Hydrolysis of the Lipoprotein Fractions of Milk by Phospholipase C

Hydrolysis of the l.ipoprotein Fractions of Milk by Phospholipase C J. P. O ' M A H O N Y 1 and W. F. SHIPE Department of Food Science, Cornell University Ithaca, New York 14850 Abstract

Phospholipase C hydrolyzed about 90% of the lipid phosphorus of both low- and high-density lipoprotein fractions of milk. Phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin were most readily hydrolyzed whereas phosphatidyl serine and inositol were only hydrolyzed after the other phospholipids had been almost completely hydrolyzed. By contrast only about 60% of the native phospholipids in milk were hydrolyzed, indicating that some are protected. That phospholipase C inhibited oxidation indicated that the phospholipids available for hydrolysis were also oxidized.

Introduction

The authors (13) have shown that phospholipase C inhibits the development of oxidized flavor in milk. Our present research was undertaken to determine the nature of phospholipase C action on milk and fractions of the fat globule membrane. I t was hoped tbat this would shed some light on the mechanism of the antioxygenic effect of the enzyme and also the structure of the fat globule membrane. Experimental Procedures

Preparation of fat globule membrane. Isolation procedures were essentially those of Brunner (2). Freshly drawn, uncooled milk from healthy individual Holstein cows was brought immediately to the laboratory and separated at 37 C in a small DeLaval separator at 10,000 rev/min. Creams were washed 4 times with 3 volmnes of 0.25 ~ sucrose at 40 C. Cream was adjusted to 35% fat with 0.25 ~ sucrose, cooled to 5 C and churned in half pint bottles in a laboratory churn. Butter-

milk was poured off and retained. Butter was melted at 40 C and centrifuged, and serum was then combined with buttermilk. Mixture was centrifuged at 25,000 × g for 1 hr. Cream plug at the top of the tnbe~ was discarded. Pellet at bottom of tubes was redispersed in 0.25 ~ sucrose and recentrifuged at 25,000 × g for 1 hr. Washing the pellet was repeated once and the supernatant was added to the original supernatant which was centrifuged at 25,000 g for 2 hr and the pellet was discarded. Supernatant was salted out in 2.2 ~ (NH~)2 S04 and the solids retained. Solids were designated low-density lipoprotein fraction and the original pellet was designated high-density lipoprotein fraction. Both fractions were dialyzed against 30 volumes of distilled water at 5 C with 5 changes of water. Dialyzed materials were freeze-dried at 0 C and stored in a darkened desiccator. Lipoprotein analysis and fraetionation. Lipid was extracted from the freeze-dried fractions by the method of Folch et al. (6). F a t content of the lipoproteins was determined by a micro adaptation of the Mojonnier method (12). About 50 mg of lipoprotein were accurately weighed into a micro-Mojonnier flask and following reagents added: 3.6 ml of water, 0.6 ml of ammonium hydroxide, 4.1 ml of ethanol, 10.25 ml of ethyl and petroleum ether. The standard Mojonnier procedure was used to complete the test. Two-dimensional, thin-layer chromatography separated lipids. Research Specialities Inc. equipment was used to prepare 0.4 mm thick layers of silica gel H R from Brinkmann Instruments. Air-dried plates were heat activated for 2 hr at 120 C and cooled. Lipid was applied to plates in suitable quantities. Plates were developed in glass chambers, lined with solvent saturated filter paper. Solven~ systems of Parsons and Patton (15) were modified to contain chloroform/methanol/water/28% aqueous ammonia, 65/37/4/.4 for the first dimension and chloroform/acetone/methanol/acetic acid/ water, 50/20/10/10/5 for the second dimension. Development was 40 to 50 rain in each direction with 10-rain drying at 20 to 25 C in between.

