Lipid Binding Properties of a Factor Necessary for Linoleic Acid Desaturation A L I C I A I. LEIKIN, A N I B A L M. NERVI 1 and RODOLFO R. BRENNER 1, Instituto de Fisiolog~a, C~tedra de Bioqufmica, Facultad de Ciencias M~dicas, Universidad Nacional de La Plata, Calle 60 y 120, 1900 La Plata, Argentina ABSTRACT

Suspension and centrifugation of crude microsomes of rat liver in low ionic strength solution separated a soluble protein fraction that is necessary for the full activity of the linoleic acid desaturase. The fraction partially purified throughSephadex G-150 still retains lipids which are mainly constituted by phosphatidylcholine. Linoleic acid predominates in the fatty acid composition. By NaC1 gradient centrifugation and electrophoresis in gelatinized cellulose acetate, the factor behaves like a lipoprotein. The factor binds linoleic acid and linoleyl-CoA that are desaturated to 3,-linolenic acid when incubated with washed microsomes. Albumin does not replace the factor. INTRODUCTION

The enzymatic system involved in stearylCoA desaturation has three components embedded in the lipid medium of the microsomes: the NADH cytochrome b s reductase, the cytochrome b s and the desaturase (1-3). All of them have been purified (4) and the system reconstituted artificially on liposomes (5). Linoteic acid is considered to be desaturated by a similar system (6). In our laboratory it has been possible to obtain a "soluble factor" either from unwashed microsomes or cytosol which is necessary to obtain maximal desaturation of fatty acid (7-9). Washed microsomes lose most of their desaturation capacity which is recovered when the microsomal washing or cytosol separated at 105,000 x g is re-added into the microsomal suspension. This factor or factors would be involved specially in the A6 desaturation of fatty acids (7). The factor has been partially purified (9), and it has been proved that a protein is involved in its activity (7). It also contains lipids (7). We have also shown that catalase can reactivate washed microsomes (9). However, there was no correlation between the catalase activity of the factor and the activating effect (9). This result was confirmed by Jeffcoat et al. (10). In this work, we have shown that the factor behaves like a lipoprotein and it is able to bind linoleyl-CoA or linoleic acid. The linoleic acid bound to the "soluble factor" can be desaturated by the desaturase of the microsomal membrane, being converted to 3,-linolenic acid. MATERIALS AND METHODS

[1-]4C]Linoleic

acid (61

~Ci/;tmol)was

1Members of the Carrera del Investigador Cientifico of the Consejo Nacional de Investigaciones Cientlficas y Tdcnicas, Argentina.

provided by the Radiochemical Center, Amersham, England. [~4C] Labeled and unlabeled linoleyl-CoA were prepared according to Kornberg and Pricer (11). Sephadex G-100, G-150 and bovine serum albumin fraction V were provided by Sigma Chem. Co., St. Louis, MO. ATP, CoA, NADH disodium salt and glutathion were obtained from Boehringer, Argentina, Buenos Aires. Microsomes

Wistar rats of 150-250 g were used. Liver microsomes were obtained by differential centrifugation as mentioned elsewhere (7). Two main fractions were obtained at 105,000 x g: the whole microsomes (M) and the cytosol (C). Soluble Factor

It was obtained from microsomes by extraction with low ionic strength solution (7) and by centrifugation at 105,000 x g for 60 min. The extracted microsomes (Me) and a supernatant (Sp) containing the soluble factor were separated. Desaturation Assay

It was performed using free fatty acids or acyl-CoA. Proteins of whole microsomes (M), 2.5 mg, were incubated with 66 /.tM [1-14C]18:2 in a total volume of 1.6 ml with the necessary cofactors (9) at 35 C for 20 rain. When the activity of the extracted microsomes (Me) was investigated, the incubated amount corresponded to the remainder after the extraction of the "soluble factor" of 2.5 mg of whole microsomal protein (M). The desaturated amount of labeled acid was measured by gas liquid radiochromatography as described previously (12). Proteins were determined either by a micro-biuret method ( 1 3 ) o r by the procedure of Lowry et al. (14).

