Purification and partial characterization of linoleoyl-CoA desaturase from rat liver microsomes

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 206, No. 1, January, pp. 21-28, 1981

Purification

TAKAKO

and Partial Characterization of Linoleoyl-CoA Rat Liver Microsomes OKAYASU,

Department

MASAKI

of Biochemistry,

NAGAO,

Hokkaido

TERUO

University

School

ISHIBASHI, of Medicine,

Desaturase

from

AND YOH IMAI Sapporo

060, Japan

Received June 10, 1980 The terminal enzyme of the linoleoyl-CoA desaturase system was purified from rat liver microsomes by Triton X-100 solubilization, DEAE-cellulose, CM-Sephadex, and affinity chromatography on cytochrome bj-Sepharose. The final enzyme preparation was homogeneous as judged by SDS-polyacrylamide gel electrophoresis and a single polypeptide of 66,000 daltons containing 49% nonpolar amino acid residues. The A6-desaturase was found to be a non-heme iron protein containing one atom of iron per one molecule of the enzyme. It was demonstrated that NADH, molecular oxygen, linoleoyl-CoA, lipid or detergent, and three enzymes, NADH-cytochrome b5 reductase [EC 1.6.2.2.1, cytochrome b,, and the terminal enzyme, were absolutely essential for A6-desaturation. In this reconstituted system the apparent K, values for linoleoyl-CoA and V were 45 PM and 83 nmoYmin/mg protein of desaturase, respectively, and the optimal pH was ‘7.0. The A6-desaturase activity in the reconstituted system was inhibited strikingly by iron chelators, cyanide, and p-chloromercuribenzene sulfonate. Furthermore, NADPH-dependent linoleoyl-CoA desaturation could also be reconstituted in the system containing NADPH-cytochrome P-450 reductase [EC 1.6.2.4.1, cytochrome b,, Afi-desaturase, and detergent. However, in this ease, the desaturase activity was only 60% that of the NADH-dependent desaturation.

It has been shown that liver microsomes catalyze at least three different fatty acid desaturations such as A’-, A6-, and As-desaturations depending upon the position of the new double bond (1-3). Other studies have indicated that these desaturations are catalyzed by distinctive desaturase systems according to the various responses occurring under certain dietary and hormonal conditions (4). All three desaturases have identical cofactor requirements for NADH or NADPH and molecular oxygen. The Ag-desaturation which converts stearoyl-CoA (C,,:,) to oleoyl-CoA (CIB:l, Ag) has been studied most extensively (5-9). The terminal enzyme, Ag-desaturase, has been purified from rat liver microsomes by StrittNADH

-

Cytochrome reductase

bg ,T- Lmoleoyl-

\ Cytochrome

NADPH-Cytochrome reductose

matter et al., and the desaturase activity has been reconstituted by NADH, stearoylCoA, oxygen, lipid, and three enzymes, cytochrome b,, NADH-cytochrome b, reductase, and Ag-desaturase (10). A similar microsomal electron transport system is thought to be involved also in the A6- and A”desaturations (4). The participation of cytochrome b5 in the A6-desaturation which converts linoleic acid (C,,,,, A6’g) to y-linolenic acid (ClsZ3, A6+‘*) in rat liver microsomes has been demonstrated by immunological inhibition studies using the anti-cytochrome b5 antibody (11,12), and the electron flow from reduced pyridine nucleotides to A6-desaturase has been represented by the following scheme.

P-450

bg +

Linolenoyl-

21

+ O2

A6 -desaturase

/

SCHEME

Cop.

CoA

1

0003-9861/81/010021-08$02.00/O Copyright D 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

OKAYASU

22

Recently, we have succeeded in solubilizing A6-desaturase from rat liver microsomes and have obtained data to show that A”- and As-desaturase responded differently to detergents (13). However, the isolation of A6-desaturase could not be accomplished because of its extreme instability. In the present study, we have purified A6-desaturase from rat liver microsomes by affinity chromatography on immobilized cytochrome bg, which was suggested by the report of Miki et al. (14). Furthermore, we have compared the enzymatic properties of A6-desaturase with those of Ag-desaturase. EXPERIMENTAL