Received for publication October 18, 1971. 1 Current address: John Stuart Research Laboratories, Quaker Oats Co., Barrington, Xllinois 60010. 408

LIPOPROTEIN

The spots were made visible by iodine vapors or by spraying with one of the following sprays: sulfuric acid 50% v/v, the specific spray of Dittmer and Lester (5) for phospholipids, and ninhydrin reagent (0.2% in ethanol) for compounds containing an amino group. Individual polar lipids were identified by specific sprays and by co-chromatography with authentic phospholipids from Applied Science Laboratories, Inc. Routinely~ the plates were first placed in tbe iodine vapor tank for 2 rain, the spots were then stipled with a sharp metal spike and the iodine vaporized. Plates were then sprayed with ninhydrin and heated to 100 C for 20 min. Ninhydrin showed position of phosphatidyl serine and ethanolamine and any lysophosphatidyl ethanolamine. This then allowed other components to be identified since relative positions of the phospholipids were constant. Phospholipid extraction and analysis were perfo~nned according to the scheme: Milk ~[ojonnier extraction

I

Lipids Solvents were evaporated under reduced pressure at 40 C, made up to 10 ml in a volumetric flask with C H C 1 J M e O t t , 2/1, v/v. 600 tditers were spotted on a preparative T L C plate and developed with hexane/ethyl ether/acetic, 75/

25/1.

T

Polar lipids (at origin)

1

Analyzed for phosphorus or eluted with CHC13/MeOH , 2/1, v/v, 3 times. Evaporated to dryness under nitrogen and .5 m] of C H C l J MeOH added. 400 ~liters were then spotted on a TLC plate.

FRACTIONS

409

percentage of the total recovered phosphorus in the individual spots. Average recoveries were from 80 to 95%. Nitrogen was determined according to the AOAC method (1).

Determination af phosphollpase C hydrolysis. The method of MeFarlane and Knight (11) as modified by Worthington Laboratories (17) was used. Unchanged phospholipids were precipitated with TCA. Liberated phosphorus containing compounds in supernatant were digested and inorganic phosphorus measured. (Since this work was undertaken, Ottolenghi (14) described various methods for the assay of phospholipase C.) To obtain the individual phospholipids, the mixture after incubation was extracted with chloroform/methanol 2/1, v/v, 3 times. The mixture was shaken with the solvents in a 100 ml separatory funnel and left until the layers separated, the bottom layer was drawn off carefully. This procedure was repeated twice and the combined extracts evaporated under nitrogen and made up to 2 ml in a 2 ml volumetric flask. Suitable aliquots were spotted on TLC plates and separated in two dimensions as described previously. Thiobarblturic acid ( T B A ) test. Basic technic of K i n g (9) was modified: Stoppered bottles containing 17.5 ml of milk samples were warmed for 10 rain in a 40 C bath. One milliliter of 50% TCA and 2 ml aldehyde,free, redistilled 95% ethanol were added. Bottles were shaken vigorously for 20 see and returned to the 40 C bath for 5 rain. Contents were filtered through number 42 Whatman paper. A 2 ml aliquot of the TBA reagent was added to 4 ml of each filtrate. (The TBA reagent contained 0.9 g TBA p e r 100 ml of aldehydefree ethanol.) A f t e r capping and shaking the contents, the test tubes were placed in a 60 C bath for 60 rain. Absorbance of the cooled samples was read at 535 nm against a distilled water reference.

1

Two dimensional TLC fi

Individual phospholipids analyzed for phosphorus content. Phosphorus of individual phospholipids was determined according to Rouser e t a ] . (16). Optical densities were corrected by subtracting the reading from a blank area eorresponding in size to that of the sample. Values were then converted to micrograms of phosphorus by factor 11.5 derived from a standard curve prepared with disodium hydrogen phosphate. Percentages of phospbolipids were expressed as a

