], who found a high degree of similarity in the amino acid compositions of erythrocyte and fat globule membranes from the bovine. Previous to this, Dowben et al. [85] found that rabbit antisera to bovine milk fat globule membranes, strongly agglutinated and hemolyzed bovine erythrocytes but not erythrocytes from rabbits or humans. Which of the many proteins of milk fat globule membrane are immunologically active is not known at present, but Coulson and Jackson [143] found that a water soluble glycoprotein(s?) containing fraction obtained from ethanol-diethyl ether-treated fat globule membranes [144] was strongly antigenic. Treatment of milk fat globule membranes with a solution of EDTA and 2-mercaptoethanol, selectively releases a major mem-

296 TABLE III AMINO ACID COMPOSITION OF BOVINE MILK FAT GLOBULE MEMBRANES AS DETERMINED IN DIFFERENT LABORATORIES Amino acid

Glu Leu Asp Arg Lys Thr Gly Ala Pro Phe Ser Ile Tyr Val His Try Cys Met

Mol ~ of total Keenan & Huang [96]

Kitchen [74]

Kobylka & Carraway [ 6 4 ]

Swope & Brunner [90]

13.6 10.6 8.0 8.1 7.6 8.9 4.6 4.8 5.5 5.9 5.7 5.4 4.2 4.4 2.6 -

13.6 9.8 10.2 6.1 6.8 6.5 4.8 5.4 5.7 5.1 8.2 4.9 3.0 6.3 1.4 0.1

12.0 9.3 9.6 4.6 5.6 6.0 7.5 7.2 6.6 4.2 8.5 4.3 2.5 6.9 1.9 -

13.4 8.6 9.0 9.0 7.7 5.2 2.1 2.9 4.8 7.4 4.7 4.8 5.6 4.3 3.1 3.9 1.6

1.7

1.7

-

-

brane glycoprotein with an apparent molecular weight o f 130 000 (K. Weber and T. W. Keenan, in preparation). This protein is strongly antigenic, as is at least one other fat globule membrane glycoprotein. Antisera to this protein are also cross reactive with extracts from bovine m a m m a r y Golgi apparatus.

IVD. Enzymes Relatively high specific activities of several different enzymes have been routinely detected in bovine milk fat globule m e m b r a n e preparations (Table IV). In general, enzymes with high specific activities in this membrane are those enzymes which are associated with plasma membranes from various tissues [123,145-147]. Mitochondrial markers such as rotenone-sensitive N A D H - c y t o c h r o m e c reductase [61, 62,85] and succinic dehydrogenase [61,62], endoplasmic reticulum markers such as glucose-6-phosphatase [74,85] and N A D P H - c y t o c h r o m e c reductase [60,96] and Golgi apparatus markers such as the galactosyltransferase o f lactose biosynthesis [30, 86,96] and glycosyltransferase o f ganglioside synthesis [87] are absent from or are present in only very low specific activities, in bovine milk fat globule membrane preparations. In addition to c o m m o n plasma membrane enzymes, h u m a n milk fat globule membranes have been reported to contain glucose-6-phosphatase and lactose synthetase activities [88]. The possiblity that this is due to greater entrainment of cytoplasmic membranes during milk fat globule secretion in this species has been discussed in a preceeding section. Since assay conditions used in different laboratories

297 TABLE IV MAJOR ENZYME ACTIVITIES IN BOVINE MILK FAT GLOBULE MEMBRANES Enzyme

References

Alkaline phosphatase (EC 3.1.3.1) Acid phosphatase (EC 3.1.3.2) 5'-Nucleotidase (EC 3.1.3.5) Phosphodiesterase (EC 3.1.4.1) Inorganic pyrophosphatase (EC 3.6.1.1) Nucleotide pyrophosphatase (EC 3.6.1.9) ATPases (Mg2+; K ÷, Mg 2+) (EC 3.6.1) Lipoamide dehydrogenase (diaphorase, EC 1.6.4.3) Cholinesterase (EC 3.1.1.8) Aldolase (EC 4.1.2.13) Xanthine oxidase (EC 1.2.3.2) Thiol oxidase (EC 1.8.3.2) ),-Glutamyl transpeptidase UDP-hexose hydrolases *

