NUTRITION RESEARCH, Vol. 2, pp. 611-618, 1982 0271-5317/82/050611-08503.00/0 Printed in the USA. Copyright (c) 1982 Pergamon Press Ltd. All rights reserved.

TRANSFER OF ~-TOCOPHEROL TO MICROSOMES MEDIATED BY A PARTIALLY PURIFIED LIVER ~-TOCOPHEROL BINDING PROTEIN. l

Willy A. Behrens 2 Ph.D., and Rend Madi~re L.T.

Bureau of Nutrit ional Sciences, Food Directorate Health Protection Branch, Health and Welfare Canada Tunney's Pasture, Ottawa, Ontario, Canada K IA 0/2

ABSTRACT

A l iver a-[3H]-tocopherol binding protein was part ial ly purified using ammonium sulfate fractionation, gel f i l t ra t ion and ion-exchange chromato- graphy from the 100,000 x g l iver supernatant of rats fed a low vitamin E diet for 3 months and administered 200 IJCi of (x-[3H]-tocopherol. The labelled protein was used to study the transfer and binding of cr to l iver microsomes. In vitro incubations showed that the transfer of a-tocopherol was directly proportional to the amount of ~-[3H]-tocopherol binding protein and to the amount of microsomai protein present. Moreover, the transfer of ~- tocopherol increased with the temperature of incubation. A 3.8 fold excess of uniabelied ~-tocopherol (bound to the same protein) inhibited the transfer of labelled ~-tocopherol by G2%. These data suggest that an cr binding protein could play a role in the intracellular transfer of ~-tocopherol.

KEY WORDS; Vitamin E, (z-tocopherol, cr binding protein

INTRODUCTION

~-Tocopherol, the most active form of vitamin E (1) is present in the membrane of intracellular organelles where i t is thought to play an important role in protecting Iipids against oxidation (2).

The mechanism by which cr is transported to intracelluiar organelies is unknown. Recent studies in vivo (3) and in vi tro ({4-7) showed that rat l iver cytoplasm contains a protein with a m ' o t ' ~ a r weight "o~,000-34,000 that binds cr with high af f in i ty and specif icity, although its function remains unknown. Some work suggests that the biological role of this protein is to transfer a-tocopherol between membranes. Mowri et at (6) showed that transfer of ~-tocopherol from liposomes to mitochondria was enhance"d" ~'~ this protein. Murphy and Mavis (7) also showed that this protein was able to transfer the vitamin from Uposomes to microsomes.

The present study was undertaken to explore factors that influence the transfer of am tocopherol from a part ial ly purified l iver a-tocopherol binding protein (0~TBP) to microsomes.

1. Bureau of Nutri t ional Sciences, publication No. 143 2. To whom correspondence and reprint request should be sent.

61 1

612 W.A. Behrens and R. Madere

MATERIALS AND METHODS

Animals end diets. Male Wistar rats (Canadian Breeding Farms, St. Constant, Qudbec) were used in all the experiments and were fed for three months on a modified AIN-76-1ow vitamin E diet (8) as follows (w/w): Casein 20.0%, DL-Methionine 0.3%, Cornstarch 15.0%, Sucrose 50.0%, Cellulose 5%, Lard 5.0% (Substituting for corn oil), AIN mineral mixture 3.5%, AIN vitamin mixture (Vitamin E-free) 1.0% Choline bi tartrate 0.2%; or a modified AIN-76-high vitamin E diet in which O-a-tocopherol acetate was added to the corn oi l to give 0.73 g per kg of diet.

~ g n of microsomal fraction. Microsomes were isolated according to the procedure ~ '~ 'B~'d-by Kornbrust and Mavis (9) with small modifications as follows; three rats (low vitamin E diet) with a body weight averaging 397 + 9 g (SO) were anesthetized with halothane (2% in oxygen) and their l ivers were perfu~d via the hepatic portal vein with chilled 0.9% NaCI. Af ter perfusion, the livers were excised, weighed and chilled with ice- cold 5 mM Tris-maleate buffer, pH 7.4, containing 0.15 M KCI, 1 mM EDTA and homogenized in the same buffer using a Teflon-glass homogenizer. The pooled homogenate was centrifuged successively at 300, 1,600, 8,000 and 30,000 x g for 10-15 rain at each speed, and each of the pellets was discarded. Microsomes were obtained from the 30,000 x g supernatant by centrifugation at 100,000 x g for 1 hr. The pellet was washed once by suspension in and resedimentation from buffered potassium chloride with no EDTA. The final microsomal fraction was suspended in 0.1 M potassium phosphate buffer, pH 7.4 at a concentration of 1 mg protein/ml and stored at -75~