1021

A L I C I A I. L E I K I N , A N I B A L M. N E R V I A N D R O D O L F O

1022

"~ instrument (Fractovap Mod. GT) with flame : detector. The column was filled with 10% O ethylene-glycol succinate in Chromosorb W (80-100 m e s h ) .

1.5

f ~

F3

1.0

Y

% ~ ;i t ',

F,

0.1 a

d i

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~

o

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i

i

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=Z

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,

30

FRACTION NUMBER F I G . 1. S p e c t r o p h o t o m e t r i c s c a n n i n g o f f r a c t i o n s obtained by gradient centrifugation of crude factor

(Sp) and human serum. Fractions 0 to 10 (d > 1.070) and 10 to 24 (D 1,020-1,070) correspond to a and j3 lipoproteins, respectively. Lipid analysis

Lipids from different fractions were extracted by the method of Folch et al. ( 1 5 ) a n d estimated by weighing and phosphorus deterruination (16). Qualitative and quantitative analysis were performed by thin layer chromatography (TLC) and photodensitometry (17). Complex lipids were separated with C13CH / CHaOH/H20 (65:25:4, v]v). Simple lipids were separated with petroleum ether (BP 3 0 4 0 C) ethyl ether/acetic acid (90:10:1, v/v). The soluble factor was dehpidated by Albutt's method (18) with ethanol/ether (3: 1, v/v) at 18 C. Fatty acid composition was determined by gas liquid chromatography on a Carlo Erba

Lipoprotein Fractionation

Lipoproteins were fractionated by different procedures. In one case, the crude factor (Sp) was centrifuged in a sodium chloride gradient according to Albutt (18) at 105,000 x g for 16 hr. Besides, the fraction obtained with Sephadex G-150 was separated by electrophoresis in 3.5 x 16 cm gelatinized cellulose acetate strips at 200 volts for 55 min. The buffer was 0.04 M sodium diethyl barbiturate (pH 8.6). Lipoproteins were stained overnight with 0.9% fat red 7B. Proteins were stained with 0.5% Amidoblack for 7 min. Fatty Acid Binding

[I-14C] Linoleic acid and [1-14C]linoleylCoA were bound to Sp or to albumin by incubation for 15 min at 25 C. 370 nmol of the free acid (8.59 mCi/mmol) or 290 nmol of the linoleyl-CoA (8.59 mCi/mmol) were incubated with 5 mg of Sp while 20.8 nmol of linoleyl-CoA (0.09 mCi/mmol) were incubated with 10 mg of albumin. The bound material was separated through a Sephadex G-100 column. The protein content was monitored by spectrophotometry at 280 nm, and the radioactivity was counted in a Packard scintillation counter. RESULTS AND DISCUSSION

Lipoprotein Structure

Previous results have shown that a soluble

TABLE I Capacity of Fractions Obtained by a Gradient Centrifugation of a Crude Protein Factor and Human Serum Protein to Reactivate the A6 D e s a t u r a t i o n C a p a c i t y o f Me a

Fractions M Me Me Me Me Me Me

+ + + + +

Sp Serum proteins F1 F2 F3

Relative % desaturation 100 17 75 16 46 86 64

Specific activity (nmol/min. mg protein) 0.16 0.03 0.12 0.03 0.08 0.15 0.11

a T h e c o n v e r s i o n o f l i n o l e i c a c i d t o 3,-linolenic a c i d w a s m e a s u r e d . The i n c u b a t i o n c o n d i t i o n s are d e s c r i b e d in t h e e x p e r i m e n t p a r t . F 1, F 2 a n d F 3 c o r r e s p o n d t o t h e f r a c t i o n s o b t a i n e d a f t e r C I N a g r a d i e n t (see Fig. 1). 0 . 2 m g p r o t e i n o f e a c h o f t h i s f r a c t i o n a n d s e r u m w e r e a d d e d t o t h e i n c u b a t i o n s y s t e m . Sp = S o l u b l e f r a c t o r o b t a i n e d b y w a s h i n g t h e microsomes. L I P I D S , V O L . 14, N O . 12

