Cholesterol exchange between microsomal, mitochondrial and erythrocyte membranes and its enhancement by cytosol

18

Biochimica et Biophysics Acta, 398 (1975) 18-27 @ Elsevier Scientific Publishing company, Amsterdam - Printed in The Netherlands

BBA 56618

CHOLESTEROL EXCHANGE DRIAL AND ERYTHROCYTE BY CYTOSOL

BETWEEN MICROSOMAL, MITOCHONMEMBRANES AND ITS ENHANCEMENT

FRANK P. BELL

Summary 1. Cholesterol exchanges between isolated rat liver microsomes and mitochondria and between erythrocytes and microsomes or mitochondria during incubation in vitro. The exchange process is temperature dependent and is not accompanied by a net movement of sterol. 2. Cholesterol exchange between the membranes was enhanced by the addition of 105 000 X g supernatant fraction (S, 0 5 ) from rat liver. The degree to which sterol exchange was enhanced was dependent on the amount of this supernatant fraction present in the incubation. 3. Enhancement of sterol exchange was not observed with heated S1 0 5 fraction, but activity was retained after dialysis or aging at 10°C; these results suggest the presence of a choles~rol~xch~ge protein in the cytosol from rat liver _

Introduction The ~xch~geab~ity of membr~e-bound cholesterol has been the subject of numerous studies and it has been observed that the cholesterol of cells such as erythrocytes [l-6] and macrophages [ 73 can exchange with cholesterol of intracellular membranes such as microsomal lipoproteins. In addition, and mitoehondrial membranes will also exchange cholesterol with lipoproteins [S] . In all these studies, however, the direct exchange of cholesterol between membr~es has not been examined. In the present studies, the direct exchange of cholesterol between mierosomes and mitochondria isolated from rat liver was examined as well as the exchange of cholesterol between erythrocyte plasma membranes and microsomes and mitochondria. It was found that cholesterol exchange between the microsomes and mitochondria is enhanced in the presence of 105 000 X g supernatant fraction (S, 0 J ) from rat liver. The en-

19

hancement of cholesterol exchange by the cytosol is not limited to exchange between intracellular organelles since it also facilitates exchange of cholesterol between erythrocytes and microsomes or mitochondria. The possibility that cholesterol exchange between intracellular organelles is of physiologic significance is discussed. Materials and Methods Preparation of su bcellular fractions

Male Sprague-Dawley rats weighing 125-150 g were billed by decapitation and the livers excised. The livers were minced with scissors and the pieces rinsed free of blood with ice-cold 0.25 M sucrose/l mM EDTA, pH 7.4. All subsequent procedures were performed at 0-4”C. A 10% (w/v) homogenate of liver was prepared in sucrose/EDTA in a glass homogenizer with three strokes of a Teflon pestle. The homogenate was centrifuged at 850 X er,. Bx to sediment whole cells, nuclei and debris. The supernatant was centrifuged 15 min at 8500 X g,, (B20 International centrifuge) to sediment mitochondria which were washed twice and suspended in half the initial volume of sucrose/EDTA (approx. 4 mg of protein/ml). The 8500 X g supernatant fraction was centrifuged for 15 min at 20 000 X g,, (B20 International centrifuge) and the pellet discarded. The resulting supernatant was centrifuged at 105 000 X g,, for 60 min (B60 Inflations centrifuge, SB283 rotor) to sediment the microsomes which were washed twice and suspended in half the initial volume of sucrose/EDTA (approx. 2.5 mg protein/ml). The 105 000 X g supernatant was filtered through glass wool to remove any traces of floating fat, recentrifuged at 105 000 X g for 60 min and designated as S1 ,, 5 . With this procedure for the isolation of subcellular particles, microsomal contamination of mitochondria is insignificant [9,10]. Microsomes and mitochondria were isolated as above from livers of rats 18 h after intracardinal injection of a suspension containing approx. 50 &i [ ’ ‘C] cholesterol (56 Ci/mol, New England Nuclear) in Tween 20/ethanol/saline [ 4] . separation

of L3H] c~o~este~o~-labeler cry thocy tes

~~throcy~s were isolated from the blood of male New Zealand rabbits (3-4 kg) 18 h after an intravenous injection with l-2 mCi E3HJ cholesterol (generally labeled, 3.2 Cilmmol, Schwarz Bio Research) [4]. Erythrocytes contained approx. 1.1 mg cholesterol/ml of packed volume with cholesterol specific activities ranging from 1000 to 1600 dpm/l_cg. Ineu ba tions

