Journol of h'rurochrmisrr?. 1978. Vol. 30. pp. 643-647. Pergamon Press. Printed In Great Britain.

SHORT COMMUNICATION Specificity of reduction of fatty acids to long chain alcohols by rat brain microsomes (Received 18 July 1977. Accepted 6 September 1977) LONGchain alcohols have been shown to be the precursor of the alkyl moiety of the glycerol ether lipids and plasmalogens (VAN DEN BOSCH,1974). The alkyl chains at the sn-1 position of these lipids are almost entirely comprised of C16:o, and CIS:, moieties (HORROCKS 1972). However, the enzyme which forms the ether bond initially by reacting acyl dihydroxyacetone phosphate with long chain alcohol, the alkyl dihydroxyacetone phosphate synthase, is relatively nonspecific. A wide variety of long chain alcohols can act as substrates for this enzyme, both in uiuo and in uitro, including the polyunsaturated alcohols which are not normally found in the ether lipids HAIRA, 1973; BANDIet al., 1971; Su & SCHMID,1972; SNYDERet al., 1973). It seemed probable that the reason for the specific alkyl chain distribution of the ether lipids is that only those specific aforementioned alcohols are available to the ether lipid-synthesizing system, and that this availability is determined, in turn, by the specificity of biosynthesis of long chain alcohols. The biosynthesis of long chain alcohol via reduction of fatty acid by NADH or NADPH has been demonstrated in several systems (KOLATTUKUDY 1970; SNYDER 1972). Most recently, an NADPH-specific reduction of fatty acids was shown to occur in developing rat & SASTRY (1976). We used a similar brain by NATARAJAN system to investigate the specificity of this conversion of fatty acids to long chain alcohols and found that the reduction is fairly specific. The results of that study are presented here. MATERIALS AND METHODS [1-14C]-Labeled lauric (C12:o),myristic (C14:0), oleic linoleic (CIS:,). linolenic and arachidonic (C20:4) acids were purchased from Amersham Corp, Arlington Heights, IL. [1-14C]-Labeled palmitic (C16:o)and stearic (C18:o) acids were from New England Nuclear Corp., Boston, MA. These fatty acids were stored in benzene containing 0.1% butylated hydroxytoluene at 4 C under an atmosphere of nitrogen. All fatty acids except linoleic and oleic acids, had a purity greater than 977; as assessed by TLC. Linoleic and oleic acids were 85-90>" pure, but since their impurities did not migrate with long chain alcohol on TLC. they were used without further purification. Nonradioactive fatty acids, ATP, NADPH, dithiothreitol, CoA, and Tergitol 15-S-9 were obtained from Sigma Chemical Corp, St. Louis, MO. Fatty acid-free bovine serum albumin was obtained from Miles Laboratories, Kankakee, IL. Silica gel (0.25 m m thick) TLC plates were from Brinkmann Instruments, Des Plaines, IL. Brains from 15 day old rats were homogenized in 0.32 M-sucrose containing 0.1 mM-EDTA (10%. w/v). The crude mitochondria1 fraction was removed by centrifuging 643

at 18,000g for 30 min. The microsomal fraction was pelleted by a 30 min centrifugation at 105,000g and was taken up in 0.32 M-Sucrose and diluted with same, as needed. Fatty acid:CoA ligase (AMP forming) (EC 6.2.1.3) was assayed by counting the radioactive acyl CoA formed from [1-I4C]fatty acid. The incubation mixture contained 100 p~-[l-'~C]fatty acid (1.7-3.1 x lo4. d.p.m./ nmol), 190 mM-Tris-HCI buffer (pH 7.4). 5 mM-MgC12, 10 mM-ATP, 5 mM-dithiothreitol, 0.025% Tergitol 15-S-9 (BANIS& TOVE,1974), 1.0mM-CoA, and 10 pg microsomal protein in a total volume of 0.4ml. The fatty acid in organic solvent was added to the incubation tube first, and evaporated to dryness under a stream of nitrogen. The remaining reagents, except for the protein, were then added and the mixture was preincubated for 5 min at 37°C. The reaction was started by addition of protein, and incubated for 5 or 10min at 37°C. Radioactive acyl CoA was extracted according to the solvent partition method of BANIS& TOVE(1974). Under these conditions, acyl CoA synthase activity was found to be linear with increasing protein up to 15pg. and also linear with time for 12min when assayed with 1Opg protein. When synthetic [1-14C]acyl CoA was added to control incubation mixtures, recovery of radioactivity by the above procedure was 92-93?", The reduction of fatty acids to long chain alcohols was assayed in an incubation mixture containing 100 p~ [1-'4C]fatty acid (1.7-3.1 x lo4 d.p.m./nmol), 50 mM-phosphate buffer (pH 7.6), 2 mM-MgCI,, 0.5 mM-MnCI,, 10 mMATP, 0.084 mM-CoA, 0.5 mg/ml bovine serum albumin, 1.25 mM-NADPH, and 1-2 mg microsomal protein in 0.32 M-sucrose (final sucrose concentration. 0.16 M). in a total volume of 0.8 ml. The fatty acid in organic solvent was added first, the solvent evaporated under a stream of nitrogen, and the remaining reagents, except for NADPH, were then added. The mixture was sonicated in an ultrasonic bath and preincubated for 5 min at 37C. The reaction being assayed was started by the addition of NADPH and was incubated at 3 7 T for 1 h under nitrogen. Lipids were extracted and washed according to the method of BLIGH& DYER(1959). The lipids were separated by TLC with CHCI3-CH30H-7.3 M-NH,OH (92:2:0.8. by vol). After localization by autoradiography. the radioactive spots were scraped out and counted in a & HAJRA1972). When liquid scintillation counter (LABELLE standard [1-14C]hexadecanol was added to control assay mixtures, recovery of this compound by the above procedure was 83-86>;, The long chain alcohols and other 14C-labeled lipid products were identified by their mobilities on TLC using the following solvent systems (v/v): CHC13-CH30H& SASTRY,1976); hexCH3COOH (92:2:1), (NATARAIAN ane-ethyl ether (90: 10); hexane-ethyl ether-CH,COOH (80:20: I ) ; and benzene.

