J. Membrane Biol. 21, 6 5 - 8 5 (1975) 9 by Springer-Verlag New York Inc. 1975

Studies on the Structure of Milk Fat Globule Membrane Ian H. M a t h e r and T. W. K e e n a n Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907 Received 8 July 1974; revised l November 1974

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

The cream fraction of milk consists of fat droplets stabilized in the milk serum by an external m e m b r a n e of protein and lipid. This m e m b r a n e layer, generally k n o w n as milk fat globule m e m b r a n e ( M F G M ) , is derived mainly f r o m the apical plasma m e m b r a n e of m a m m a r y secretory cells at the time of milk fat secretion. D u r i n g this process, fat droplets within the secretory cells a p p r o a c h and are progressively enveloped by plasma m e m b r a n e . T h e fat droplets are finally expelled into the gland lumen entirely s u r r o u n d e d by apical plasma m e m b r a n e . Evidence for this process has been obtained f r o m electron-microscopy ( B a r g m a n n & K n o o p , 1959; B a r g m a n n , Fleischauer & K n o o p , 1961) and b y biochemical c o m p a r i s o n of M F G M with isolated m a m m a r y plasma m e m b r a n e . B o t h m e m b r a n e s have a similar 5

J. Membrane Biol. 21

66

I . H . Mather and T. W. Keenan

lipid composition (Keenan, Morr6, Olson, Yunghans & Patton, 1970) and MFGM contains enzymes characteristic of plasma membrane preparations (Dowben, Brunner & Philpott, 1967; Patton & Trams, 1971). In addition to the plasma membrane component, milk fat globules also appear to be surrounded by a layer of material absorbed from the cytoplasm prior to secretion (Brunner, 1969; Wooding, 1971; Bauer, 1972). This layer may in part be derived from endoplasmic reticulum (Stein & Stein, 1967; Martel-Pradal & Got, 1972) and cytoplasmic surfactant molecules (Patton, 1972) and lies between the fat globule core and the plasma membrane layer. The relationship between these two layers and the molecular architecture of the plasma membrane layer has been the subject of much speculation. Electron-microscope studies and thermodynamic considerations suggest that the proteins of mammary plasma membrane undergo a structural rearrangement during and after the formation of MFGM. The cytoplasmic face of the plasma membrane undergoes an abrupt change of environment during fat secretion, from predominantly hydrophilic to hydrophobic conditions. The fat globule core is composed largely of triglycerides (at least 98 %). Plasma membrane proteins, particularly those with predominantly hydrophilic characteristics, might therefore be expected to rearrange to face the aqueous phase. Patton and Trams (1971), however, suggested that at least some of the membrane proteins may retain an asymmetric distribution in the MFGM lipid bilayer, as in other membrane bilayers (e.g., erythrocytes, Hubbard & Cohn, 1973; hepatocytes, Evans, 1974). This conclusion was based on the measurement of 5'-nucleotidase and Mg 2+-activated ATPase activities from intact and disrupted fat globules. The ATPase activity was significantly higher in isolated membrane fractions. This paper presents biochemical evidence that MFGM contains elements of protein asymmetry. Many of the MFGM proteins are resistant to trypsin hydrolysis in whole cream, but they are rapidly hydrolyzed in isolated membrane. Further, the lactoperoxidase labelling technique of Phillips and Morrison (1970) reveals significant differences between the accessibility of MFGM proteins to lactoperoxidase-125I in whole cream and isolated MFGM. Materials and Methods Preparation of Washed Cream and M F G M Fresh milk was obtained from a herd of Holstein cows at the time of normal milking. Random samples were collected from individual cows and pooled into batches (3 liters) for cream separation and M F G M isolation.

Structure of Milk Fat Globule Membrane

67

Cream was separated from the milk serum at room temperature by centrifugation at 12,000 x g for 10 min. The cream fraction was washed twice with sucrose solution (0.28 M) containing Tris-HC1 buffer (10 raM, p H 7.5) and MgC12 (1.0 mM). The total volume of the washings was 3 liters and the yield of washed cream approximately 40 g (wet weight). M F G M was prepared by resuspending the washed cream in Tris-HCl buffer (50 mM, p H 7.5) and by churning the suspension in a Waring blendor until butter formed. The mixture was then warmed to 37 ~ to release trapped membrane from the butter granules. M F G M was obtained as an orange-brown pellet after centrifugation (95,000 x g , 1.0 hr).