PROCEDURES

Materials. The following chemicals were obtained from commercial sources: [l-‘4C]linoleic acid (56 mCi/ mmol) (New England Nuclear Corp., Boston, Mass.); coenzyme A and NAD(P)H (Kyowa Hakko Co., Tokyo, Japan); bovine serum albumin (fatty acid poor) (Calbiothem, La Jolla, Calif.); Triton X-100 (Wako Chemical Co., Tokyo); Sepharose 6B and CM-Sephadex (C-50) (Pharmacia, Sweden); DEAE-cellulose (Brown Co., Berlin, N. H.); Bio Beads SM-2 (Bio-Rad, Richmond, Calif.); trypsin (Type XI), egg yolk lecithin, bathophenanthroline sulfonate, p-chloromercuribenzene sulfonate, and tiron (Sigma Chemical Co., St. Louis, MO.). The fat-deficient diet was obtained from Oriental Co. (Tokyo, Japan), and [ l-‘4C]linoleoyl-CoA was prepared as described previously (13). Enzyme assay. Linoleoyl-CoA desaturase activity was determined as described previously (13). In the reconstitution experiments of the linoleoyl-CoA desaturase system, 1.5 nmol cytochrome b,, 1 unit NADHcytochrome b, reductase, l- 10 fig A6-desaturase protein, and 0.5 mg Triton X-100 were mixed at 4”C, and 30 nmol [l-‘4C]linoleoyl-CoA, 50 pmol Tris-HCl (pH 7.2), and 0.5 pmol NADH were added to a total volume of 0.5 ml and incubated for 5 min at 30°C. The subsequent procedures followed those given in the report cited above. Preparation

of microsomal

electron

transport

com-

Cytochrome b, was purified from detergent (15) or trypsin (16) extracts of rabbit liver microsomes. NADH-cytochrome b, reductase and NADPH-cytochrome P-450 reductase were purified from rat liver microsomes by the method of Mihara and Sato (17) and Masters et al. (18), respectively. Analytical methods. Protein was determined by the method of Lowry et al. (19) or Wang and Smith (20) using bovine serum albumin as the standard. Liposomes were prepared from egg lecithin by the method of Strittmatter et al. (lo), and phosphorus was estimated by the method of Marinetti (21). The iron in the purified enzyme was analyzed by the bathophenanthro-

ponents.

ET AL. line sulfonate procedure (22). Amino acid analysis was performed by the technique of Spackman et al. (23) after hydrolysis with 6 N HCl at 110°C for 24 h in a sealed evacuated tube. Tryptophan was determined by the spectral procedures of Beaven and Holiday (24), and the carbohydrate was estimated by the anthroneH&SO, method (25). Lipid analysis for tracing the reaction products was carried out according to the method of Holloway and Holloway (26). Electrophoresis. Sodium dodecyl sulfate (SDS)‘polyacrylamide gel electrophoresis was performed according to the method of Maize1 (27) with 10% resolving gel (pH 8.8) and 3% stacking gel (pH 6.8) each containing 1% SDS, and the gels were stained with 0.05% Coomassie blue. Preparation of cytochrome b,-Sepharose. Sepharose 6B was suspended in 0.1 M sodium bicarbonate (pH 8.3) and activated by cyanogen bromide according to the method of Axen and Ernback (28). Approximately 50 ml of activated gel was suspended in 50 ml of 0.1 mM trypsin-solubilized cytochrome b, in 0.1 M sodium bicarbonate (pH 8.3), and stirred gently for 16 h at room temperature in a nitrogen atmosphere in the dark. About 100 nmol of cytochrome b, were fixed in 1 ml of the packed gel. After blocking the residual reactive groups with 1 M glycine, the gel was washed thoroughly with 0.1 M ice-cold sodium bicarbonate (pH 8.3) containing 1 M NaCl, 0.1 M sodium acetate (pH 4.1) containing 1 M NaCl, and finally 20 mM Tris-HCl buffer (pH 8.1) containing 0.5 mM EDTA, and stored in the same buffer at 4°C. The immobilized cytochrome b, reduced cytochrome c in the presence of NADH, purified NADH-cytochrome b, reductase, and remained stable for at least 1 year. RESULTS