The exchange of isotopic cholesterol between labeled donor erythrocytes, microsomes and mitochondria and initially unlabeled microsomes or mitochondria was examined in 0.25 M sucrose/l mM EDTA, pH 7.4, in the presence and absence of S1 0 5 . All incubations were done in stoppered flasks at 37°C unless otherwise stated and contained streptomycin and penicillin (50 pg and 50 units/ml, r~p~tively). After various time intervals, the membrane fractions were separated by ~en~ugation for analysis. Separation of erythrocytes, mitochondria and microsomes after incubation was performed as above by

centrifugation at 1000 X g, 8500 X g and 105 000 X g, respectively, and the isolated fractions washed twice with sucrose/EDTA.

Lipids of mi~rosomes, mitoch~~d~a and S1 0 $ were extracted ove~ight with 20 vol. chloroform/methanol (2 : 1, v/v) and the extracts washed according to Folch et al. [ll] . Cholesterol of the lipid extracts was isolated by thin-layer chroma~~aphy 143. Erythroeytes were hydroly~d in 15% alcoholic KUII and the non-~po~i~iable fraction extracted with n-hexane [4] . Cholesterol radio~~t~~ty was assayed by liquid s~int~lat~o~ count~g [3] and cholesterol was measured as cholesteryl acetate with a Hewlett-Packed Model 400 gas chromatograph equipped with a flame ionization detector using 5a-cholestane as an internal standard [ 12]. The column was a 6 ft glass U-tube, 4 mm internal diameter packed with 1% DC-560 on 100-200 mesh Gas Chrom & ~Applied Science Laboratories). ~pe~~ti~g ~rnperat~~s were: column 230” C; injection heater 235°C and detector 240°C. Flow rates (mI~min) were: nitrogen carrier, 45; air, 300; hydrogen, 30. In one set of experiments (Fig. 6), cholesterol exchange from labeled erythrocytes to microsomes was determined by directly assaying radioactivity of the rnicrosomes following digestion in a commercial tissue solubilizer (MCS reagent, Amersh~~Se~le) [ 31. Protein was ~timated by the method of Lowry et al, fl3] s Labeled membranes incubated in the absence of membrane acceptor or soluble protein (S, ,, 5 ) transferred only trace amounts of radioactivity into the incubating medium suggesting that disintegration of the membranes was not occurring, ResuIts When isolated microsomes and mitochondria were incubated in the absence of cytosol proteins, a bidirectional flux of cholesterol (unesterified) was observed. Figs 1 and 2 show the time-related changes in cholesterol specific

and microsames (e------e 1 labeied Fig. 1. The exchange of cholesterol between mitochondria fo -----0) in vivo with [14Clcholesterol, The incubation mixtures consisted of 24.5 mg of mitoohondrial protein containing 70.5 fig cholesterol and [ 14CI cholesterol-labeled miorosomes containing 12.0 mg protein and 443.1 pg cholesterol (spec. act. 349 dpm/pg) in a total volume of 17.5 ml of 0.26 M sucrose/l mM EDTA, pH 7.4. Incubation was at 87°C. Mierosomes and mitochondria were isolated from aliquots of the incubation mixture at various time inter~aIs and ckolesterd speeifle wSitities determined.

"0

2

4

6

HOURS labeled and mitoehondria (a -----a) Fig. 2. The exchange of cholesterol between microsomes (0 ------Q) in viva with [l%]cholesterol. The incubation mixture consisted of 12.2 mg of microsomat Protefn eontahting 416.1 &g eholastaro~ and [14C]cholesterol-labaled mitochondria containing 20.4 m&IPmtain and 52.7 pg chalestexol fspec.set. 412 dpm/pg) in a totat volume of 17.5 mf of 0.25 x4 sucrose/l mM EDT& pII 7.4. Incubation was at 37-C. M~erosomes and mitochondria were isoiated from "s of the incubation mixture at various time interval and cho&sterol. sp8cific activities debennined.