Preparation of Samples for Polyacrylamide Gel Eleetrophoresis Cream and M F G M samples for electrophoresis were extracted with sodium dodecyl sulfate (SDS) (5: l, SDS: membrane protein, w/w) and mercaptoethanol (10 m ~ ) f o r 30 rain at 37 ~ The samples were then centrifuged (100,000 x g , 1.0 hr) at 25 ~ With extracted cream samples, the upper layer of butter fat was removed and the aqueous fraction retained for electrophoresis. After centrifuging the samples, pellets were present in both cream and M F G M fractions. Table 1 compares the protein levels in the supernatants and pellets after solubilizing M F G M with SDS in the absence and presence of mercaptoethanol. In the presence of SDS and mercaptoethanol almost 100% of the membrane protein is solubilized, compared with reference samples digested in sodium hydroxide. This is an indirect estimation from the protein content of the pellet, since mercaptoethanol interfered with the protein assay in supernatant fractions giving anomalously high values.

Polyaerylamide Gel Eleetrophoresis Membrane proteins were separated by electrophoresis in polyacrylamide gels (10 %, w/v) containing methylene bisacrylamide (0.135%, w/v), phosphate buffer, SDS and mercaptoethanol according to the method of Weber and Osborn (1969). Before electrophoresis all samples were dialyzed overnight, at room temperature, against a mixture of sodium phosphate buffer, p H 7.0 (50 mM), SDS (0.1%, w/v) and mercaptoethanol (10 raM). Samples of solubilized membrane were mixed with sucrose solution (10%, w/v, final concentration) and bromophenol blue. After electrophoresis, the gels were Table 1. Solubilization of isolated milk fat globule membrane with sodium dodecyl sulfate and mercaptoethanol Treatment

Sample

Total protein 1Tlg

% a

SDS (1.0%, w/v)

supernatant pellet

10.2 0.56

95.3 5.2

SDS (1.0 %, w/v) + mercaptoethanol (10 raM)

supernatant pellet

(15.1 ) 0.11

(140) 1.0

M F G M was resuspended in distilled water and aliquots (10.7 mg membrane protein) were treated with SDS and mercaptoethanol in a total volmne of 5.0 ml for 30 min at 37 ~ Pellets after centrifugation (100,000 x g , 1.0 hr) were dissolved in N a O H (1.0 M, 3.0 ml) at 100 ~ and aliquots taken for protein determination. a Expressed as a % of the total protein measured before detergent extraction. 5*

68

1. H. Mather and T. W. Keenan

cut at the bromophenol blue front and stained for protein with coomassie blue. After destaining electrophoretically in acetic acid (7 %, w/v), gels were scanned with a Beckman gel scanner in an Acta III spectrophotometer at 650 nm. Carbohydrate-containing material was stained with periodic acid-Schiff (PAS) reagent according to the methods of Fairbanks, Steck and Wallach (1971). Proteins containing no carbohydrate were located in PAS-stained gels by soaking the gels overnight in a mixture of Na2S20 5 (1 g) and 8-anilinonaphthalene sulfonic acid (ANS) (10 rag) in 100 ml of HC1 (0.1 N) (Glossmann &Neville, 1971). The gels were then washed with a solution of Na2S2Os (0.1%, w/v) in HC1 (0.01 N) to remove excess ANS and scanned at 400 nm. The location of PAS-positive mateiial was recorded by scanning the same gels at 550 nm.

Trypsin Hydrolysis Washed cream, prepared as described above, was resuspended in three times the volume of Tris-HC1 buffer, pH 7.5 (50 mM). The suspension contained 2.48 mg/ml M F G M protein. Trypsin (10 mg/ml) in HCI (0.001 N) was added to samples (9.9 ml) of the cream suspension at 30 ~ The final volume was made to 10 ml in each case with HC1 (0.001 N). The concentration of trypsin for each sample was varied from 20 gg to 100 lag/ml and proteolysis was stopped at desired intervals by the addition of sufficient trypsin inhibitor (4 mg/ml) to obtain a concentration ratio of trypsin to trypsin inhibitor of 1:4 (w/w). After a further 30-min incubation the samples were washed three times (3 • 35 ml) with the sucrose washing medium (see above) at room temperature. The cream was separated from the washings each time by centrifugation (12,000 x g, 10 min). The packed cream samples were then incubated with SDS (10%, w/v; 0.5 ml) and mercaptoethanol (100 mM; 0.5 ml) fo~ 2.0 hr at 37 ~ Sodium phosphate buffer, p H 7.0 (50mM; 4.0ml) was added to the mixture before centrifugation (100,000 x g , 1.0hr) at 25 ~ Aliquots of the aqueous fractions obtained after centrifugation were dialyzed against a mixture of sodium phosphate buffer, p H 7.0 (50 mM), SDS (0.1%, w/v) and mercaptoethanol (10 mM) before electrophoresis. M F G M , prepared as described above, was resuspended in Tris-HC1 buffer, p H 7.5 (50 mM). The concentration of M F G M protein was 4.4 mg/ml. Aliquots (1.9 ml) of this suspension were treated with trypsin and trypsin inhibitor as described above. After proteolysis, the M F G M samples were washed three times with the sucrose washing medium (3 x 38 ml) and the membrane collected each time by centrifugation (95,000 x g , 1.0 hr). Preparation of the membrane samples for electrophoresis was as described for the trypsin-treated cream samples.