Purijication

of A6-Desaturase

Male, Wistar strain rats weighing lOO120 g were fed a fat-free diet, and the liver microsomes were prepared as described previously (13). The purification procedures are summarized in Table I. All procedures were conducted at 0 to 4°C. Ten percent (w/v) Triton X-100 was added to a suspension of microsomes (20 mg of protein/ml) in 0.1 M Tris-HCl buffer (pH 7.5) to give a final concentration of 2%. The mixture was stirred gently for 20 min, diluted with 20 mM Tris-HCl buffer (pH 7.5) to lower the concentration of the detergent to 0.5%, and centrifuged at ‘77,OOOgfor 90 min. All subsequently used buffers contained 0.5% Triton X-100 and 10% glycerol. This ’ Abbreviation

used: SDS, sodium dodecyl sulfate.

RAT LIVER

LINOLEOYL-COENZYME

step resulted in an excellent recovery and 1.5fold purification. After equilibration with 20 mM Tris buffer (pH 7.5), the supernatant fluid was applied to a DEAE-cellulose column (4 x 20 cm). Seventy percent of the desaturase activity emerged in the void volume with 2.7-fold purification. The active fraction was adjusted to pH 7.2 with 0.3 N HCl and passed over a CM-Sephadex column (2.8 x 18 cm) which had been equilibrated with 20 mM Tris buffer (pH 7.2) and eluted with the same buffer. Although this step gave only 2.0-fold purification, all of the cytochrome P-450 was retained by the column. The void volume, containing 70% of the desaturase activity, was adjusted to pH 8.1 by 20 mM Tris base and loaded on a cytochrome b,-Sepharose column (2.8 x 10 cm) which had been equilibrated with 20 mM Tris buffer (pH 8.1). After washing with 4 column vol of the same buffer, elution was carried out with 0.3 M KC1 in the same buffer at a flow rate of 8 ml/h, and 5-ml fractions were collected (Fig. 1). The final enzyme preparation was purified about 200- to 230-fold with a lo-15% yield, and stored in small aliquots under nitrogen at 70°C. The enzyme was unstable and lost 80% of its activity after repeated freezing and thawing. Purity

and Molecular Weight

The final enzyme preparation was homogeneous upon SDS-gel electrophoresis (Fig. 2). The molecular weight of the A6-desaturase was 66,000 i 1000 under denaturating conditions (Fig. 3), and that of the native form was about 65,000-68,000 as determined by gel filtration of the crude fraction by Sephadex G-200 in the presence of 0.2% of deoxycholate (data not shown). TABLE PURIFICATION

Total protein (mg) Microsomes Triton X-100 DEAE-cellulose CM-Sephadex b,-Sepharose

1617 1295 321 116 0.86

Specific activity (nmoUmidmg) 0.15 0.22 0.61 1.20 34.4

DESATURASE Total activity (nmollmin) 243 235 1% 139 30

PURIFICATION

23

FIG. 1. Affinity chromatography of A6-desaturase on the cytochrome &Sepharose column. The enzyme was eluted with 0.3 M KC1 in 20 mM Tris-HCl buffer (pH 8.1) containing 0.5% Triton X-100 and 10% glycerol. Fractions of 5 g were collected at a flow rate of 8 ml/h.

These findings suggested that the enzyme was a single polypeptide. Iron, Phospholipid, and Ca,rbohydrate Determinations

The A6-desaturase contained 15.1 t 1.4 nmol ironlmg of protein, which corresponded to a minimum molecular weight of 66,000 5 6000. Since the purified enzyme did not reveal any distinct absorption spectra in the visible region (data not shown), A”desaturase obtained presumably a nonheme iron protein. The final enzyme preparation contained 20-40 mol of phospholipidsl mol of protein but no detectable carbohydrates (~0.5 mol/mol of protein). Amino Acid Analysis

The amino acid composition of the present A6-desaturase is shown in Table II. Fortynine percent of the amino acid residues were nonpolar, as determined by the procedure of Capaldi and Vanderkooi (29). Reconstitution of NADH-Dependent A6-Desaturase System