61, 62, 74, 85, 151, 152 74, 85, 151,152 74, 157, 162 85 74 74, 157 74, 85, 157, 158 61, 74, 152 85 85, 152 61, 62, 74, 85, 152 74 74 65

* Activity detected with UDPgalactose and UDPglucose. Release of free sugar may be due to action of a hydrolase releasing sugar phosphatase and the subsequent action of a phosphatase.

have varied widely, specific activity values reported for the major globule membrane enzymes cannot be meaningfully compared. Unfortunately, it is also not possible to compare fat globule and m a m m a r y plasma membrane enzymes since there has been no enzymological characterization of the latter. The fact that there is constant flow of membrane into milk and the probability that all membranes of the m a m m a r y cell originate mainly from endoplasmic reticulum may also explain the presence of residual enzyme activities of other cell membranes in the milk fat globule membrane. Although few investigators have used milk fat globule membrane as starting material, some of the enzymes known to occur in this membrane have been extensively purified from whole milk. Prime examples are xanthine oxidase, which has been crystallized from bovine milk [148] and both acid [149] and alkaline [150] phosphatases, which have been purified several thousand-fold from milk. Relevant literature on the properties of these enzymes has been reviewed and will not be repeated here [151,152]. Alkaline phosphatase isozymes occur in milk and at least some of these isozymes are believed to be glycoproteins [153,154]. Alkaline phosphatase seems to be unique among milk fat globule membrane enzymes in that it is unusually stable in the presence of sodium dodecyl sulfate [155], as are alkaline phosphatases from other mammalian sources [155] and from Escherichia coli [155,156]. This property allows detection of alkaline phosphatase in sodium dodecyl sulfate-polacrylamide gels [155]. Milk fat globule membrane preparations avidly liberate glucose or galactose from the respective UDP-sugar [86]. Whether this activity, which has a distinctly alkaline p H optimum, is due to a nucleotide pyrophosphatase of the type recently

298 purified from hepatocyte plasma membranes [141] remains to be determined. Adenosine triphosphatases of the milk fat globule membrane have also been characterized [85,157,158]. This activity is markedly stimulated by Mg 2+ and slightly stimulated by K ÷ but is not affected by Na ÷ and is insensitive to the glycoside ouabain [157,158]. The latter two parameters serve to distinguish this activity from the N a ÷ATPase found in several tissues [159]. This lack of a Na+-ATPase correlates with the demonstration of this enzyme in the basal and lateral, but not apical, plasma membrane of mammary secretory cells [160] and with electrophysiological data showing that Na ÷ is passively distributed across the apical plasma membrane [161]. That milk fat globule membrane shows no energy-dependent ability to accumulate calcium suggests that a CaE+-stimulated ATPase may not be present in this membrane [54]. In contrast, Golgi apparatus-rich fractions from mammary gland have both a Ca 2+stimulated ATPase and an ATP-dependent calcium accumulation system [54]. Two separate fractions containing 5'-nucleotidase activity have been resolved by gel filtration of detergent extracts of fat globule membranes [162]. Based on different Km values and certain other differences in properties, it was concluded that these two fractions contained different 5'-nucleotidase enzymes. One of these two fractions was found to contain primarily sphingomyelin in the chloroform-methanol extractable phospholipids [162]. Widnell and Unkeless [163] similarly noted 5'nucleotidase from rat liver plasma membranes to be a sphingomyelin-associated enzyme. However, Evans and Gurd [140] found mouse liver plasma membrane 5'-nucleotidase to be a glycoprotein with a marked tendency to aggregate and to lack phospholipid in the purified state, indicating that activity of the enzyme is not lipid-dependent. The other membrane-bound enzymes listed in Table IV have received little study other than simple measurement of specific activities in the membrane. Moreover, there have been few attempts to detect the presence of these enzymes in mammary subcellular fractions; the exceptions being the use of certain of these enzymes to monitor fraction purity [7,89,96]. Kitchen [74] found nearly all of the globule membrane enzymes to be present in higher specific activities in the skim milk membrane fraction. In contrast to Plantz and Patton [ll6], he was able to demonstrate MgE+-ATPase activity in skim milk membranes. The reason for this discrepancy remains to be resolved. Glycosyltransferases are concentrated in Golgi apparatus [30,87,96,164-166] but are also reported to be present in plasma membranes (e.g. refs 167-170) where they are believed to function in recognition and intercellular adhesion [170,171]. Recent observations have cast doubt on the existence of surface glycosyltransferases [172] and we have been consistently unable to detect glycosyltransferase activities in bovine milk fat globule membrane fractions [86,87]. We have also been unable to detect adenyl cyclase, a known plasma membrane enzyme [145-147] in bovine milk fat globule membranes (E. D. Jarasch and T. W. Keenan, unpublished). Adenyl cyclase activity occurs in mammary gland [173] and it would be of interest to determine if this activity is present in the lateral or basal regions of the secretory cell plasma membrane.