Results and Discussion

Data in Table 1 show that phospholipase C inhibits oxidation as measured by the TBA test. Extent of inhibition depended on the sample source. Phospholipolysis of the lipoprotein fractions from the fat globule merebrahe is in F i g u r e 1. Hydrolysis was r a p i d in the early stages with about 80% of the phospholipid being hydrolyzed in 2 hr. The pattern of hydrolysis was similar for both lowand high-density lipoproteins. The same pattern of hydrolysis was obtained in fractions from a large number of Friesians in the same JOURI~'AL OF DAIRy SCIEI~CE VOL. 55, NO. 4

410

0'~¢IAHONY

TABI~ 1. Effect of phospholipase C treatment on thiobarbituric acid test of various milk after 3 days storage at 5 C. Increase in TBA X 10 s Cow

Control milk

Treated milk

Suppression

1 2 3 4 5 6

145 65 31 146 94 43

47 25 18 72 30 0

63 58 32 47 60 49

(%)

herd. To determine whether the reduction in rate of hydrolysis was due to enzyme inhibition an additional 0.1 ml of the lipoprotein homogenate was added to the mixture and incubated for another 30 rain. The lipids were extracted and separated but no phosphatidyl choline was detected, indicating that the enzyme was still active and that the rate of hydrolysis was primarily dependent on the concentration of available substrate. Percentages of hydrolysis of phospholipids extracted from low-density lipoprotein mixtures after incubation for 0.5, 1.0, 1.5, and 2.0 hr are in Table 2. The results show complete hydrolysis of phosphatidyl choline (PC) and almost complete hydrolysis of the sphingomyelin (Sph) and phosphatidyl ethanolamine ( P E ) . However, phosphatidyl serine (PS) and inositol ( P I ) were hydrolyzed only after the other phospholipids had been almost completely hydrolyzed. Thin-layer chromatography revealed lysophosphatidyl ethanolamine ( L P E ) and phosphatidic acid. These were identified by

AND

SHIPE

specific sprays and co-chromatography with authentic standards. Lysophosphatidyl ethanolamine occasionally chromatographed along with PS. Its occurrence however was always less than 1% of the total phospholipid. Lysophosphatidyl ethanolamine was not present in all milks and its significance is not clear. To determine the rate and extent of phospholipolysis in native milk, 2.5 mg of enzyme were added to I8 g of milk and the mixture incubated for 0.5 to 2 hr. Then the reaction was stopped with ammonium hydroxide and the lipid was extracted by the Mojonnier method and analyzed. Amount of hydrolysis was determined by subtracting the remaining lipid phosphorus from the initial lipid phosphorus. Results are in F i g u r e 2. The rate of phospholipolysis was initially high with 40% of the phospholipid being hydrolyzed in 0.5 hr followed by a slower rate up to 2 hr. A t the end of 2 hr 40% of the initial phospholipid remained unhydrolyzed. Relative amounts of hydrolysis of individual phospholipids are in Table 3. Phosphatidyl choline was hydrolyzed more rapidly than the others with no hydrolysis of PS or PI. The relative rates of hydrolysis of phospholipids indicate that p a r t of the phospholipid substrate in native milk is protected from phospholipase, since only about 60% of the phospholipid in native milk was hydrolyzed in 2 hr whereas in both the low- and the highdensity lipoprotein fractions almost 90% was hydrolyzed. Itayashi and Smith (7) reported that the deoxycholate released lipoproteins conrained 67% of the total lipid phosphorus. I t TABLE 2. P e r cent of initial phospholipids in low density lipoproteins of f a t globule membrane hydrolyzed by phospholipase C.a Time (hr)

10(

Phospholipid

-'- 3( n~' (3_

Sphingomyelin Phosphatidyl choline Phosphatidyl ethanolamine PROTEIN Phosphatidyl serine o = HIGH DENSITY LIPOPROTEIN Phosphatidyl inositol

o /.