I

OF LINOLEOYL-COA

A DESATURASE

Recovery (%I 100 117 81 57 12

Linoleoyl-CoA desaturase activity was reconstituted with NADH, molecular oxygen, lipid or detergent, and three enzymes: NADH-cytochrome b, reductase, cytochrome b,, and A6-desaturase (Table III). The omission of one of these components from the system led to an almost complete loss of activity. The reaction products was

24

OKAYASU

ET AL.

the concentration of three enzymes. Figure 4

indicated that the rate of A6-desaturation in the reconstituted system was proportional to the concentration of cytochrome bg, reductase, and A”-desaturase under conditions in which each of the other enzymes was present in excess. However, trypsin-solubilized cytochrome b, showed only slight activity, and moreover this activity was not dependent on the amount of cytochrome b,. Effect of substrate concentration, incubation time, and pH on the A6-desa.turase activity. Linear increases in the rate of A6-de-

saturation were observed in amounts up to about 50 PM linoleoyl-CoA (Fig. 5A). Apparent K, and V values for linoleoyl-CoA were calculated to be 45 PM and 83 nmol/ minlmg protein, respectively, from linear, double-reciprocal plots. The rate of A6-desaturation was constant for up to 5 min of incubation time (Fig. 5B), and the subsequent increase in incubation time resulted in a striking decline of activity. The optimal pH was 7.0 with a narrow range (Fig. 50

BPB )

FIG. 2. Polyacrylamide disc gel electrophoresis of the purified A”-desaturase in the presence of SDS. Electrophoresis was carried out as described under Experimental Procedures, except that approximately 10 Fg of protein was added.

identified as y-linolenic acid by gas-liquid radiochromatography as described previously (11) and the A6-position was further defined by characterizing the radioactive dicarboxylic acid after obtaining oxidative cleavage with sodium periodate and potassium permanganate (30). Furthermore, an analysis of the total lipids extracted from the reaction mixture by thin-layer chromatography revealed that the major product was y-linolenoyl-CoA. Dependence of A6-desaturase activity on

Reconstitution of NADPH-Dependent A6-Desa,tura,seSystem

Linoleoyl-CoA desaturase activity was reconstituted with NADPH, molecular oxygen, lipid or detergent, and three enzymes: NADPH-cytochrome P-450 reductase, cytochrome b,, and A6-desaturase. No activity occurred without one of these components. Figure 6 shows that the A”-desaturase activity was dependent on the amount of reductase. In the presence of sufficient

0

0.2

0.4

0.6

0.8 Rf

FIG. 3. Molecular weight estimation of the A6-desaturase by SDS-gel electrophoresis. The standard proteins were 1, human transfer-in (80,000); 2, bovine serum albumin (68,000); 3, catalase (58,000); 4, ovalbumin (43,000); and 5, aldolase (40,000).

RAT LIVER

LINOLEOYL-COENZYME

TABLE AMINO

ACID COMPOSITION LINOLEOYL-COA

Lys His Arg Asp Thr Ser

37 12 24 97 30 42

A DESATURASE

II

TABLE

OF RAT LIVER MICROSOMAL DESATURASE”

Glu Pro Gly Ala cys Val

51 29 52 55 4 36

Met Ile Leu Tyr Phe Try

10 23 47 15 23 11

Total residues = 598 u Based on a molecular weight of 66,000 determined by iron analysis and disc gel electrophoresis.

amounts of reductase and cytochrome b5, NADPH-dependent A6-desaturase activity was only 60% of that of NADH-dependent desaturation with the same amounts of the terminal enzyme. The following investigations were carried out in the NADH-dependent reconstituted system. Requirement for Detergent or Lipid in Reconstituted System Since the final enzyme preparation of A6desaturase still contained 0.5% Triton X-100, we studied the effect of detergent or lipid on the enzyme activity (Table IV). When Triton was removed by Bio Beads

25

PURIFICATION III

RECONSTITUTION OF LINOLEOYL-COA DESATURASE SYSTEM

Desaturase activity (nmol/min/mg)

System Complete” Complete Complete Complete Complete Complete

-

35.0 N.D.b N.D. N.D. 3.1 N.D.

F,, d-b, Desaturase NADH O*

n The complete system contained 2 units NADHcytochrome b, reductase (F,,), 2 nmol detergentsolubilized cytochrome b, (d-bs), 5 Fg A”-desaturase protein, and 0.5 pmol NADH in a final volume of 0.5 ml, and was incubated as described under Experimental Procedures. b Less than 2.4 nmol/min/mg desaturase protein.