LIPID-BINDING FACTOR OF LINOLEATE DESATURATION protein factor, present in the cytosol and loosely b o u n d to the microsomes, is necessary to obtain full activity of the A6 desaturase of f a t t y acids (7-9). The crude factor contains lipids (7). A f t e r partial purification through Sephadex G-150 gel filtration or D E A E cellulose c h r o m a t o g r a p h y (9), lipids extractable by the procedure of F o l c h et al. (15) were still f o u n d fixed to the factor. In this e x p e r i m e n t it was shown that whereas the l i p i d / p r o t e i n ratio (w/w) o f cytosol was 0 . 0 1 7 : 0 . 0 3 8 , the ratios f o u n d for Sp and the partially purified factor through Sephadex G-150 were 0.95:1.1 and 0.17:0.33, respectively. These results suggest a lipoprotein structure. Therefore, it was decided to c o m p a r e the properties of the factor to those of the serum lipoproteins. Both the crude factor (Sp) separated from rat liver microsomes and h u m a n serum were centrifuged in a NaC1 gradient at 10 C for 16 hr at 105,000 x g (18). The UV scanning at 280 n m is shown in Figure 1. It was f o u n d that the fractions F I , F 2 and F 3 of the factor that correlated c~, the b o u n d a r y between e~ and 13 and t3 serum lipoproteins activated microsomal desaturation of linoleic acid to 74inoleic acid (Table I), but the highest activity was found in fraction F 2. Therefore, this fraction would have a h y d r a t e d density b e t w e e n a and /3 serum lipoproteins. However, serum c o m p o n e n t s were inactive. The lipoproteic behavior of the factor was also investigated by gelatinized cellulose acetate electrophoresis. The results c o m p a r e d to h u m a n serum are shown in Figure 2. The partially purified factor (G-150) o b t a i n e d by chromatography of Sp through Sephadex G-150, according to Figure 3, showed several protein bands, but only one band was stained by the lipid reagents. The lipoprotein band ran between t3- and a-lipoproteins of serum. The c o m p o s i t i o n of the lipids b o u n d to the factor was also investigated. A factor partially

1023

FIG. 2. Electrophoresis is gelatinized cellulose acetate. 10 /~1 of human serum and t08 ~g of the factor purified with Sephadex G-150 were applied. Experimental conditions are described in Materials and Methods.

ochrome

C

150 v Lkl

vine serum a l b u m i n

D 100

C~~;I:~e f a c t o r 9 D e x t r a n blue

~_ 5o .J

0

0

i

i

i

i

m

2

4

6

8

"tO

MOLECULAR WEIGHT Log

FIG. 3. Tentative molecular weight of "soluble factor" determined by filtration through a Sephadex G-150 (1.5 x 20 cm) equilibrated with 0.02 M Phosphate buffer (pH 7.4) and 0.1 mM EDTA, standards of Cytochrome C (14,000 dtns), bovine serum albumin (68,000 dtns), catalase (244,000 dtns) and dextran blue (2 x 106 dtns) were used.

TABLE II Lipid Composition of Cytosol, Me, Sp and Sp Partially Purified through Sephadex G-150 (G 150) Lipids a Free fatty acids Phosphatidylethanolamine Phosphatidylcholine Sphingomyelin Lysophospholipids Triacylglycerols Cholesterol

Diacylglycerols

Cytosol (%) 13.4- 13.9 6.7 - 9.6 19.5 - 20.6 . . . 14.6 - 38.1 9.8- 32.6 7.0 - 12.2

Me (%)

.

3.316.1 28.1 . . 9.3 11.3

7.8 - 16.2 - 30.4 - 22.3 - 15.5

Sp (%) 12.7- 22.1 1.86.9 5.4 - 6.4 0.0 - 1.5 37.5 - 64.0 10.3 - 25.4

18.7 - 20.9

1.6 -

4.5

G-150 (%) 15.27.728.0 0.0 6.4 7.9 4.2 8.2

-

15.9 9.4 42.5 1.1 1.3 15.5 7.6 16.9

aCholesteryl esters were minor components in all the fractions. LIPIDS, VOL. 14, NO. 12