25 / / /

-.r-

'~

0

o

o5

1.o

1.5

z0

z5

3.o

5.o

~TIME (HR)

:FIO. 1. Phospholipolysis of fat globule membrane fractions by phosphollpase C. J O U R N A L OF D A I R Y S C I E N C E V O L . 5 5 ,

NO. 4

0.5

1.0

1.5

2.0

78

83

83

86

89

98

99

100

68

88

86

86

0

18

24

51

18

43

35

34

a Values are means of two replications. Coefficient of variability was 7%. Identical patterns were obtained for the high density lipoproteins.

LIPOPROTEIN

8 °6O

t.aJ CO

°

r'~

~J D rY O

i o

45

o

0

t'Y"

>"-r"

u) O I f3_

30 N a..

I.J_

__1 0 212

O

"nF-._J

0.5

1.0

1.5

2.0

TIME (HR) Fro. 2. Ph0sph01ipolysis of native milk by phospholipase C. may be significant that this is about the same as the percentage of lipid phosphorus hydrolyzed by phospholipase C in native milk in our study. Conceivably, the enzyme attacked the same phospholipids released by deoxycholate, assuming that loosely bound phosphoIipids are involved in both cases. The f a t emulsions in milks treated with enzyme for 4 hr at 37 C were not destabilized as indicated by no oiling-off on the surface of hob coffee. This is compatible with observation (7) that removal of the deoxycholate released material does not cause oiling-off, indicating that the polar groups of the phospholipids on the outer lipoproteins are not critical to physical stability of the fat globule membrane. This has been observed by Lenard and Singer (10) for red blood cells and more recently by Chuang et al. (4) for reconstituted cytochrome oxidase membranes. I n the latter work hydrolytic release of 80% of the phosphorus present did not destroy the membrane structure. Their conclusion that hydroph0bic interactions between phospholipid and protein constituents of the membrane play an important role in

411

FRACTIONS

maintaining integrity of the membrane also are applicable to our study. The role of neutral lipid components in the structure of the fat globule membrane is unclear but hydrophobic interactions between these and the proteins are undoubtedly also important. Compositional data on the lipoproteins (Table 4) indicated that protein to phospholipid ratios of 1.2 and 2.7 for low- and highdensity lipoproteins are in the ranges reported by Jenness and Palmer (8) for butter serum and buttermilk. This information also supports the theory that high-density lipoprotein is not firmly bound to the fat core. Furthermore, the high-density fraction was reddish brown which is the same color as the high-density outer fraction obtained by Chien and Richardson (3). However, it should be pointed out that the lipoprotein fractions prepared in this work were heterogeneous. Sucrose gradient separations revealed that the high-density lipoprotein contained at least two classes of lipoproteins as shown by layers appearing in the gradient at different densities. Even though phospholipase C activity did not affect the physical stability of the fat emulsion, it did alter oxidative stabiliby. This suggests that change in oxidative stability does not involve a significant change in components responsible for emulsion stability. Possibly, components involved in oxidative stability are loosely bound in the outer portion of the fat globule membrane. That phospholipase inhibited oxidation indicates that the phospholipids available for hydrolysis were also involved in oxidation. Apparently, hydrolysis caused a positional rearrangement of constituents. Perhaps the diglycerides formed by hydrolysis migrated towards the hydrophobic TABL~ 3. Per cent of initial phospholipids in native milk hydrolyzed by phospholipase C.a Time (hr) Phospholipid

0.5

2.0

Sphingomyelin Phosphatidyl choline Phosphatidyl ethanolamine Phosphatidyl serine Ph0sphatidyl inositol