SM-2 according to the method of Halloway (31), A6-desaturase activity was completely lost; however, the readdition of Triton, liposome, or both to the detergent-removed enzyme, restored it to 65, 57, or 94% of the activity of original enzyme, respectively. Effect of Inhibitors A’-Desaturase activity of the reconstituted system was extensively inhibited by

I-

4

B d-b,

t-b, f: Cyt.

b, (nmol)

Reductase

(unit)

a’ -desaturase

( W,

FIG. 4. Dependence of the NADPH-initiated A6-desaturase activity of the reconstituted system on concentration of cytochrome b, (A), NADH-cytochrome b, reductase (B), and A6-desaturase (C). The reaction mixtures contained: 5 pg A’-desaturase protein (A, B); 1.5 nmol cytochrome b, (B, C); 1 unit reductase (A, C); 0.5 mg Triton X-100; 0.5 pmol NADH; 30 nmol [l-‘4C]linoleoyl-CoA; and 50 pmol Tris-HCl (pH 7.2) in a total volume of 0.5 ml. (d-b,: detergent solubilized cytochrome b,; t-b,: trypsin solubilized cytochrome b,).

26

OKAYASU

20

40

LINOLEOYL-COA

60

80

100

( pM

)s

ET AL.

FIG. 5. Effect oflinoleoyl-CoA concentration (A), incubation time(B), and pH (C) on NADH-initiated A6-desaturation catalyzed by the reconstituted system. Incubations were carried out for 5 min (A, C) in the presence of 60 /.LM [1-L4C]linoleoyl-CoA (B, C) at pH 7.2 (A, B) as described in the text.

potassium cyanide (Table V). A catalytic role for the non-heme iron of A6-desaturase was implied by the decrease in activity upon the addition of iron chelators. Since Tiron had a more inhibitory effect than bathophenanthroline sulfonate, the iron in the A6desaturase appeared to be ferric. Thioldirect reagents such as p-chloromercuribenzene sulfonate strikingly inhibited the enzyme activity, whereas, N-ethylmaleimide had little effect. Dithiothreitol and

/.%mercaptoethanol enzyme activity.

mildly

The purification procedures employed herein produced a reasonable yield of homoTABLE REQUIREMENTS OF TRITON THE RECONSTITUTED DESATURASE

IV X-100 OR LIPOSOME LINOLEOYL-COA SYSTEM

Original” Triton removal” Triton removal + Triton (0.05%)” Triton removal + Triton (0.1%) Triton removal + Triton (0.3%) Triton removal + liposomes (45 nmol EL)‘,” Triton removal + Triton (0.1%) + liposomes (45 nmol EL)’ ADDED

c unit)

6. Dependence of NADPH-initiated As-desaturase activity catalyzed by the reconstituted system on NADPH-cytochrome P-450 reductase concentration. The reaction mixtures contained 5 pg A6-desaturase protein, 1.8 nmol cytochrome b,, 0.5 mg Triton X-100, 0.5 pmol NADPH, 30 nmol [l-14C]linoleoyl-CoA, 50 pmol Tris-HCl (pH 7.2), and the indicated amounts of reductaae in a total volume of 0.5 ml. FIG.

the

DISCUSSION

Enzyme

REDUCTASE

inhibited

FOR

Relative activity (So) 100’ 0 50 65 59 57 94

n Original enzymes contained 0.5% Triton X-100. b Triton was removed from the original enzyme by the methods described in the text. c After the readdition of Triton X-100 or liposomes, the reaction mixtures were preincubated for 5 min at 4°C. d EL, egg lecithin. p 100% activity corresponded to 51.9 nmol linoleoylCoA desaturatediminimg desaturase protein.