activity when [ *4 C] cholesterol-labeled micrasomes or [I 4 C] cholesterallabeled mitochondria were incubated in vitro with unlabeled mitochondria or microsomes, respectively. In each case, the specific activity (dpm/pg) of cholesterol in the initially labeled fraction decreased with time while the cholesterol specific activity of the initially unlabeled fractions increased with time. No change in cholesterol content of the fractions occurred indicating that the bidirectional flux of cholesterol was via an exchange process. Isotopic equilibration between labeled mitochon~a and microsomes (Fig. 2) was virtually complete by 6 h while equ~ibration between labeled microsom~ and mitochondria was established more rapidly, reaching eq~libration in approx. 3 h (Fig. I). These differences are probably related to the low levels of cholesterol in the mitochandria (approx. 3 pg/mg protein) compared to the microsames (approx. 35 jfg/mg protein). Mitochondrial and microsomal cholesterol also exchanged readily with the cholesterol of erythrocytes. The specific activity of erythrocyte cholesterol (dpm/pg) decreased with time when the cells were incubated with either isolated mitochondria or microsomes as shown in Figs 3 and 4, respectively, while the specific activity of cholesterol in the organelles increased with time. The specific activity of mit~hondrial cholesterol increased at a faster rate than that of microsomal cholesterul and, once more, probably reflects the lower cholesterol content of the mito~on~a compared with the microsomes (2.03 vs 28.14 fig cholesterol/mg protein, respectively). Isotopic ~uilib~~o~ was not complete within 4 h {Figs 3 and 4) suggesting perhaps that cholesterol ex-

1500

G ?

1200

E ; 900 P d is 6 600 E iI 300 0

C 0

2

4

/

P

0

HOURS

4

2 HOURS

Fig. 3. The exchange of cholesterol between mitochondria (o-----o ) and erythrocytes (e----O) labeled in viva with [3H1 cholesterol. The incubation mixture consisted of 2.5 ml (packed volume) of [3H3cholesterol-labeled erythrocytes (spec. act. 1400 dpm&g) and 34 mg mitochondrial protein containing 69 #g cholesterol in a total volume of 17.5 ml of 0.26 M sucrose/l mM EDTA, pH 7.4. Incubation was at 37’C. At hourly intervals, erythrocytes and mitochondria were isolated from aliquots of the incubation mixture and cholesterol specific activities determined. Fig. 4. The exchange of cholesterol between microsomes (@-----o ) and erythrocytes <+-----O ) labeled in viva with f3Hlcholesterol. The incubation mixture consisted of 2.5 ml (packed volume) of [3H3cholesterol-labeled erythrocytes (spec. act. 1400 dpm/pg) and 21 mg microsomal protein containing 546 ug cholesterol in a total volume of 17.5 ml of 0.25 M sucrose/l mM EDTA, pH 7.4. Incubation was at 37’C. At hourly intervals, erythrocytes and microsomes were isolated from aliquots of the incubation mixture and cholesterol specific activities determined.

TABLE I THE EFFECT OF TEMPERATURE ON THE EXCHANGE OF (3HlCHOLESTEROL LABELED ERYT~ROCYTES AND UNLABELED MITOCHONDRIA AND MICROSOMES

BETWEEN

The incubation mixture consisted of 0.3 ml (packed volume) of (3H~cholesterol-labeled erythrocytes (spec. act. 1000-1500 dpm&ggf, 4.3-5.3 mg mitochondrial protein containing 10.7-17.5 @#Icholesterol or 2.5-3.3 mg microsomal protein containing 51.8-68.4 /.q~cholesterol, in a total volume of 2.1 ml of 0.25 M sucrose/l mM EDTA, pH 7.4. Incubations were for 1 h. Temperature (‘C)

&g r3Hl cholesterol exchanged/mg organelle cholesterol* Mitochondria

Microsomes

--.--

Mitochondria Microsomes

37 0

466 * 123** 39 (2849)‘*+

144 i: 41 17 (11-21)

3.2 2.5

* Calculated from radioactivity (dpm) recovered in the mito~hond~a or microsomes and the initial specific activity (dpmlrrg) of erythrocyte cholesterol. * * Values are the mean of six experiments + S.E. of the mean. * * * Values are the mean of two experiments with the range in parentheses.