Purif ication of labelled ~-tocopherol binding protein (oFj~H]-TBP). Two rats (average body weight: 386 g) kept on a'~ow vitami~ E diet were use~ l~-this experiment. Each rat was injected intraperitoneally with 200 IJCi of D-0~[5-methyl-=H]-tocopherol (SA: 19.7 Ci/mmol, Amersham Corp. Arlington Heights IL) dissolved in 1.5 ml of saline solution containing 0.1 ml of ethanol and 0.05 ml Tween 80. 24 hrs after dosing, the rats were anesthetized with halothane and the l ivers perfused as indicated above. Af ter perfusion, the livers were excised, weighed and chilled in 0.25 M sucrose containing 10 mM Tris-HCl buffer at pH 7.4 and 1 mM EDTA. A 50% l iver homogenate was prepared in the same medium using a Teflon-glass homogenizer. The homogenate was centrifuged at 4~ for 20 min at 15,000 x g. The supernatant was recentrifuged in a Beckman LS-75B ultracentrifuge at 4~ for 1 hr at 100,000 x g to obtain a clear supernatant. The l iver supernatant was then fractionated using ammonium sulfate. The fraction that precipitated between 30 and 60% of saturation was applied to a Sephadex G-I00 column. The conditions of chroma- tography were identical to those reported previously (3). The radioactive fraction that contained 0Ftocopherol and that eluted at a molecular weight of 32,000 was collected and concentrated at 75% ammonium sulfate saturation. The resulting pellet was resuspended in 0.02 M Tris HCI buffer, pH 7.5 and dialyzed overnight in the same buffer, followed by ion- exchange chromatography on a 2.6 x 23 cm column of DEAE-Sephacel (Pharmacia, Uppsala, Sweden). The column was washed with 3 volumes of the same buffer (15 ml/hr), and subsequently the sample was eluted with a linear gradient of 0 to 0.5 M NaCI. Only one radioactive peak eluted from the column and was associated with the bulk of (z-tocopherol. The peak eluted at a concentration of 0.14 M NaCI. The fraction was collected and used without further treatment.

An identical purif ication of the protein was conducted from rats kept on a high- vitamin E diet, without the administration of labelled a-tocopherol. The purif ication steps were monitored by estimating ~-tocopherol, as indicated below.

(~-Tocopherol binding to microsomes. The binding of (l-[SH]-tocopherol to microsomes was performed acconJing to the rP~Ocedure described by Murphy and Mavis (10) as follows; A known concentration of cr H]-TBP was added to microsomes suspended in 0.01 M potassium phosphate buffer, pH 7.4 and incubated at 37~ (unless otherwise indicated) in a shaking water bath. The final volume of the incubation medium was 1 mL Following incubation the a-tocopherol bound to microsomes was separated from 0~-TBP by negative pressure f i l t rat ion with 0.22 IJm mixed cellulose acetate and nitrate f i l ters (Mill ipore