RAT

LIVER

LINOLEOYL-COENZYME

geneous A6-desaturase from a workable quantity of materials. Although the final enzyme preparation retained KC1 and Triton X-100, further procedures to remove them led to a drastic loss of activity. The purified enzyme yielded a turnover of 5.5 mol of substrate/mol of A6-desaturase/min at 30°C. The observed turnover number and total activity in the induced microsomes indicated that the A6-desaturase may represent only about 0.18% of the total microsomal protein. The reconstitution of the A6-desaturase system in this report has established the involvement of cytochrome bj on A6-desaturation, and confirmed the electron flow route postulated in Scheme 1 from other immunological experiments in microsomes (11, 12). It has been reported that cytochrome bj may participate in many other lipid metabolisms such as cholesterol biosynthesis (32,33), plasmolagen biosynthesis (34), phospholipid desaturation (35), fatty acid elongation (36), etc. We have found that the A6-desaturase has a high affinity for immobilized cytochrome b5 providing that adequate concentrations of detergent, ion strength, and pH are present. Continued investigations are needed to determine the interaction between cytochrome b, and the terminal enzymes in the various reactions described above. Enoch and Strittmatter have reported on the participation of NADPH-cytochrome P-450 reductase in stearoyl-CoA desaturation on the reconstituted system (37), and have suggested that NADPH might be the actual physiological reductant for fatty acid desaturation under certain metabolic conditions. Although the NADPH-dependent A6-desaturation was also reconstituted in this study, its activity was only 60% of that initiated by NADH, which led us to suppose that the NADH-dependent system might be preferable in A6desaturation. Recently, we have reported that A”-desaturase was solubilized with Triton X-100 or deoxycholate at a concentration where Aydesaturase was scarcely solubilized, and we postulated that the formation of the two desaturases differed significantly in the microsomal membrane (13). The polarity index of the purified A”-desaturase was 49% and

A DESATURASE

27

PURIFICATION TABLE

V

EFFECT OF VARIOUS CHEMICALS ON THE RECONSTITUTED LINOLEOYL-COA DESATURASE SYSTEW

Addition

Concentration ew

Relative activity (%I

1 5

100 60 9

1 1 1’ 0.4’ 1 1

82 21 100 25 86 68

None KCN Bathophenanthroline sulfonate Tiron N-Ethylmaleimide p-CMBS P-Mercaptoethanol Dithiothreitol

” The purified linoleoyl-CoA desaturase was reconstituted as described under Experimental Procedures. All chemicals were added to the assay mixture without preincubation. 100% activity corresponded to 35 nmoYmin/mg desaturase protein. * p-Chloromercuribenzene sulfonate. r 10 pmol ascorbic acid instead of 0.5 pmol NADH was added.

resembled that of cytochrome b5, which is an amphipathic protein containing a hydrophilic, catalytic segment, and a smaller hydrophobic sequence involved in the attachment to microsomal vesicles (15). On the other hand, the polarity index of AY-desaturase has been reported to be 38% (lo), suggesting that Ag-desaturase is buried more deeply than A6-desaturase in the microsomal membrane. Moreover, A”-desaturase has been shown to be larger in molecular weight than AY-desaturase in rats (53,000) (10) and in chickens (33,000) (38). In the present study, stearoyl-CoA was not desaturated by our A6-desaturase system (data not shown). Considering the discrepancy of their size and properties, we have confirmed that A6- and Ag-desaturase are different enzymes, despite their analogous character of having one molecule of non-heme iron (10). ACKNOWLEDGMENT

This research was supported in part by a grant from the Ministry of Education, Science and Culture of Japan.

28

OKAYASU

ET AL.