ALICIA I. LEIKIN, ANIBAL M. NERVI AND RODOLFO R. BRENNER

1024

TABLE III Fatty Acid Composition of Cytosoi, Me, Sp and Partially Purified Factor through Sephadex G-150 a Cytosol Acids

(%)

16:0 16:1 18:0 18:1 18:2 20:4

10.3-21.5 1.0- 3.2 14.8- 17.2 4.9- 10.8 25.8 - 51.7 11.5- 19.4

Me (%) 13.8 - 18.0 0.64- 2.7 32.0 -33.1 5.8 - 11.1 20.6 - 21.6 18.6-21.0

Sp (%)

G-150 Fraction (%)

3.8- 13.6 0.3- 3.3 2.1- 11.9 1.2- 6.8 41.5 - 90.0 1.5- 2.8

9.3- 14.8 0.9- 1.1 10.1- 16.7 5.4- 5.6 59.0 - 63.0 3.0- 4.4

aResults are the extreme values of three experiments. Other minor components make for 100%. p u r i f i e d t h r o u g h S e p h a d e x G - 1 5 0 was e x t r a c t e d b y t h e p r o c e d u r e o f F o l c h et al. (15). T h e lipids analyzed by TLC showed the composition s t a t e d in Table II. T h e y were c o m p a r e d t o t h e lipid c o m p o s i t i o n of c y t o s o l , Sp ( c r u d e f a c t o r ) a n d w a s h e d m i c r o s o m e s (Me). T h e c o m p o s i t i o n of t h e S e p h a d e x G - 1 5 0 f r a c t i o n varied b e t w e e n c e r t a i n limits f r o m e x p e r i m e n t to e x p e r i m e n t , a n d b o t h p o l a r a n d n o n p o l a r lipids were f o u n d . Phosphafidylcholine, phosphatidylethanola m i n e , l y s o p h o s p h o l i p i d s a n d free f a t t y acids a m o u n t e d to 60 to 75.5% of t o t a l lipids. N o n p o l a r lipids c o n s t i t u t e d b y triacylglycerols, diacylglycerols, c h o l e s t e r o l a n d c h o l e s t e r y l esters c o m p l e t e d t h e lipid c o m p o s i t i o n . It is r e m a r k a b l e t h a t p h o s p h a t i d y l c h o l i n e largely p r e d o m i n a t e d a n d in s o m e e x p e r i m e n t s constit u t e d 4 2 . 5 % o f all t h e lipids. On t h e o t h e r h a n d , p h o s p h a t i d y l e t h a n o l a m i n e was a m i n o r c o m p o n e n t . Free f a t t y acids were also p r e s e n t in a r a t h e r c o n s t a n t p r o p o r t i o n of t h e o r d e r o f 15%. The lipid c o m p o s i t i o n of t h e partially p u r i f i e d f a c t o r was u n d o u b t e d l y r e l a t e d t o t h e c o m p o s i t i o n s o f t h e c y t o s o l a n d Sp, b u t s o m e differences were f o u n d . Sp was r e m a r k a b l y rich in l y s o p h o s p h o l i p i d s , b u t t h e p u r i f i c a t i o n of t h e f a c t o r e v o k e d a n o u t s t a n d i n g e n r i c h m e n t in p h o s p h a t i d y l c h o l i n e at t h e e x p e n s e of o t h e r fractions, m a i n l y l y s o p h o s p h o l i p i d s a n d triacylglycerols. The lipid c o m p o s i t i o n was also d i f f e r e n t f r o m t h e w a s h e d m i c r o s o m e s (Me). It c o n t a i n e d less p h o s p h a t i d y l e t h a n o l a m i n e a n d m o r e free acids. T h e r e f o r e , it is possible to c o n s i d e r t h a t t h e p r o t e i n of t h e f a c t o r is m a i n l y a n d specifically b o u n d t o p h o s p h a t i d y l c h o l i n e , b u t is also carries o t h e r m i n o r lipids, specially free f a t t y acids (Table II). T h e f a t t y acid c o m p o s i t i o n s of c y t o s o l , w a s h e d m i c r o s o m e s (Me), Sp a n d partially p u r i f i e d f a c t o r t h r o u g h S e p h a d e x G - 1 5 0 are s h o w n in T a b l e III. The f r a c t i o n s s h o w e d LIPIDS, VOL. 14, NO. 12

--.