Lactoperoxidase-lZSI Labelling Washed cream and M F G M samples were prepared as described above and resuspended in sodium phosphate buffer, pH 7.5 (50 m~). Suspensions of cream (3.6 ml) and M F G M (2.4 ml) were incubated at 30 ~ in final volumes of 4.0 ml and 2.8 ml, respectively. Each sample contained lactoperoxidase (200 ~tg, 9 units), Na125I (0.97 mC, 0.56 nmole) and cold carrier (3,44 nmoles). Protein iodination was initiated by the addition of H20 2 to a final concentration of 1.33 i~r~. The H20 2 solution was added in aliquots (10 ~tliters) to maintain a low concentration of free peroxide throughout the labelling procedure. Account was taken of the volume occupied by the cream when estimating reagent concentrations. Iodination of membrane proteins was quenched by the addition of bovine serum albumin (10 mg/ml, 2.0 ml) after 2.0 rain. Both cream and M F G M samples were washed with the sucrose washing medium three times, as described for the trypsin experiments. Samples of both cream and M F G M were treated with SDS (2.5 %, w/v) and mercaptoethanol (20 raM) in a final volume of 2.0 ml for 1.0 hr at 37 ~

Structure of Milk Fat Globule Membrane

69

The SDS supernatants were then centrifuged (100,000 • 1.0h r) and the aqueous fractions dialyzed overnight, against a mixture of sodium phosphate buffer, pH 7.0 (50 raM), SDS (0.1%, w/v) and mercaptoethanol (10 raM). The solubilized proteins were separated by polyacrylamide gel electrophoresis and the gels stained with coomassie blue and destained as described above. The gels were scanned and then sliced into 1.0 mm sections with a Macrotome GTS-I (Yeda Ltd., Rehovot, Israel). The radioactivity in each gel section was determined with a Packard Auto-Gamma Spectrometer (Model 3001). Extraction of Cream with MgCIz

Six samples of cream which had been labelled by lactoperoxidaseYS1 as described above were collected together and washed with the sucrose washing medium. The wellpacked cream suspension was dispersed in Tris-HC1 buffer (50 raM, pH 7.5) in the ratio 1 : 1 (w/v). The suspension was then treated with MgC12 at a final concentration of 1.5 M at room temperature for 15 rain and the cream separated by centrifugation (12,000 xg, 10 min). The supernatant was centrifuged at 4 ~ (100,000 xg, 1.0 hr) and the residual cream removed by aspiration. A small pellet was also obtained. The clear supernatant was dialyzed overnight at 4 ~ against Tris-HC1 buffer (50 raM, pH 7.5). A slight precipitate which formed during dialysis was removed by centrifugation (100,000 xg, 1.0 hr). Samples of labelled washed cream, labelled MgClz-extracted cream and the above dialyzed and centrifuged supernatant were analyzed by SDS-polyacrylamide gel electrophoresis. Chemical Determinations

Protein was determined by the method of Lowry, Rosebrough, Farr and Randall (1951) using bovine serum albumin as a standard. Sialic acid was determined by the method of Jourdian, Dean and Roseman (1971). Chemicals

Acrylamide and N,N'-methylene-bisacrylamide were obtained from Eastman Organic Chemicals. The trypsin preparation was Sigma Type XI, treated with diphenyl carbamyl chloride to remove chymotrypsin activity. Soybean trypsin inhibitor (Type I-S), cytochrome c, ovalbumin, bovine serum albumin, /~-galactosidase and lactoperoxidase were from Sigma Chemical Company. Neuraminidase extracted from Clostridium perfringens was obtained from Boehringer Mannheim GmbH. NaX2SI ( > 14 racing I) was obtained from Amersham Searle Corp.