REFERENCES 1. MARSH, J. B., AND JAMES, A. T. (1962) Biochim. Biopkys. Acta 60, 320-328. 2. BRENNER, R. R., AND PELUFFO, R. 0. (1966) J. Biol. Chem. 241, 5213-5219. 3. CASTUMA, J. C., CATALA, A., AND BRENNER, R. R. (1972) J. Lipid Res. 13, ‘783-789. 4. BRENNER, R. R. (1974) Mol. Cell. Biockem. 3, 41-52. 5. OSHINO, N., IMAI, Y., AND SATO, R. (1966) Biockim. Biopkys. Acta 128, 13-28. 6. JONES, P. D., HOLLOWAY, P. W., PELUFFO, R. O., AND WAKIL, S. J. (1969) J. Biol. Ckem. 244, 744-754. 7. HOLLOWAY, P. W., AND WAKIL, S. J. (1970) J. Biol. Chm. 245, 1862-1865. 8. HOLLOWAY, P. W., AND KATZ, J. T. (1972) Biochemistry 11, 3689-3695. 9. OSHINO, N., AND OMURA, T. (1973) Arch. Biockem. Biopkys. 157, 395-404. 10. STRITTMATTER, P., SPATZ, L., CORCORAN, D., ROGERS, M. J., SETLOW, B., AND REDLINE, R. (1974) Proc. Nat. Acad. Sci. USA 71, 45654569. 11. OKAYASU, T., ONO, T., SHINOJIMA, K., AND IMAI, Y. (1977) Lipids 12, 267-271. 12. LEE, T. C., BAKER, R. C., STEPHENS, N., AND SNYDER, F. (1977) Biockim. Biopkys. Acta 489, 25-31. 13. OKAYASU, T., NAGAO, M., AND IMAI, Y. (1979) FEBS Lett. 104, 241-243. 14. MIKI, N., YAMANO, T., ANDTAKAHASHI, T. (1978) J. Tozicol. Sci. 3, 289. 15. SPATZ, L., AND STRITTMATTER, P. (1971) Proc. Nat. Acad. Sci. USA 68, 1042-1046. 16. KAJIHARA, T., AND HAGIHARA, B. (1968) J. Biockem. (Tokyo) 63, 453-461. 17. MIHARA, K., AND SATO, R. (1975) J. Biohem. (Tokyo) 78, 1057-1073. 18. YASUKOCHI, Y., AND MASTERS, B. S. S. (1977) J. Biol. Ckem. 251, 5377-5344. 19. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.,

20. 21. 22. 23. 24. 25. 26. 27.

28. 29. 30.

31. 32. 33. 34.

35. 36.

37. 38.

AND RANDALL, R. J. (1951) J. Bi01. Chem. 193, 265-275. WANG, C. S., AND SMITH, R. L. (1975)Anal. Biockem. 63, 414-417. MARINETTI, G. V. (1962) J. Lipid Res. 3, l-20. ATKIN, C. L., THELANDER, L., AND REICHARD, P. (1973) J. Biol. Chem. 248, 7464-7472. SPACKMAN, D. H., MOORE, S., AND STEIN, W. H. (1958) Anal. Ckem. 30, 1190-1199. BEAVEN, G. H., AND HOLIDAY, E. R. (1952) Advan. Protein Chem. 7, 319-325. ROE, J. H. (1955) J. Biol. Ckem. 212, 335-343. HOLLOWAY, C. T., AND HOLLOWAY, P. W. (1974) Lipids 9, 196-200. MAIZEL, J. V., Jr. (1969) In Fundamental Techniques in Virology (Habel, K., and Salzman, N. P., eds.), pp. 334-362, Academic Press, New York. AXEN, R., AND ERNBACK, S. (1971) Eur. J. Bio&em. 18, 351-360. CAPALDI, R. A., AND VANDERKOOI, G. (1972) Proc. Nat. Acad. Sci. USA 69, 930-932. MORTON, R. E., HARTZ, J. W., REITZ, R. C., WAITE, B. M., AND MORRIS, H. P. (1979) Biockim. Biopkys. Acta. 573, 321-331. HOLLOWAY, P. W. (1973) Anal. BiocZzem. 53, 304-308. GAYLOR, J. L., AND MASON, H. S. (1968) J. Biol. Ckem. 243, 4966-4972. REDDY, V. R., KUPFER, D., AND CASPI, E. (1977) J. Biol. Ckem. 252, 2797-2801. PAULTAUF, F., PROUGH, R. A., MASTERS, B. S. S., AND JAHNSTON, J. M. (1974) J. Biol. Ckem. 249, 2661-2662. PUGH, E. L., AND KATES, M. (1977) J. Biol. Chem. 252, 68-73. KEYES, S. R., ALFANO, J. A., JANSSON, I., AND CINTI, D. L. (1979) J. Biol. Ckem. 254, 77787784. ENOCH, H. G., AND STRITTMATTER, P. (1979) J. Biol. Ch.em. 254, 8976-8981. PRASAD, M. R., AND JOSHI, V. C. (1979) J. Biol. Ckem. 254, 6362-6369.