=-Tocopherol Binding Protein 613

Intertech, Inc. Bedford, Mass). The f i l ters were pretreated for 18 hrs in unlabelled 0~-TBP to minimize adsorption of the protein to the f i l ter . To quantitate the amount of ~- tocopherol bound to microsomes, 0.5-0.7 ml of the suspension was applied to a f i l te r . The f i l te r was immediately washed with 15 ml cold 0.].M potassium phosphate buffer, pH 7.4, and placed into a scint i l lat ion vial containing 15 ml of Aquasol-2 (New England Nuclear, Boston, Mass). A f te r 45 rain, the scint i l lat ion vial was vortexed, and the radioact iv i ty was measured to determine the amount of (x-[SH]-tocopherol bound to microsomes. The radioact iv i ty of a 0.1-0.2 mI aliquot of the same suspension was also measured to determine the total cr Al iquots of 0.1-0.2 ml were used to estimate ~- tocopherol at the end of the incubation period. These two measurements indicated that the puri ty of the ~-[SH]-tocopherol at the end of the incubation period was greater than 92%. The amount of ~-[SH]-TBP adsorbed to the f i l te r in the absence of mierosomes was 4 to 6% of the amount applied and equal to the amount wi th microsomes present in an incubated control (Time 0), indicating that cr was not being trapped by the microsomal particles. The loss of microsomes during f i l t ra t ion was determined by prelabelling the microsomes: 200 mg of microsomal protein was labelled by incubation in the presence of ~-[SH]-TBP~ as indicated above for 180 rain. The labelled microsomes were separated from the free ~-[=H]-TBP by centr i fugation at 100,000 x g followed by washing and resuspension in 0.1 M potassium phosphate buffer, pH 7.4. Al iquots of the labelled microsomes were f i l tered as usual and the percentage of the total radioact iv i ty retained by the f i l ters determined. The retention was 99.4 + 0.3 (SD, n = 3).

Other analyt ical methods. Protein was estimated by the method of Bradford (11) using a protein assay kit (Bio-Rad Laboratories, Richmond, CA.). o~tocopherol was estimated using the high performance liquid chromatography (HPLC) method of Thompson and Hatina (12) as previously indicated (3).

RESULTS

Puri f icat ion of labelled and unlabelled ~-TBP. Ammonium sulfate fract ionat ion followed by gel f i l t ra t ion and ion exchange chromatography produced a part ia l ly purif ied ov-TBP. When (}-[SH]-tocopherol was injected prior to sacrif ice, the f inal protein preparation had a specific ac t iv i ty equal to 0.518 mCi/mg, and 0.072 1Jg cr and 600 pg of protein per ml of solution. A preparation of 45 ml was obtained. When ~-TBP was purif ied using the same procedure from livers of rats kept on a high-vi tamin E diet, the f inal preparation contained 0.284 pg cr and 500 IJg of protein per ml of solution. A more detailed method of pur i f icat ion wi l l be published elsewhere.

cr binding to microsomes. The time course of binding of ~-tocopherol to l iver microsomes in the presence of cr is shown in Figure 1. Apparent equil ibrium was reached at three hours under the conditions of incubation described in the f igure legend. A l l subsequent incubations were carried out at this t ime. The amount of ~-[SH]-tocopherol bound to microsomes was direct ly proportional to the amount of labelled protein present in the incubation medium (Figure 2). Moreover, the amount of 0r bound to microsornes was also direct ly proportional to the amount of microsomal protein as can be seen in Figure 3. The transfer of the vi tamin was dependent on the temperature of incubation. A t 0~ the transfer was completely inhibited but increased l inearly as the temperature was raised (Figure 4). Incubation at 56~ results in no transfer due to destruction of ~-tocopherol at this temperature. Figure 5 shows that increased amounts of unlabelled cr added as 0~-TBP were able to compete with the transfer of the labelled vi tamin to microsomes. Increasing unlabelled tocopherol by 3.8 fold resulted in an inhibi t ion equal to 62%.

614 N.A. Behrens and R. Madere

flO. i

Time course of ~-tocopherol binding to l iver microsomes. Microsomes were incubated at 37~ in 1 ml of a medium con- taining 100 1Jg of microsomal protein and 83.5 nM of cr tocopherol as ~-[SH] TBP (0.223 pCi/nmol) in 0.1 N1 po- tassium phosphate buffer, pH 7.4. Each point represents the mean + SD of three determina- tions. -

Z F., k- O ==

Z 15 E= .J O n- uJ Z ,-= 0

O.15 -

O.IO-

(.> o

0.05-

(3 z

O nn

I I I I 6 0 120 180 2 4 0

INCUBATION T I M E ( r a i n )

FIG. 2

Effect of di f ferent amounts of o~[SH]-TBP in the transfer of cr to l iver micro- somes. Microsomes were incubated at 37oC, for 1B0 rain in 1 ml of a medium containing 100 pg of microsomal protein and di f ferent amounts of a- [SH]-TBP in 0.1 M potassium phosphate buffer, pH 7.4. Each point represents the mean + SD w of three determinations.