,

644

Short communication RESULTS

We found that rat brain microsomes will form radioactive long chain alcohol from '4C-labeled fatty acid when incubated with NADPH and other cofactors, thus confirm& SASTRY (1976). Figure ing the findings of NATARAJAN 1 shows an autoradiogram of the radioactive products formed from [l-14C]palmitic acid after their separation on TLC. It is seen that NADPH is absolutely necessary for the formation of alcohol, but not for most other products of the assay. The identity of the long chain alcohol was confirmed by its co-migration on TLC with standard hexadecanol in four different solvent systems, and also by comigration of its acetate ester (ROUSER et al., 1969) with hexadecyl acetate on two different solvent systems. Most of the radioactivity added to the assay mixture remains at the origin (Fig. 1) and consists largely of unconverted fatty acid with some phospholipid. A major radioactive by-product was identified as triglyceride (RF 0.90, Fig. I), and was presumably formed from endogenous substrate and acyl CoA generated in the assay mixture. Minor products were tentatively identified as cholesterol ester, wax, and diglyceride. Only trace amounts of radioactivity migrating with standard palmitaldehyde accumulated after 1 h incubation, amounting to 2-3% of the radioactive hexadecanol formed during the same incubation. This contrasts & SASTRY (1976). sharply with the findings of NATARAJAN who reported the levels of long chain aldehyde formed to be 6-7 fold higher than that of hexadecanol.

FIG. 2. Relative rates of formation of [1-'4C]acyl CoAs (a,) and of '4C-labeled long chain alcohols (b.) from the [1-'4C]fatty acids indicated on the abscissa, when assayed as described in the Methods section. The indicated fatty acids are, from left to right; lauric (12:0), myristic (14:O). palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2), linolenic (18:3), and arachidonic acids (20:4). Mean values for each assay have been expressed as a ratio to the value obtained for the conversion of [1-'4C]palmitic acid to 1 S.D. product (see text for details). Each value is a mean

Figure 2 shows the rates of formation of acyl CoAs and long chain alcohols from eight different [1-'4C]fatty acids. In each case, the amount of product formed was norqalized to the value obtained when [1-'4C]palmitic acid was used as substrate. For formation of palmityl CoA, this value is 8.3 & 0.4 nmol/min/mg protein. For reduction of palmitic acid to hexadecanol, the value is 8.6 0.2 pmol/ min/mg protein. DISCUSSION These results show that microsomes from 15 day old rat brain contain the enzyme systems necessary to convert fatty acids to both acyl CoAs and to long chain alcohols. Apparently, acyl CoA rather than free fatty acid is the immediate substrate for the reduction reaction. This is supported by both a strict requirement for the cofactors necessary for acyl CoA generation (Bishop & Hajra, unpublished results), and by work in other systems in which acyl CoA was used directly as substrate (SNYDER1972). Due to an active brain microsomal palmityl-CoA hydro& VANCE 1976) and to the low lase (EC 3.1.2.2) (BROPHY activity of the reductase, acyl CoA could not be used directly as substrate in these assays. The findings of this study show that the enzymatic system which reduces fatty acids to long chain alcohols is fairly specific for palmitic, stearic, and oleic acids, as shown by Fig. 2. This is similar to that reported by NATARAJAN & SCHMID(19776) in a preliminary communication. The specificity of this system resides with the enzyme which actually performs the reduction rather than with the acyl CoA synthase. All fatty acids tested formed their CoA derivatives readily, the rate for formation of palmityl CoA being nearly lo00 fold higher than its subsequent reduction, under the assay conditions used. The lack of reactivity of the reduction system with arachidonic acid cannot be due to inhibition by a minor contaminating component of the fatty acid, since arachidonic acid was found not to inhibit the reduction of [1-'4C]oleic acid (Bishop & Hajra, unpublished experiment). The specificity of this reduction system can explain, at least in part, the specific alkyl group distribution in the ether lipids. While alkyl DHAP synthase will incorporate both polyunsaturated alcohols and shorter chain alcohols (Clo to C14) into the ether linkage (HAJRA1973),the reduction system will not form them from the corresponding fatty acids to any appreciable degree. Linoleic and linolenic acids are exceptions in that they are reduced to their alcohols at a low, but significant rate. The complete exclusion of these two moieties from the ether lipids in brain is probably due to this lower reduction rate, and also to the near et absence of these two acids in brain tissue (KISHIMOTO a/., 1964). These findings are consistent with the endogenous long chain alcohol levels reported recently by & SCHMID(1977a). These authors found that NATARAJAN the C16:0, CIS:^, and Cls,l alcohols constituted greater than 90% of the total alcohol in 15 day old rat brain. Further aspects of this system are under investigation in this laboratory. Acknowledgement-This work was supported by Grant NS08841 from the National Institutes of Health and by USPHS Training Grant GM00187. Department of Biological Chemistry and the Mental Health Research Institute, Ann Arbor, M I 48109, U.S.A.