Results

T h e p u r p o s e of this study was to d e t e r m i n e the spatial distribution of the proteins of M F G M within the lipid bilayer of the m e m b r a n e . It was therefore i m p o r t a n t to establish a washing p r o c e d u r e t h a t effectively r e m o v e d c o n t a m i n a t i n g s e r u m proteins w i t h o u t d a m a g i n g the integrity of the m e m b r a n e . C r e a m fractions were w a s h e d at r o o m t e m p e r a t u r e , as in o u r experience c r e a m is m o r e difficult to r e s u s p e n d in washing m e d i u m at 0 ~ a n d s o m e M F G M p r o t e i n s are lost f r o m the fat globules at low tern-

70

I.H. Mather and T. W. Keenan

1

2

3

4

5

6

Fig. 1. Comparison of milk serum proteins, washed cream preparations and MFGM on SDS-polyacrylamide gels. 1, milk serum protein; 2, unwashed cream; 3, 4 and 5 represent cream washed once, twice and three times, respectively; 6, isolated MFGM

peratures (unpublished observations). Fig. 1 shows the protein patterns obtained in SDS-polyacrylamide gels after washing cream one, two or three times; milk serum and isolated M F G M samples are also shown. The major milk serum proteins were absent from both cream and M F G M samples after two washes. The protein components 18 and 19 (see Fig. 2 for a numbering key) migrated more slowly than es- and/~-casein, the two major serum caseins. In longer gels component 21 was shown to have a lower molecular weight than c~-lactalbumin, the other major protein in milk (see Table 2 and Gordon, 1971b). Mouse anti-sera to isolated M F G M did not co-precipitate with skim milk proteins in Ouchterlony diffusion tests. Anderson and Cheeseman (1971) obtained essentially similar results with a smaller volume wash (5 : 1, buffer/cream) performed three times.

Structure of Milk Fat Globule Membrane

71

12 0.8

m

16 0.7~

0.6~

0.5 E

O

~

0.4

0.3~

0.2

21

554

18 19

20

0.1 1 2

0

0

0.2

0.4

Mobility

0.6

0.8

1.0

Fig. 2. Separation of MFGM proteins on SDS-polyacrylamide gels after extraction of a whole cream sample washed twice. Protein bands routinely seen in gels after electrophoresis are numbered in the gel scan from the top of the gel. A photograph of the same gel is shown above the scan

72

I.H. Mather and T. W. Keenan

16 ~

~

1

16

2

Fig. 3. Comparison of the protein patterns of whoie cream and isolated MFGM after extraction with SDS and electrophoresis on polyacrylamide gels. 1, whole cream; 2, isolated MFGM

For all experiments, the initial cream fraction obtained was washed twice. Fig. 2 shows the protein pattern routinely obtained after electrophoresis of a twice-washed cream sample which had been directly extracted with SDS and mercaptoethanol. Protein components regularly observed in such samples are numbered consecutively from the top of the gel. Components 13 and 15 appear in the gel scan as shoulders to the major peaks 12 and 16, respectively, but were separate components in the gel. Component 21 was sometimes present as a more diffuse band than the sharp band shown in Fig. 2 (see, for example, Fig. 3). The molecular weights of the major protein components were determined by the method of Weber and Osborn (1969) and are given in Table 2. The results are in reasonable agreement with some recent independent molecular weight determinations for the major M F G M proteins (Kobylka & Carraway, 1972; Anderson, Cawston &

Structure of Milk Fat Globule Membrane

73

Table 2. Molecular weights of milk fat globule membrane proteins Component No.

Apparent molecular weight This study

(a)

(b)

3 4 9 10 11 12 14 15 16 17 18 19 20 21

155,000 138,000 98,000 89,000 74,000 62,500 51,500 (48,000) 43,500 37,500 31,000 27,000 17,500 11,000

155,000 -92,000 80,000 65,000 53,000 ----

169,000-t-26,200 -94,200__+10,600 74,600• 7,200 54,600• ---

Molecular weights in SDS-polyacrylamide gels were determined by comparison of the MFGM protein bands with the following standards: cytochrome c, cytochrome e dimer, ovalbumin, bovine serum albumin and/r MFGM and control gels were run simultaneously in triplicate. See Fig. 2 for numbering key. (a) Kobylka and Carraway (1972); (b) Anderson, Cawston and Cheeseman (1974).