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ELUTION VOLUME (ml) FIG. 4. Sephadex G-t00 column profiles of (a) [1-14]linoleyl-CoA binding to crude factor (Sp); (b) [1-14]linoleic acid binding to crude factor (Sp) and (c) linoleyl-CoA binding to albumin. Experimental conditions are described in Materials and Methods.

LIPID-BINDING FACTOR OF LINOLEATE DESATURATION

1025

TABLE IV Reconstitution of the 2x6 Desaturation Activity by Addition of the Complexes: Sp-Linoleyl-CoA, the Sp-Linoleic A c i d and Albumin-Linoleyl-CoA to Extracted Mierosomes (Me) a Specific activities (n mol 3' 18: 3]min. mg prot.) Fractions

Sp4inoleyl-CoA

Sp-linoleic acid

M Me Me + Sp Me+(Sp-linoleyl-CoA)b Me+(Sp-linoleic acid) c Me+ (Alb umin-lin oleyl-CoA) d

0.16 NMe 0.16 0.22 --. .

0.13 0.02 0.10 . . 0.14 .

.

Aibumin-linoleyl CoA 0.14 0.03 0.13

.

.

.

.

. --0.01

aThe incubation conditions are described in the experimental part. b 1.2 mg of Sp protein ; 35 nmol [ 1-14 C ] linoleyl-CoA. el.2 mg of Sp protein ; 59 nmol [ 1-14C]linoleic acid. dl.1 mg of albumin; 28 nmol [ 1-14C]linoleyl-CoA. eNM not measurable. different compositions, but in cytosol, Sp, and G-150, linoleic acid p r e d o m i n a t e d . The highest c o n c e n t r a t i o n of arachidonic acid was only f o u n d in Me and cytosol. The purification of Sp through Sephadex G-150 c o n c e n t r a t e d a lipoprotein with a less variable fatty acid c o m p o s i t i o n in which linoleic acid c o n s t i t u t e d ca. 61% of all the fatty acids, whereas the saturated acids, palmitic and stearic, contributed 25%. This rather typical c o m p o s i t i o n o f the lipids would suggest that the protein structure of the factor would determine a selective binding o f lipids specifically rich in linoleic acid. This specificity for linoleic acid might also be related with the preference of the factor to activate the A6 desaturation of linoleic acid and similar acids (7). To investigate the c o n t r i b u t i o n of lipids to the activity of the factor, delipidation experiments were performed. In earlier e x p e r i m e n t s diethylic ether was e m p l o y e d . The loss of 70% of the lipids did n o t inactivate the soluble factor and lipids " p e r se" did not activate the desaturation (7). Different procedures were tested using sodium d e o x y c h o l a t e , p h o s p h o lipase A 2 and solvent e x t r a c t i o n ( e t h a n o l / e t h e r ; 3:1, v/v). However, little success was obtained and deactivation was shown together with protein precipitation. Therefore, it was impossible to discriminate if the effect was due to the lipid e x t r a c t i o n or to protein deactivation and precipitation. A n y h o w , it was f o u n d that t h e lipids were tightly b o u n d to the protein of the factor, and it was difficult to obtain a totally

delipidated protein. It could be possible that lipids were necessary to stabilize the proteins and a protein/lipid structure w o u l d be involved in the active form of the factor. The insolubility of the protein of the factor in an aqueous m e d i u m when it was deprived of the lipids w o u l d suggest a l i p o p r o t e i n structure, similar in some way to the /3 t y p e of serum, since the proteins of this last class have also p o o r solubility in aqueous buffers in the lipid-free form. The molecular weight of the factor tentatively estimated by filtration through a Sephadex G-150 c o l u m n was a bit higher than 260,000 (Fig. 3) and, therefore, lower than the molecular weight of fl-lipoproteins of serum. Binding of Linoleic Acid and LinoleyI-CoA