J. E. BISHOP A. K. HAJRA

645

FIG.1. Autoradiogram of products of the assay for formation of long chain alcohol fiom [1-'4C]fatty acid after their separation on TLC with CHC13-CH30H-7.3 M - N H ~ O H(92:2:0.8, by vol). Lane 6 shows the products of the complete assay mixture, in which 1.34 x 106d.p.m. [1-'4C]palmitic acid was incubated for 1 h (see text for details). The radioactivity in the hexadecanol (RF 0.42) was 1.15 x 104d.p.m. In lane 5, NADPH had been deleted from the assay mixture. Lane 4 is a zero time control. Standards are: (1) palmitaldehyde, (2) 1,2-diolein, (3) [1-'4C]hexadecanol, and (7) triolein.

Short communication REFERENCES BAND1 Z. L., AAES-JORGENSEN E. & MANGOLD H. K. (1971) Biochim. biophys. Acta 239, 357-367. BANISR. J. & TOVES. B. (1974) Biochim. biophys. Acta 348, 21G220. BLIGHE. G. & DYERW. J. (1959) Can. J. Biochem. 37, 91 1-917. BROPHYP. J. & VANCE D. E. (1976) Biochem. J. 160, 247-251. H A l R A A. K. (1973) in Tumor Lipids. Biochemistry and Metabolism (WOOD R., ed.) pp. 183-199. American Oil Chemists’ Society. Champaigne, IL. HORROCKS L. A. (1972) in Ether Lipids, Chemistry and BioF., ed.) pp. 177-272. Academic Press, New logy (SNYDER York. KlsHiMoTo Y.,DAVIES W. E. & RADINN. s. (1964) J . Lipid Res. 5, 329-338. KOLATTUKUDY P. E. (1970) Biochemistry 9, 1095-1 102.

647

LABELLE E. F., JR & HAJRAA. K. (1972) J. biol. Chem. 247, 5825-5834. NATARAJAN V. & SASTRY P. S. (1976) J. Neurochem. 26, 107-1 13. NATARAJAN V. & SCHMID H. H. 0.(1977a) Lipids 12, 128-130. NATARAJAN V. & SCHMID H. H. 0. (1977b) Fedn Proc. Fedn Am SOCSexp. Biol. 36, 851. ROUSERG., KRITCHEVSKY G., YAMAMOTO A., SIMON G., GALLI C. & BAUMAN A. J. (1969) in Methods in Enzymology (LOWENSTEIN J. M., ed.) VOI. XIV, pp. 272-317. Academic Press, New York. F. (1972) in Ether Lipids, Chemistry and Biology SNYDER (SNYDER F., ed.) pp. 122-157. Academic Press, New York. SNYDER F., CLARKM. & PIANTADOSI C. (1973) Biochem. biophys. Rex Commun. 53, 350-356. SU K. L. & SCHMID H. H. 0. (1972) J. Lipids Res. 13, 452-457. VAN DEN BOSCHH. (1974) A. Rev. Biochem. 43, 243-278.

Specificity of reduction of fatty acids to long chain alcohols by rat brain microsomes.

Journol of h'rurochrmisrr?. 1978. Vol. 30. pp. 643-647. Pergamon Press. Printed In Great Britain. SHORT COMMUNICATION Specificity of reduction of fat...
295KB Sizes 0 Downloads 0 Views