Cheeseman, 1974). The membrane preparations contained three major size classes of polypeptide of 155,000, 62,500 and 43,500 daltons (components 3, 12 and 16, respectively). The separation of isolated M F G M proteins under similar conditions generally gave a similar pattern of protein bands apart from the loss of one major component (Fig. 3). In isolated M F G M , component 16 was often present in only minor amounts whereas this was a major protein of intact washed cream. The loss of this component appeared to occur during the isolation of M F G M since component 16 was still present in cream samples that had been washed at least five times before extraction with SDS. There were at least five major PAS-positive components in both whole cream and isolated membrane (Fig. 4a and b). Labelling of the major protein components with ANS enabled the positions of the PAS-bands to be directly compared with the positions of the major protein components in the same gel. Components I and II were intensely stained with PAS but poorly stained with coomassie blue and they did not bind ANS in detectable amounts. Components III, IV and V appeared to have identical mobilities to the coomassie blue-positive components 10, 12 and 16. Component V

74

I . H . Mather and T. W. Keenan

(b)

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2

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Fig. 4. Comparison by SDS-polyacrylamide gel electrophoresis of the protein and glycoprotein components of (a) whole cream, (b) isolated MFGM, (c) trypsin-treated whole cream, and (d) trypsin-treated MFGM. The protein concentrations for both whole cream and isolated M F G M were 4.0 mg/ml before trypsin treatment (20 lag/ml, 20 rain). ( ) extinction of PAS-positive components at 550 nm; ( . . . . . . . ) extinction of ANS at 400 nm. The arrows mark the positions of the major coomassie blue-positive components. See the legend to Fig. 2 for a numbering key. Components A and B are defined in Fig. 5

also a p p e a r e d to be p r e s e n t in l o w e r a m o u n t s in isolated M F G M ,

compared

with w h o l e c r e a m , in the s a m e m a n n e r as c o m p o n e n t 16. I n a d d i t i o n to the P A S - p o s i t i v e c o m p o n e n t s , a large b a n d of t u r b i d material was p r e s e n t t o w a r d s the b o t t o m ( a n o d e end) of the gels c o n t a i n i n g w h o l e c r e a m samples. This m a t e r i a l stained o n l y w e a k l y with P A S a n d

Structure of Milk Fat Globule Membrane

75

coomassie blue and its identity is at present uncertain. The turbid material was not visible in gels fixed and stained for protein according to Weber and Osborn's method (1969) (compare Fig. 2 with Fig. 4a).

Trypsin Hydrolysis of MFGM Proteins The hydrolysis of certain membrane proteins by trypsin or other proteases has frequently been used as a criterion for protein asymmetry in membranes. Membrane proteins that are externally located at the surface of an organelle or cell membrane are generally accessible to hydrolysis by trypsin or other proteases (Bender, Aaran &Berg, 1971; Triplett & Carraway, 1972). Proteins on the internal face or within the membrane are more resistant to proteolytic attack because proteases do not readily penetrate lipid bilayers. Washed cream and isolated membrane were treated with trypsin at three different concentrations for 30 rain (Fig. 5). Many of the membrane proteins of whole cream appeared to be resistant to trypsin hydrolysis. Components 1-8, 10, 11, 14 and 21 were still present in whole cream after treatment with trypsin (100 rtg/ml) for 30 rain. The minor components 4-8 appeared to be partially removed. The fate of components 18, 19 and 20 is difficult to determine as they become obscured by products formed during trypsin hydrolysis (labelled A and B in Figs. 4 and 5). Time course studies showed that the most rapidly hydrolyzed proteins were 9, 12, 15, 16 and 17. Components 12 and 16 are major components in intact washed cream and component 16 is the protein often partially lost during M F G M isolation. In contrast, the proteins of isolated M F G M were rapidly hydrolyzed except for component 10. Component 3 was also still present after trypsin hydrolysis, although in reduced amounts, and appeared to give rise to a polypeptide of lower molecular weight, coincident with component 4. Higher concentrations of trypsin (50 and 100 ~tg/ml) gave similar results to those for 20 ~tg/ml shown in Fig. 5. Time course studies between 0-60 rain with both whole cream and M F G M preparations incubated with trypsin (20 ~tg/ml) confirmed that the proteins of isolated M F G M are more rapidly hydrolyzed by trypsin than the proteins of whole cream. The fate of the PAS-positive components of whole cream and isolated M F G M during trypsin hydrolysis are shown in Fig. 4c and d. Components I and III were resistant to trypsin hydrolysis and components II, IV and V were hydrolyzed in both whole cream and isolated MFGM. Thus, the coomassie blue-positive components 10, 12 and 16 behave in a similar fashion to the PAS-positive components III, IV and V, respectively, on

76

I.H. Mather and T. W. Keenan

3

12 16 A "------~

B "-'--~

1

2

3

(a)

4

(b)