37 75 55 0 0

51 82 63 0 0

a Values are means of two replications on two samples. The coefficient of variability averaged 5%, where CV -- . -100s -

s = standard deviation ~ -- mean

JOURI~AL O~ DAIRY SCI]~I~CE VOL. 55, NO. 4

O'MAHONY

412 TABLE 4. Composition membrane. Cow

Lipoprotein

421 438 530

Low density

of the low and

H i g h density

Average

SHIPE

high density lipoprotein fractions of the f a t globule

Protein a,1

Total a Lipid

Neutral a Lipid

Phospholipid a,2

Protein: phospholipid

21 21 24 22

79 79 76 78

65 65 61 64

14 14 15 15

1.2 1.2 1.2 1.2

43 41 41 41

57 59 59 58

42 44 43 43

15 15 16 15

2.9 2.7 2.6 2.7

Average 421 438 530

AND

a Values are average of duplicates expressed as p e r cent dry weight. 1 P e r cent nitrogen × 6.25. 2 Lipid phosphorus × 25. center of the globules and thereby became less susceptible to oxidation. I t is also possible that hydrolysis was accompanied by a partial redistribution of copper. Possibly, some of the copper associated with the fat globule membrane was released to the serum phase.

(9)

References (1) Association of Official Agricultural Chemists. 1960. Official and Tentative Methods of Analysis, 9th ed., Washington, D.C., p. 643. (2) Brunner, J. R. 1965. Physical Equilibria in Milk: The Lipid Phase in Fundamentals of Dairy Chemistry. ed. B. H. Webb and A. H. Johnson. Avi Publishing Co., Westport, Conn. p. 403. (3) Chien, It. C., and T. Richardson. 1967. Gross structure of the fat globule membrane of cow's milk. J. Dairy Sci., 50" 451. (4) Chuang, T. F., Y. C. Awasthi, L. Funk, and F. L. Crane. ]970. Involvement of hydrophobic and hydrophi]ic groups of phospholipids in membrane formation. Bioehim. Biophys. Aeta, 211:599. (5) Dittmer, J. K., and R. L. Lester. 1964. Specific spray for phospholipids. J. Lip. Res., 5: 126. (6) Folch, J., M. Lees, and G. It. S. Sloane. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem., 226: 497. (7) Hayashi, S., and L. M. Smith. 1965. Membranous material of bovine milk f a t globules. L Comparison of membranous fractions released by deoxyeholato and by churning. Biochemistry, 4 : 2550. (8) Jenness, R., and L. S. Palmer. 1945. Substances adsorbed on the fat globules in

JOURNAL OF DAIRY SCIENCE VOL. 55, lqO. 4

(16)

(11)

(12) (13)

(14)

(15)

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(17)

cream and their relation to churning. V. Composition of the "membrane" and distribution of the adsorbed substances in churning. J. Dairy Sci., 28: 611. King, R. L. 1962. Oxidation of milk fat globule membrane material. I. Thiobarbituric acid reaction as a measure of oxidized flavor in milk and model systems. ft. Dairy Sci., 45: 1165. Lenard, J., and S. J. Singer. 1968. Structure of membranes: Reaction of red blood cell membranes with phospholipase C. Science, 159: 738. MacFarlane, M. G., and B. C. J. G. Knight. 1941. Biochemistry of bacterial toxins. I. Leeithinase activity of C. wetvhii toxins. Biochem. J., 35: 884. Mojonnier, J., and 1=[. C. Troy. 1922. The Technieal Control of Dairy Products. 1st edition. Chicago, Illinois. O'Mahony, J. P., and W. F. Shipe. 1970. Effect of variations in phospholipid composition of f a t globule membrane fractions and the oxidative stability of milk. ft. Dairy Sci., 53: 636. Ottolenghi, A. O. 1969. I n Methods in Enzymology, XIV. ed. Colowick and Kaplan. Academic Press, New York City, p. 188. Parsons, J. G., and S. Patton. 1967. Two dimensional thln-layer chromatography of polar liplds from milk and mammary tissue. J. Lipid Res., 8: 696. Rouser, G., A. N. Siakotos and S. Fleiseher. 1966. Quantitative analysis of phospho]ipids by thin layer chromatography and phosphorous analysis of spots. Lipids, 1: 85. Worthington Biochemical Corporation, Pub. 4. 1968. Freehold, New Jersey.