299 V. CRITIQUE OF THE MEMBRANE The milk fat globule membrane has merits as a model and source for biomembrane studies. By virtue of the secretion mechanism, it tends to be a fairly pure form of plasma membrane or a useful derivative thereof. If can be obtained from a great variety of mammalian species and this may be of particular value for immunological purposes. It envelopes the surface of a fat droplet not unlike the way in which it encompassed the cell, i.e., with the same side exposed. It can be obtained without danger of artifacts attending membrane preparation by tissue disintegration. On the other hand, this membrane has limitations. It is probably not typical of plasma membrane from the basal and lateral surfaces of the mammary epithelial cell. Since it is a membrane involved in secretion it may not be typical of membranes with other primary functions. Deposition of the membrane on a fat droplet would seem to limit its utility, at least in that condition, for transport studies. Moreover, this structural configuration renders uncertain which of the globule surface constituents are part of its original (intracellular) surface and which are components of the membrane and subsequently superimposed on that surface. There also is the possibility of post-secretion changes in the membrane which may render it misleading as a natural model. This matter relates to the origin of membrane material in skim milk discussed following and elsewhere [6,8].

VA. Origin of skim milk membranes The identification of the lipoprotein material in bovine and caprine skim milks as consisting of membranous vesicles is reviewed in Section II. The origin of this material is the subject of current research and discussion. Wooding [18,174-176], using morphologic evidence from the electron microscope, claims that very soon after secretion, the initial milk fat globule membrane is lost from the globule by vesiculation and that this phenomenon accounts for virtually all of the membrane material in skim milk [176]. Bauer [ 177] has compared the appearance of milk fat globules after preparation by conventional fixation and embedding with freeze fracture procedures. He concludes that the former produces some loss of membrane from the globules. It is generally recognized that preparation of large fat droplets for viewing by electron microscopy poses difficulties. However, a number of investigators have noted post-secretion changes in the appearance of the membrane on milk fat globules [3,18,174, 178]. Regarding the contention that membrane material in skim milk is derived largely from the surface of milk fat globules, there is evidence indicating at least three other contributing sources. As previously mentioned the appearance and dimensions of certain membranous sacs strongly indicates the presence of sloughed microvilli [56]. The occurrence of membrane bound viruses in the fraction is also quite likely. During exocytosis, viruses frequently adopt plasma membrane from the host cell. Without going into this tangential matter we draw attention to an interesting recent study [107] of membranes from a milk-born mammary tumor virus and to interest in milk-born viruses in relation to breast cancer [179].