Fig. 5. Proteolysis of MFGM proteins in (a) whole cream and (b) isolated membrane. (a) Whole cream (2.5 mg MFGM protein/ml) was treated with trypsin for 30 rain as follows: 1, control-no trypsin added; 2, 20 gg/ml; 3, 50 gg/ml; 4, 100 gg/ml. (b) Isolated MFGM (4.4 mg protein/ml) was treated with trypsin (20 gg/ml) for 20 min

exposure to trypsin. These results support the proposal made above that components 10, 12 and 16 are identical to components III, IV and V, repectively. A new PAS-positive component (labelled (i) in Fig. 4 d ) appears in trypsin-treated M F G M samples. It is possible that this is a product of the hydrolysis of component 3. Component 3 stains weakly with PAS. Component (i) may be a peptide fragment which stains more strongly with PASI than the parent protein because the carbohydrate residues are in a more exposed state. Incubation of whole cream and isolated M F G M with neuraminidase led to similar rates for the release of about 70 % of the membrane-bound sialic acid (Fig. 6). Sialic acid bound to ganglioside is resistant to neuromini-

Structure of Milk Fat Globule Membrane 100

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75 0)

,.o

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r,t3

50

25

00

I

50

100 150 Time of incubation (nfin)

I

200

Fig. 6. Release of sialic acid from whole cream and isolated MFGM during treatment with neuraminidase. Whole cream (10 mg MFGM protein) or isolated MFGM (15 mg protein) were separately incubated in acetate buffer (20 mrs, pH 5.5) containing NaC1 (150 raM) in final volumes of 50ml. Neuraminidase (0.01 unit/rag MFGM protein) was added and samples were taken for sialic acid assay at the times indicated in the figure. (--9 whole cream; (--z~--zx--) isolated MFGM

dase in both whole cream and isolated M F G M (Keenan, Schmid, Franke & Wiegandt, 1975). Approximately 10% of the membrane-bound sialic acid is associated with ganglioside. Neuraminidase therefore releases over 70 % of the protein-bound sialic acid in both whole cream and isolated M F G M .

12Si.Labelling of M F G M Proteins The lactoperoxidase-125I method of labelling cell surface proteins (Phillips &Morrison, 1970, 1971; Huang, Tsai & Canellakis, 1973; Tsai, Huang & Canellakis, 1973) was applied in this present study to the labelling of the proteins in whole cream and isolated M F G M . A comparison of the 125I-labelling pattern obtained with whole cream and isolated M F G M samples is given in Fig. 7. In whole cream, components 15 and 16 were significantly labelled above background levels. Components 10 and 12 were also labelled but to a lesser extent. In addition,

78

I . H . Mather and T. W. Keenan

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1

I

--1---

~

I

- - ~

6

7

2000

1ooo

0 0.4

i 0.3

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1

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Fig. 7. Iodination of whole cream and isolated M F G M with lactoperoxidase. The protein concentration for both the cream and the M F G M sample was 4.0 mg/ml. Account was taken of the volume occupied by the cream when estimating protein concentrations. (--Q-- 9 whole cream; ( _ A _ _ ~ _ ) isolated M F G M ; ( ) scan of the gel containing the whole cream sample before sectioning. A sample of skim milk solubilized with SDS and mercaptoethanol was subjected to SDS-polyacrylamide gel electrophoresis at the same time as the radioactive samples. The arrows mark the positions of %-casein (es) , /?-casein (/?) and ~.-lactalbumin (e-L)

a peak of radioactivity, of relatively high mobility was present in such preparations. This material runs coincidentally with the turbid material shown in Fig. 4a and e. Although also coincident with component 21, it is most unlikely that the radioactive material is protein in nature (see Discussion), in the absence of H202 and lactoperoxidase the incorporation of 12sI into cream samples was 8 % of the level in samples with all necessary additions. Labelling of isolated M F G M with lactopero• led to

Structure of Milk Fat Globule Membrane

79

an increased incorporation of 115I into the membrane compared with whole cream samples. The specific activities were 867 cpm/gg protein for whole cream and 2,196 cpm/gg protein for isolated MFGM. Essentially all the major polypeptides were labelled in isolated MFGM samples and there were significant increases in the specific activities of components 10 and 12. Component 16 was only partially lost from the membrane during isolation of this particular sample for labelling. The observed increase in the specific activity of the isolated membrane, therefore cannot be ascribed to the loss of protein material during MFGM isolation. Similar results were obtained with higher concentrations of iodide (up to 10 -4 M) and H 2 0 2 (up to 10 -3 M). Extraction of Cream with M g C l 2 By three criteria, component 16 appears to be a surface p r o t e i n - the protein is hydrolyzed by trypsin and labelled by lactoperoxidase-125I in whole cream. Also the component is often partially released into the supernatant during membrane isolation. Attempts to wash this protein off the surface of fat globules by a variety of methods (EDTA, 10 rag, citrate, 10 mM, and osmotic shock) were unsuccessful. However, high concentrations of MgC12 (at least 1.0 M) led to the release of components 15 and 16 and also relatively minor amounts of components 3, 10 and 11 (Fig. 8). Extraction of 12SI-labelled whole cream with MgC12 (1.5 M) confirmed that the major bands in Fig. 8 were derived from components 15 and 16 and were not merely artifacts consisting of denatured protein (Fig. 9). 0,5