0 , 3 -

Z

O,.

~- 0.2- O E

J O n- t d Z n

o OJ- o

z

o lll

0 0 I00

r TOCOPHEROL (nM)

.... I 2O0

~-Tocopherol Binding Protein 615

FIG. 3

Effect of dif ferent am- ounts of microsomes in the binding of r Di f ferent amounts of mi- crosomes were incubated at 37~ for 180 rnin in 1 ml of a medium containing 83.5 nM of (~-[SH]-toco- pherol as ~-[SH]-TBP in 0.1 M potassium phosphate buffer, pH 7.4. Each paint represents the mean + 5D of 3 to 6 experiments.-

50-

o ~40-

_ J 0

~ 30- -i- 11. 0

0 ~- 20- I "d

Q Z ~ I0- m

0 0

/ I I I 2 4 6

MICROSOMAL P R O T E I N ( ~ g ) x 10 -2

015-

FIG. 4

Figure 4. Temperature- dependence of a-toco- pherol transfer to l iver microsomes. Microsomes were incubated at the in- dicated temperatures, for 180 rain in 1 rnl of a me- dium containing 100 p g of microsomal protein and 83.5 nM of ~-[SH]-toco- pherol, as u-[3H]-TBP in 0.I M potassium phosphate buffer, pH 7.4. Each point represents the mean + SO of 3 to 4 determinations.

I-- 0 Iv O.

OAO- E

o

. J 0 a : W

~ 0.05- r

!

Z

0

0 0

I i 20 40

TEMPERATURE (=C)

616 W.A. Behrens and R. Madere

FIG. 5

Effect of unlabelled a-toco- ,T, pherol on the transfer of a- [SH]-tocopherol to l iver micro- ~O.lOO- somes. Microsomes were in- cubated at 37~ for 180 rain in 1 ml of a medium containing =~

v 100 1Jg of microsomal protein and 83.5 nM of a-[SH]-toco- r pherol, as o~-[3H]-TBP, in the presence of different amounts ~ o.o5- of unlabelled a-tocopherol o (added as ~-TBP) in 0.1 M po- tassium phosphate buffer, pH 7.4. Each point represents the z mean + SD of 3 experiments =~ with the exception of the in i t ia l value (6 experiments), o

I I I I I oo 2oo 500 4oo

UNLABELED ~-TOCOPHEROL (nM)

DISCUSSION

The results reported herein indicate that a purified preparation of ~-[SH]-TBP, labelled in vivo with ov[3H]-Tocopherol, is able to transfer the vitamin to l iver micro- S o m e s .

The nature of the bond between a-tocopherol and 0~TBP is not known, but appears to be rather strong, as assessed by its persistance even after ammonium sulfate fractionation, as well, gel f i l txat ion, ion exchange chromatography, and extensive dialysis. Even though the incubation conditions are probably not the optimal ones the transfer of m-tocopherol to microsomes occurs. This could indicate that the af f in i ty of low-vitamin E microsomes, or membranes in general, for the vitamin is even greater than that of ~-TBP.

Under the conditions of incubation the amount of bound ~-[SH]-tocopherol was l inearly proportional to the concentration of 0~[~H)-TBP and to the amount of microsomal protein. The transfer of vitamin was dependent on temperature of incubation, indicating that the mechanism of transfer is a specific process. A 3.8 fold increase of unlabelled ~- tocopherol (bound to the same protein) was enough to produce a decrease of 62% in the binding of a-[~H]-tocopherol to micresomes. Therefore the binding of a-tocopherol to microsomes is saturable. These findings are more consistent with the view that a- tocopherol is bound to microsomal proteins, as had been suggested by Timberg and Barber (13), rather than in agreement with mere solubil i ty in, or non-especific intercalation in the membrane phospholipid bilayer of micremes as suggested by Murphy and Mavis (10).