300 Perhaps of greatest interest is evidence that skim milk contains endoplasmic reticulum. This membrane is known to be the site of milk triglyceride synthesis within the cell [15-17]. The capacity for net synthesis of triglycerides in skim milk of several species has been established [180-184]. More precisely glycerol kinase and all the enzymes for production of triglycerides via the glycerol-3-phosphate pathway have been demonstrated [183,184]. Likewise de novo synthesis of phosphatidylcholine has been accomplished with this system using either 14C-glycerol [183] or 14C-fatty acid [181,184]. When the distribution in skim milk of label incorporated into triglycerides and phosphatidylcholine was analyzed following incubation with 14C-palmitate, the activity was found mainly in the membrane-rich (fluff) fraction [181]. We feel that this constitutes supporting evidence of endoplasmic reticulum in skim milk and that this system may be useful for studying fat droplet formation at the membrane level. In summary a number of kinds of membrane may exist in ruminant skim milks including: vesicles derived from plasma membrane, milk fat globule membrane, secretory vesicles and endoplasmic reticulum; sloughed microvilli and membraneenclosed viruses. Methods of separating and characterizing these quantitatively would be useful for fundamental studies and industrial purposes (see ref. 58). Such research should take into account that frequency of milking [58] and mastitis [106] are variables influencing membrane yield and that turbulence and foaming during milking may denude fat globules of membrane. VB. Membrane sidedness The milk fat globule provides a unique opportunity to study the molecular architecture of the plasma membrane since the secretion mechanism exposes the outer surface of this membrane on the fat globule (Fig. 1 and 4). By reacting or applying specific probes to the intact globule and comparing the result with total membrane composition or released membrane reactivity, symmetry of lipid and protein distribution in the two faces of the membrane may be adduced. VB-1 Lipids. Treatment of intact milk fat globules with 2,4-dinitrofluorobenzene failed to yield evidence for reactive ethanolamine and serine groups ([97] and R. Newman, personal communication), implying that the respective phosphatides may be inaccessible to the reagent. Further, ethanolamine and serine phosphatides show reactivity when intact fat globules are treated with the membrane-impenetrable reagent, trinitrobenzene sulfonate [185], but react to a larger extent when isolated milk fat globule membranes are treated with this reagent (T. W. Keenan, in preparation). These phosphatides are also unreactive when intact fat globules are treated with phospholipases A or C but do react when isolated membranes are so treated. In contrast, sphingomyelin and phosphatidylcholine are degraded to the same extent when either intact globules or isolated membranes are treated with phospholipases (T. W. Keenan et al., in preparation). This suggests that the lipid bilayer of the milk fat globule membrane is similar to those of erythrocytes and the influenza virus in that the choline containing phospholipids are concentrated in the outer half

302 milk fat globule before and after release of the membrane by churning. Activities of the enzymes were such as to suggest that virtually all of the 5'-nucleotidase activity is in the outer surface, whereas activities for nucleotide pyrophosphatase and Mg 2+activated ATPase may at least partly occur on the inner membrane surface. There is now considerable evidence that 5'-nucleotidase, a glycoprotein as purified from rat liver [140], occurs more or less exclusively in the exterior surface of plasma membranes from a variety of mammalian cells [147,208]. The fact that sphingomyelin has been shown to co-isolate with this enzyme from both the milk fat globule membrane [162] and the plasma membrane of the rat hepatocyte [163], indicates the close proximity of these two components in the outer surface of the plasma membrane. Kobylka and Carraway [136] have questioned the integrity of the milk fat globule membrane and consequently the validity of observations regarding its structure. They observed that all major membrane proteins were cleaved when intact fat globules were treated with trypsin or pronase. This led them to conclude that the membrane surrounding the fat globule does not represent a significant permeability barrier to proteolytic enzymes and to suggest that the membrane does not exist on the globule in an intact form. This latter suggestion was based on Wooding's [174, 176] morphological observation that the milk fat globule membrane is partially sloughed from the globule after secretion. In distinct contrast, Mather and Keenan [65] found major differences in trypsin-catalyzed rates of hydrolysis of proteins in intact fat globules and isolated membranes. Further, lactoperoxidase-catalyzed iodination revealed that several of the membrane proteins were much more accessible in isolated membranes than they were in intact fat globules. These observations clearly suggested that the membrane does present a permeability barrier in the intact fat globule. Based on their results, Mather and Keenan concluded that polypeptide constituents 10, 12, 15, 16, II, IV and V of Fig. 6 were externally disposed and that constituents 3, 5-8, 11, 14 and 21 were localized along the inner face of the membrane. Constituent 16 could be eluted from fat globules with 1.5 M magnesium chloride. This, and results from trypsin treatment, indicated that constituent 16 partially masks constituent 12 on the face of the membrane. Further, both isolated membranes and intact fat globules were found to react to the same extent with concanavalin A [202] and with neuraminidase [65], inferring that the carbohydrate portions of membrane glycoproteins are externally disposed. The above observations strongly suggest that there is asymmetry in distribution of at least certain protein constituents of the membrane. In retrospect it appears that the erythrocyte and the lactating mammary cell have limiting membranes with different structural functional characteristics and that the proteolysis treatments used by Kobylka and Carraway [136] were too extensive to reveal elements of protein asymmetry in the fat globule membrane. These observations on distribution of membrane proteins are difficult to rationalize with rapid degradation and loss of membrane from the globule. The fact that there is no net decrease in the amounts of phospholipids, 5'-nucleotidase activity and ATPase activity associated with the globules for as long as 96 h after procurement of milk from the animal, also argues against extensive shedding of the globule membrane