1

P

3

HOURS Fig. 5. The ixQm&?ement of isotwrpic cholesterol ercbatigs between WtWocyte, mi~xwamal arrd m&ochondrial mxrmbranes in the ptaasnce of SlS3* AD incubations were performed at 87’C in O,25 M sucroaefl. il~M EDTA, PH 7.4. ~3~l~~~er~-l~ed erytbroeytes 12.b mf packed v&me. spee, act_ 14of) dpmf& were incubated in R tot& v&e of 17.8 r& with (a) 34 mg mitoebondriaf protebx contaznhq 69 FtB;ehole8~~ in the presemze #k---e ) aBd sbsence @-.B a$ 32 mg of &es; lxotein, 0.0 21 mg micresumal protein conta?n&g WG gg &ok&era]. in the preaenm (*---+ snd absence (o----41 ) of 32 mg of Siog protein. 114CIchoiest%rol-labeled microsomes oontaining 18.7 mg protein sad 429 gg cholesterol f#pec. act. 3996 dpm&g) were inuubaSedIn 8 total vokzme of 27 ml with 32.3 mg m~t#ebond~~ protein cantaining 46.2 &g cholesteEo1 in tbh$pmsenee (%------a) and absence P-j aP 32 mg 0% 320s protein. Cbolestexol specific a&v&a wexa dekmtned 0x1khhe membranes sep%r%W frp*n the ineubason mixtxxms at bon&y Wervala. Fig. 6. The effect of differing ~o~~~~tio~ of Slag on [5Hleh&esterol excbwa between IaWed erythrocytes and unlabeled mW%xames.The inek&bationmixture eonajatefl of 0.3 mX @&cked volumes of [3Hlcholes.Clt~oN%beled arytbr~rtes, 1.7 mg m&xwmmai protein coata$nSng53.6 pg ahokasteroi and up to 4.3 mg Slo$ protefn in a total volume of 2.1 ml a.25 M swmse/X adi4 EDTA, pH 7.4, After incubation! for 1 h at 87’C, the mierosomss were isolated firrrm the incubation m&Ware. dissolved in % tisaue aolubilizer and assayed for radIoactivity (see Bfr&eri&a ;rutdMes&&&. The r&&eWp is &rrwn by t&e Wear regression tine*

24

TABLE

11

THE EFFECT

OF VARIOUS

TREATMENTS

ON THE ACTIVITY

OF SIo5

Membranes labeled with isotopic cholesterol were incubated with unlabeled membranes for 1 h at 37’C in 0.25 M sucrose/l mM EDTA, pH 7.4, as shown below. Incubation mixtures for each specific membrane combination were identical except for the addition of SIo5 that was either fresh. dialyzed 18 h at 10°C. aged 96 h at 10°C or heated for 10 min at 65 or 100°C.,Incubation mixtures were: 0.3 ml (packed volume) of labeled erythrocytes containing 300 fig cholesterol (spec. act. 1347 dpm/pg), microsomes (2.3 mg protein; 72 j.& cholesterol) or mitochondrla (6.1 mg protein; 27.4 kg cholesterol) in a total volume of 2.1 ml; labeled mitochondria (6.5 mg protein; 12.4 pg cholesterol. spec. act. 2904 dpm/Fg) and unlabeled microsomes (2.3 mg protein: 51.2 fig cholesterol) in a total volume of 4.0 ml; labeled microsomes (2.5 mg protein; 58.6 l.(g cholesterol. spec. act. 2862 dpm/kg) and unlabeled mitochondria (6.0 mg protein; 9.2 pg cholesterol) in a total volume of 4.0 ml. Radioactivity (dpm) recovered in the mitially unlabeled fraction of incubations wlthout SIo5 added (control) was set at 100% and radioactivity (dpm) recovered in the initially unlabeled fraction of incubations receiving S105 was expressed relative to the 100% value. Additions

of

Relative

percent

exchange

of isotopic

cholesterol

s105* f3Hl Erythrocytes/ Mitochondria

[3Hl Erythrocytesl Microsomes

100 229 158 196 111 -

100 222 213 205 43 55

-

___

None (control) Fresh Dialysed Aged 1OO’C heated 65’C heated

[ 14Cl Microsomesl Mitochondria .- ~____

[ 14Cl Mitochondrlal Microsomes

100 206

104 98

* Incubations with erythrocytes received 0.6 ml of Slos or 0.6 ml of sucrose/EDTA other incubations received 1.6 ml of SIo5 or 1.6 ml of sucrose/EDTA (control).