The capacity of the factor partially purified through Sephadex G-100 filtration to bind linoleic acid and linoleyl-CoA was also investigated by i n c u b a t i o n with the substrates at 25 C for 15 min. After incubation, the material was fractionated in Sephadex G-100. In Figure 4a and b, it is s h o w n that the label of either [ 1-14C] linoleyl-CoA or [ 1-14C] linoleic acid was f o u n d in the peak of the factors. The i n c u b a t i o n o f albumin with [ 1 - 1 4 C ] l i n o l e y l CoA showed a similar curve (Fig. 4c). However, the albumin b o u n d linoleyl-CoA, was n o t desaturated in the same conditions (Table IV). Only the linoleyl-CoA or linoleic acids b o u n d to the factor were desaturated to 7-1inolenic acid when t h e y were incubated with Me and the suitable cofactors (Table IV). Therefore, these LIPIDS, VOL. 14, NO. 12

1026

ALICIA I. LEIKIN, AN1BAL M. NERVI AND RODOLFO R. BRENNER

results show that the factor did not accomplish the mere function of binding the substrate, but it would be involved very probably in some direct interaction between the substrate and the desaturating system of the microsomes. Besides, the present experiments also show that the partially purified factor has some properties that are different from other proteins, such as albumin and catalase, in spite of the possibility that some of the catalase activity found in the factor (9) may contribute to its activating effect. If we consider that the factor contains ca. 15% (Table II) of free acids, and it is remarkably rich in linoleic acid (Table III), the addition of Sp evokes an increase of unlabeled substrate. Therefore, it would be necessary to correct the values shown in Table IV with Sp by a positive coefficient. This concentration has not been calculated, since it would not modify the meaning of the experiment and it would only demonstrate higher potency of the factor than that one calculated. ACKNOWLEDGMENTS This work was supported by grants from the Comisidn de Investigaciones Cientificas de la Provincia de Buenos Aires, Consejo Nacional de Investigaciones Cientlficas y T6cnicas, and Secretarla de Ciencia y Tecnologla, Argentina.

LIPIDS, VOL. 14, NO. 12

REFERENCES 1. Oshino, N., Y. Imai, and R. Sato, J. Biochem. 69:155 (1971). 2. Holloway, P.W., and S.J. Wakil, J. Biol. Chem. 245:1862 (1970). 3. Holloway, P.W., Biochemistry 10:1556 (1971). 4. Strittmatter, P., L. Spatz, D. Corcoran, M.J. Rogers, B. Setlow, and R. Redline, Proc. Natl. Acad. Sci. USA 71:4565 (1974). 5. Enoch, H.G., A. Catalg, and P. Strittmatter, J. Biol. Chem. 251:5095 (1976). 6. Brenner, R.R., Moll. Cell. Biochem. 3:41 (1974). 7. Catal~, A., A.M. Nervi, and R.R. Brenner, J. Biol. Chem. 250:7481 (1975). 8. Nervi, A.M., R.R. Brenner, and R.O. Peluffo, Lipids 10:348 (1975). 9. Catal~, A., AA. Leikin, A.M. Nervi and R.R. Brenner, Adv. Exp. Med. Biol. 83:111 (1977). 10. Jeffcoat, R., P. Dunton, and A.T. James, Biochim. Biophys. Acta 528:28 (1978). 11. Kornberg, A., and W.E. Pricer, J. Biol. Chem. 204:329 (1953). 12. Brenner, R.R., and R.O. Peluffo, J. Biol. Chem. 241:5213 (1966). 13. Munkres, K.D., and F.M. Richards, Arch. Biochem. Biophys. 109:466 (1965). 14. Lowry, O., N. Rosebrough, A.S. Farr, and R.J. Randall, J. Biol. Chem. 193:265 (1951). 15. Folch, J., M. Lees, and G.H. Sloane-Stanley, J. Biol. Chem. 226:497 (1957). 16. Chen, P.S., T. Toribana, and W. Huber, Anal. Chem. 28:1756 (1956). 17. Nutter, L.J., and O.S. Privett, J. Chromatogr. 35:519 (1968). 18. Albutt, E., J. Med. Exp. Tech. 23:61 (1966).

[Revision received August 10, 1979]