Under our conditions of incubation 3 hours were needed to reach equilibrium. However, Murphy and Mavis (7) reported that only 4 rain were necessary to reach equilibrium, with spontaneous transfer, in the absence of o~TBP, reaching the same level in 20 rain. Mowri at al (6) reported that 20 rain were needed for equilibration but spontaneous transfer act iv i ty was much lower than that reported by the previous authors. These discrepancies could well be due to dif ferent methods used to study the transfer act iv i ty of m-TBP. As well, both Murphy and Mavis (7) and Mowri et al. (6) used m-[SH]-tocopherol bound to liposomes to study this transfer act iv i ty . This approach has been questioned by

-~ocopherol Binding Protein 617

Conrad and Singer (14) who have east serious doubt on the use of liposomes as models for biomembranes.

The molecular weight of l iver ~-TBP was estimated by several workers (3-7) to be in the range of 30,000 to 34,000. I t could be detected only in l iver. However, Mowri et al (6) reported that cytosol preparations from l iver, heart, spleen and lung were able to ~ 'a~ fe r a-tocopherol from labelled liposomes to l iver mitochondria. A more precise determination of the molecular weight and other physico-chemical properties of this protein must await a pure preparation of ~-TBP. Also, this could lead to the development of immunoehemical methods for the assay of the protein and faci l i tate determination in other tissues.

The purification described hem is an abbreviation of a more detailed method developed in this laboratory. This longer method has enabled us to produce only small amounts of a nearly homogenous protein fraction. By increasing the amount of starting material and modifying the protocol, we hope to overcome this problem.

The data presented herein, together with those of Mowri et al (6) and Murphy and Mavis (7) strongly suggest that the metabolic role of this ~ E ~ i n is to act as an intraceilular carrier of a-tocopherol. However, other possibilities, such as this protein playing a more specific metabolic role, or the existence of an enzyme with (~-tocopherol as a prosthetic group, must also be considered.

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Scott, M.L. Vitamin E. In: The Fat-Soluble Vitamins. H.F. DeLuca (ed). Plenum Press, New York and London 1978, Chapter 3. pp. 133-210.

Witting, L.A. Vitamin E and lipid antioxidants in free-radical- init iated reactions. In= F'ree Radicals in Biology. W.A. Pryor (ed). Academic Press, New York, 1980, Vol. 4, pp. 295-319.

Behrens, W.A., and Madere, R. Occurrence of a rat l iver (z-Tocopherol Binding Protein in vivo. Nutr. Rep. Inter. 25, 107-112, 1982.

Catignani, G.L. An cr Binding Protein in Rat Liver Cytoplasm. Biochem. Bioph),s. Res. Comm. 67, 66-72, 1975.

Catignani, G.L., and Bieri, 3.G. Rat l iver ~-Tocopherol Binding Protein. Biochem. Biophys. Acta. 497,349-357, 1977.

Mowri, H., Nakagawa, Y., Inoue, K., and Nojima, S. Enhancement of the Transfer of (x-TocopheroI between Liposomes and Mitochondria by Rat-Liver Protein(s). Eur. 3. Biochem. 117, 537-542, 1981.

Murphy, D.3., and Mavis, R.D. Membrane Transfer of ~-Tocopherol Influence of Soluble a-Tocopherol-binding Factors from the Liver, Lung, Heart, and Brain of the Rat. 3. Biol. Chem. 256, 10464-10468, 1981.

AIN Ad Hoc Committee on Standards for Nutritional Studies. 3. Nutr. 107, 1340- 1348, 1977.

Kornbrust, D.3., and Mavis, R.D. Relative Susceptibility of Microsomes from Lung, Heart, Liver, Kidney, Brain and Testes to Lipid Peroxidation: Correlation with Vitamin E Content. Lipids I_~5,315-322, 1980.

618 W.A. Behrens and R. Madere

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Bradford, M.M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantitites of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 7-2, 248-254, 1976.

Thompson, J.N., and Hatina, G. Determination of Tocopherols and Tocotrienols in Foods and Tissues by High Performance Liquid Chromatography. 3. Liquid Chromat. 2, 327-344, 1979.

Timberg, H.M., and Barber, A.A. Studies on Vitamin E Action: Peroxidation Inhibition in St ructura l Prote in- l ip id Micelle Complexes Derived from Rat Liver Micmsomal Membranes. 3. Nutr. 100, 413-418, 1970.

Conrad, M.3. and Singer, 5.3. The solubility of Amphipathic Molecules in Biological Membranes and Lipid Bilayers and its Implications for Membrane Structure. Biochem. 2._00, 808-818, 1981.