(control);

all

terol-labeled microsomes and mitochondria was increased almost 2-fold. Differences in the rate of exchange observed as early as 1 h of incubation were retained over the 3-h incubation period. The facilitation of cholesterol exchange observed in the presence of S1 o 5 bears a linear relationship to the concentration of S1 o 5 added to the incubations (Fig. 6). The influence of various treatments on the ability of S1 o 5 to enhance membrane cholesterol exchange is shown in Table II where [” H] cholesterol exchange in the absence of SOS was set at 100% and exchange in the presence of S1 o s expressed relative to the 100% value. The activity of S1 o s was found to be destroyed upon heating (65 and 100°C) but not by cold since S1 o 5 that was aged 4 days at 10°C retained activity. Activity was also retained in S1 o 5 that was dialysed for 18 h. Discussion Cholesterol is synthesized by the microsomal fraction (endoplasmic reticulum) of mammalian cells [14] and must by necessity be redistributed to other parts of the cell such as to the mitochondria and plasma membrane for structural roles as well as to serve as a precursor for biosynthesis of such compounds as corticosterone by adrenal mitochondria. While the mechanism of intracellular cholesterol translocation is unknown, the studies presented here demonstrate that cholesterol exchange between isolated microsomes and mito-

25

chondria occurs in vitro. This exchange appears to be the result of a direct interaction of the membranes since the presence of soluble proteins or lipoproteins is not necessary for exchange to take place (Figs 1 and 2). From these studies one cannot say that cholesterol exchange also occurs between these organelles in vivo; however, if the endoplasmic reticulum and the outer mitochondrial membrane are contiguous structures as has been suggested [ 151, in vivo exchange would seem feasible. The exchange of cholesterol between the microsomes and mitoehondria observed in these studies is not the result of a unique interaction between these membranes since each also exchanges cholesterol directly with that of plasma membranes of erythrocytes (Figs 3 and 4); the exchange process is probably not different from the exchange of cholesterol between erythrocytes and iipopro~~s which is also ~rn~rat~e dependent {16]. The exchange of cholesterol between erythrocytes and mitochondria was affected to about the same extent by temperature as the exchange between erythrocytes and microsomes since the ratios of cholesterol exchanged to mitochondria vs microsomes were similar for both 0 and 37°C incubations (Table I). An approximate Z-fold increase in the exchange of cholesterol between the microsomes and mitochondria was observed when St 0 5 was added to the incubations (Table II, Fig. 5); St 0 s was equally active in promoting cholesterol exchange between erythrocyte membranes and mitochondria and microsomes ~dicating that its exch~ge~~~~~g property is a feature not restricted to exchange of sterol between in~a~ellul~ organelles. The striation of exwas linear over a $-fold range of S, o 5 concentrations in change by SXOS incubations of [ 3 H] cholesterol-labeled erythrocytes and microsomes (Fig. 6). The nature of the S, o 5 component that stimulates cholesterol exchange has not been examined in detail in these studies but, has been found to be nondialysable, heat labile but stable at 10°C (Table II), suggesting that the component is a protein or lipoprotein. Data from the incubations of labeled microsomes with mitochondria in the presence of S1 ,, 5 (Fig. 5) raises the possibility that microsomal cholesterol is contained in multiple pools. With S 1 o 5 present, the specific activity of mitochondrial cholesterol exceeded the initial specific activity (3996 dpm/pg) of microsomal cholesterol reaching 4825 dpmjpg after 3 h incubation; these data could arise only if the apparent specific activity of microsomal cholesterol was less than the specific activity of an actively exchanging cholesterol pool. It. is possible that similar results would have been observed in incubations without S1 o J (Figs 1 and 5) if incubations times had been increased. The S1 o s used in these studies contained approx. 1.6 pg cholesterol/mg protein and also became labeled with [3 H] cholesterol in the incubations. However, it is unlikely that the S, 0 5 component stimulates cholesterol exchange by acting as a carrier between membranes since S 1 o s cholesterol specific activity, measured after incubation of [ 3 H] cholesterol-labeled erythrocytes with microsomes, was less than 10% of the microsomal cholesterol specific activity after 1, 2, 3 and 4 h of incubation. After 1 h of incubation, the radioa~ti~ty of the S 1 o s fraction was 3400 dpm and increased steadily to 4700 dpm by 4 h. During the same time interval, microsomal radioactivity increased from 126 000 to 430 000 dpm. This observation reduces the possibility that a pro-