303 [209]. Moreover, although skim milk membranes and fat globule membranes are compositionally very similar, they are metabolically clearly distinguished [60,67,116, 181]. The older literature frequently alludes to the ease of dislodging xanthine oxidase and alkaline phosphatase by washing fat globules (see pp. 20, 2 l, 50 in ref. 1). Z~ttle et al [210] observed that 85 ~o of the xanthine oxidase and alkaline phosphatase activities were removed from fat globules by four successive washings with water. Mere cooling of the milk releases a substantial proportion of these enzymes from the globules indicating relatively weak hydrophobic binding. The increased activity of these enzymes on their release from the globule implies an interior location in the membrane. Briley and Eisenthal [21 l] have investigated the catalytic properties of free, purified bovine milk xanthine oxidase in comparison to its milk fat globule membrane-bound form. The bound enzyme exhibited enhanced oxidase activity toward NADH relative to that toward xanthine, when compared with the free form of the enzyme. One glycoprotein known for several decades to occur on the surface of bovine milk fat globules is a globulin. It produces clumping (agglutination) of the globules, thus causing their rapid rise to form a cream layer [212]. Recent innovations that cross link membrane constituents to establish near neighbor locations represent a promising approach to membrane structure (e.g. refs 213-215). Release by cooling of materials hydrophobically bound in the milk fat globule membrane, such as xanthine oxidase and alkaline phosphatase [210], followed by gel column separation and analysis should yield much valuable information regarding lipid-protein-enzyme associations in the membrane. Regarding the question of how stable the membrane is, such factors as: the freshness of the milk including time in the gland, species variations and methods of stabilizing the membrane on the fat globule warrant investigation. The findings of Ray [216], that Ca ÷2 addition improves recovery (stability) of rat hepatocyte plasma membrane is suggestive.

VI. SUMMARY The milk fat globule is essentially an oil droplet enclosed in plasma membrane from the lactating cell. Envelopment in this membrane is the mechanism by which milk fat globules are expelled from the cell. This remarkable secretory process provides the membranologist with a unique source of plasma membrane; one that is oriented on an inert (glyceride) core with the same exterior exposure it had on the cell; also one that can be obtained in substantial quantity and purity by relatively mild manipulations. The literature on the milk fat globule membrane is reviewed in the following areas: membrane isolation, preparation, composition (lipid, protein, enzymes), stability including the presence and nature of membrane material in the skim milk phase, and "sidedness" from the standpoint of lipid and protein distributions in the exterior and

304 inner surfaces. The emerging evidence in this latter area agrees closely with findings on limiting membranes from other types of cells; i.e. glycoproteins, 5'-nucleotidase, sphingolipids, and phosphatidylcholine are concentrated in the outer (exposed) surface, while the amino lipids (phosphatidylethanolamine and phosphatidylserine) are on the interior side of the membrane. How unstable the milk fat globule membrane is and the origin and identity of an interesting array of membrane material in skim milk are matters requiring further research. The latter material includes a variety of membranous vesicles, sloughed microvilli and on occasions membrane enclosed viruses. While there are yet many gaps in the knowledge of the globule membrane, research interest in it has been strong because of its importance in properties of milk and milk products. The value of this research interest is now further fortified by basic considerations in cytology, biochemistry and the relevance of mammary cell membranes to the breast cancer problem.

ACKNOWLEDGEMENTS Preparation of this review and the research of the authors were supported in part by grants GB 251 l0 from the National Science Foundation and G M 18760 and H L 03632 of the National Institutes of Health. T. W. K. is supported by Public Health Service Research Career Development Award G M 70596, from the National Institute of General Medical Science. We thank Bridget Stemberger for assistance with electronphotomicrographs and Professor W. W. Franke and Drs E. D. Jarasch, I. H. Mather and K. Weber for valuable discussions and for providing results in advance of publication. Paper No. 4838 and 5860 in the journal series of the Pennsylvania and Purdue Agricultural Experiment Stations, respectively.

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