I

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}

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1

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5 6 7 Length of gel (cm)

8

9

10

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Fig. 8. SDS-polyacrylamide gel electrophoresis of the supernatant after MgC12 (1.5 ~) extraction of whole cream. The MgC1 z supernatant was dialyzed and centrifuged before preparing a sample for electrophoresis. Numbered arrows refer to the mobilities of the major proteins of M F G M . See the legend to Fig. 2 for a numbering key

80

I.H. Mather and T. W. Keenan 2000 !

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3

4 5 6 7 Length of gel (cm)

8

9

10

Fig. 9. The extraction of whole cream with MgC12 (1.5 M). (--o--o--) whole cream before MgC12 treatment; (--zx--A--) whole cream after MgCI2 treatment; (--e--t--) supernatant after MgCl z extraction, dialysis and centrifugation

Discussion

Previous attempts to define the structure of M F G M have relied to a large extent on results from electron-microscopy. This has led to several proposals for the structure of M F G M . The main area of disagreement is the extent to which the membrane proteins rearrange within the lipid bilayer after milk fat secretion and the overall stability of the resulting structure. Keenan et al. (1970) have suggested that the membrane proteins rearrange following expulsion of the fat droplets into the gland lumen. This proposal followed the observation that M F G M is apparently morphologically different from plasma membrane. Henson, Holdsworth and Chandan (1971) suggested that M F G M is only similar to mammary plasma membrane when the membrane borders a cytoplasmic inclusion. Such in-

Structure of Milk Fat Globule Membrane

81

clusions are formed at the time of fat secretion. Between 1-5 % of the fat globules in goat's milk were estimated to contain cytoplasmic material trapped between the outer membrane and the fat globule core (Wooding, Peaker &Linzell, 1970). Bauer (1972) concluded that M F G M has the structure of a typical unit membrane. He also suggested that the substantial loss of unit membrane observed in some electron-micrographs occurs during the preparation of the samples for electron-microscopy. A time dependent loss of M F G M into the milk serum was not completely discounted. The evidence presented in this paper tends to support a model for M F G M in which the membrane retains at least some elements of protein asymmetry after formation from mammary plasma membrane. At least 12 of the 21 membrane polypeptides separated by polyacrylamide gel electrophoresis are resistant to trypsin hydrolysis, compared with isolated M F G M controls. Of the other polypeptides several are obscured in the gel by protein fragments formed during trypsin hydrolysis. Two major polypeptides are hydrolyzed rapidly-components 12 and t6 in Fig. 2. Following a similar study, Kobylka and Carraway (1973), concluded that all the major proteins of the membrane are hydrolyzed by trypsin in whole cream. However, proteolysis was stopped by the addition of SDS and heat treatment. In a study on the denaturation of proteins by SDS, Nelson (1971) concluded that several proteins, including trypsin, are active for some time after the addition of detergent. In Kobylka and Carraway's study it is therefore possible that proteolysis was not stopped immediately after detergent addition, thus leading to general hydrolysis of all the membrane proteins. In this present study, three of the five PAS-positive components (II, IV and V) are hydrolyzed by trypsin in both whole cream and isolated MFGM. At least 80 % of the protein-bound sialic acid is accessible to neuraminidase in whole cream and the rate of sialic acid release is the same with either whole cream or isolated membrane as substrate. Thus, a substantial proportion of the carbohydrate residues in the membrane appear to be on the surface of the fat globule. The observation that components I and III were resistant to trypsin digestion in both whole cream and isolated membrane does not exclude the possibility that these glycoproteins are also at least partially exposed on the fat globule surface. Recent work on the binding of Concanavalin A to M F G M indicates that essentially all the lectinbinding sites are exposed on the surface of intact fat globules (Keenan, Franke & Kartenbeck, 1974). Both whole cream and isolated M F G M bound very similar amounts of Concanavalin A over a range of protein concentrations. Nicolson and Singer (1974) have recently shown that the Concanavalin A and Ricinus communis agglutinin binding sites for a number of 6