26

tein such as the sterol carrier protein found in S I o 5 is tr~slocating cholesterol between membranes as had been suggested [17] . It is interesting that 105 000 X g supernatant from rat liver stimulates the exchange of phospholipids between microsomes and mitochondria in vitro [9,10,1$,19]. This phospholipid exchange activity of the supernatant appears not to be lipoprotein in nature [lo] ; in fact, fractionation of beef heart [ 201 and beef liver [21] cytosols have yielded 21 000 and 22 000 molecular weight protein fractions, respectively, that will stimulate phospholipid exchange between membranes. A recent study by Ehnholm and Zilversmit [22] showed that a partially purified fraction (pH 5.1 supernatant) from beef heart cytosol stimulated 3 2 P-labeled-phosphatidylcholine but not f1 4 C] cholesterol exchange between labeled liposomes and mitochond~a. These data suggest that the choles~rol~xchange component of is not identical with the phospholipid-exch~ge protein. S 105 The exchange of microsomal cholesterol with mitochondria and plasma membranes may be of physiological importance in distributing cholesterol from the site of synthesis to other areas of the cell. Cholesterol exchange between membranes in this study tended toward equilibration of isotope (Figs l-4) between membrane fractions without evidence of net transfer. This is not to say, however, that exchange cannot lead to net movement, particularly if one of the membrane systems involved in an exchange process was metabolizing cholesterol and thus offsetting the balance of the bidirectional flux of sterol. In this regard, it is noteworthy that simple cholesterol exchange between plasma lipoproteins and e~thro~ytes can be “converted” to a net transfer of sterol to the lipoproteins when the enzyme lecithin : cholesterol acyltransferase (EC 2.3.1.43) is present in the system [2,23]. The significance of the operation of in vivo would serve to further facilitate cellular cholesan “SI 0 5 component” terol distribution. Acknowledgements This work was made possible by support from The Medical Research Council of Canada (MT-3067) and the Canadian Heart Foundation. The author is grateful to Mr D. Archer and Mr W. Nusca for technical assistance and to Mrs Swift and staff for secretarial assistance. F.P.B. is a Research Scholar of the Canadian Heart Foundation. References 1 2 3 4 5 6 7 8 9 10 11 12 13

Hagerman, J.S. and Gould, R.G. (1951) Proc. Sot. Exp. Biol. Med 78.329-332 Murphy, J.R. (1962) J. Lab. Clin. Med. 60, 571-678 Bell, F.P. and Schwartz, C.J. (1971) Biochim. Biophys. Acta 231, 553-557 Bell, F.P. (1973) Exp. Mol. Pathol. 19. 293-303 Quarfordt, S.H. and Hilderman. H.L. (1970) J. Lipid Res. 11, 628-535 Ashworth, L.A.E. and Green, C. (1964) Biochim. Biophys. Acta 84.182-187 Werb, 2. and Cohen, Z.A. (1971) J. Exp. Med. 134.1545-1569 Graham, J.M. and Green, C. (1967) Biochem. J. 103,16C-18C Wirtz, K.W.A. and ZiIversmit. D.B. (1968) J. Biol. C&em. 243.3596-3602 Wii, K.W.A. and Zilversmit, D.B. (1969) Biochim. Biophys. Aeta 193.105-116 Folch, J., Lees, M. end Sloane-Stanley. G.H. (1957) 3. Biol. Chem. 226.497-509 Vela, B.A. and Acevedo. H.F. (1969) Steroids 14.499-517 Lowry, O.H., Rosebrough. N.J., Fan, A.L. and Randall, R.J. (1961) J. Biol. Chem. 198.265-275

14 Chesterton. C.J, (1968)

J. BioI. Chem. 243, X147-1161

15 Parsons, D.F., WiUiams. G.R., Thampsrxx, W.. W&m, ture and Compartmentation Adriatica Editrice, Beri 16 17 18 19 20 21 22 23

ff&rag&rieHo,

D. and Chance, B. (1967)

E., Papa, S., SIater,

Mitochondrial Struc-

E.C. and Tager,

J.M.,

e&f,

pp. 29,

Bell, F.P., Somer, J.B., Craig, I.H. and Schwartz, C.J. (1972) Pathology 4,206-214 Scallen, T.J.. Schuster, M.W. and Dhar, A.K. (1971) J. Biol. Chem. 246,224-230 Aldyama, M. end Sakagami, T. (1969) Biochim. Biophys. Acta 187, 106-112 McMurray. W.C. and Dawson, R.M.C. (1909) Biochem. J. 112,91-108 Wirtz, K.W.A. and Zilversmit, D.B. (1970) FEBS Lett. 7.4446 Wirtz, K.W.A., Kamp, H.H. and Van Deenen, L.L.M. (1972) Biochim. Biophys. Acta 274.606-617 Eimhohn, C. and Zilversmit, D.B. (1973) Y. Biol. Chem. 248,1719-1724 Glomeet. J.A. (1970) Am J. Ciin. Nutr. 23, X129--1136