J. Membrane Biol. 21

82

I.H. Mather and T. W. Keenan

mammalian cell lines are exclusively located on the external surface of the plasma membranes. The lactoperoxidase-125I labelling technique of Phillips and Morrison (1970) has been used previously to label the surface proteins of erythrocytes (Phillips & Morrison, 1970, 1971 ; Tsai et al., 1973), mouse lymphocytes (Marchalonis, Cone & Santer, 1971), L cells (Poduslo, Greenberg & Glick, 1972) and HeLa cells (Huang etal., 1973). In this present study, major peaks of radioactivity that coincided with components 15 and 16 were obtained on labelling intact whole cream (Fig. 7). A third major radioactive component with a relatively high mobility did not appear to stain with coomassie blue and its identity remains unknown. It is possible that traces of ~-lactalbumin were still present in the preparations of washed cream. However, this seems unlikely since antibody-antigen tests did not reveal the presence of any milk serum proteins. Also the tyrosine content of ~lactalbumin (Gordon, 1971a) is not high enough to explain the apparently high specific activity of this component. The radioactivity migrated coincidentally with the turbid material shown in Fig. 4a and c. Lipids are known to have approximately the same mobility in SDS-polyacrylamide gels and have been reported to be labelled with lactoperoxidase- 1251by some workers (e.g., Poduslo et al., 1972). The nature of this material is currently under investigation. Low incorporation of 1251 into washed cream preparations could be due to the presence of extraneous neutral lipid coating the external surface of the fat globules. However, there is little evidence for this; an electrophoretic study by Newman and Harrison (1973) indicated that many charged groups are associated with the surface of washed fat globules. No evidence could be obtained for the presence of neutral lipid on the fat globule surface. The possibility that traces of lipid in the washed cream preparations were inhibiting the activity of lactoperoxidase (or trypsin) was tested by separately labelling samples of isolated M F G M and a mixture of whole cream and isolated MFGM. The combined specific activity for the mixture (whole cream and isolated MFGM) was only 3 % lower than the separately determined specific activity for isolated MFGM. Direct inhibition of enzyme activity by lipids in the cream preparations is therefore unlikely. In contrast to whole cream, the labelling of isolated M F G M with lactoperoxidase-~25I resulted in the incorporation of radioactivity into essentially all the major MFGM proteins. The low incorporation of ~25I into component 3 in isolated M F G M may be partly due to the presence of triglyceride in the preparations. Triglyceride has been shown to adhere

Structure of Milk Fat Globule Membrane

83

to the inner surface of M F G M after separation of the membrane from the butter fat (Keenan et al., 1970; Keenan, Olson & Mollenhauer, 1971). Such M F G M preparations do not readily form vesicles, presumably because of the adhering triglyceride. The low incorporation of 125I into component 3, is not therefore due to the formation of sealed "right-side out" vesicles of isolated MFGM. The results of both the trypsin and lactoperoxidase experiments indicate that components 15 and 16 are surface polypeptides. In addition, both polypeptides 15 and 16 can be eluted from the surface of fat globules with high concentrations of MgC12. Proteins from other membranes can also be removed by raising the ionic strength of the suspending medium, e.g. acetylcholine esterase from erythrocytes (Mitchell & Hanahan, 1966) and (Na § K+)-dependent ATPase from pig kidney membranes (Rendi, 1970). The possibility that components 15 and 16 are polypeptides adsorbed onto the fat globules from the milk serum cannot be entirely discounted. However, these polypeptides could not be detected when the proteins of milk serum were separated by SDS-polyacrylamide gel electrophoresis (Fig. 1). If components 15 and 16 were originally serum proteins then the fat globule surface must contain sites which selectively bind these polypeptides with a high affinity. Also the levels of protein bound are presumably at or below the binding capacity of the membrane surface; otherwise excess protein would be present in the milk serum. The above results strongly suggest that the proteins of MFGM retain elements of asymmetry in the membrane lipid bilayer after formation of M F G M from mammary plasma membrane. However, this study does not exclude the possibility that the membrane proteins rearrange during and after M F G M formation from plasma membrane. A reorganized protein layer may be present underneath the major surface polypeptides. We thank Dr. Wayne Yunghans for help with the photography and Dr. Carola Weber for conducting the Ouchterlony diffusion tests. This work was supported by grant GM 18760 from the National Institute of General Medical Science. T.W.K. is supported by Public Health Service Research Career Development Award GM-70596 from the National Institute of General Medical Science. Purdue University AES Journal paper No. 5582.

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