Biochimica et Biophysics @ Elsevier/North-Holland
Acta, 488 (1977) Biomedical Press
WAX ESTER SYNTHESIS IN THE MOUSE PREPUTIAL GLAND TUMOUR
M.R. GRIGOR Department (Received
of Biochemistry, January
L. HARRIS University
of Otago, Dunedin
Summary The microsomal fraction from the mouse preputial gland tumour contains an acyltransferase which catalyzes the synthesis of wax esters. The enzyme is inhibited by moderate concentrations of free fatty acids (40 J.&I or more) but the inhibition is relieved by the addition of bovine serum albumin. The specific activity of the enzyme increases markedly between the 20th and 30th days of tumor growth. A number of other lipid synthesizing enzymes show similar trends for specific activity as related to tumour age.
Introduction The mouse preputial gland tumour has been shown to accumulate large quantities of wax esters during the latter part of tumour growth, after 25-30 days following inoculation [ 11. Previous to this the wax esters are not detectable, or are present only in trace amounts, although the other lipid classes found in older tumours are present. These include the alkyldiacylglycerols which also require fatty alcohols for their synthesis . We have examined the enzymes involved in wax ester synthesis, fatty alcohol synthesis and glyceride synthesis in the tumour in an endeavour to elucidate factors which might control the accumulation of wax esters. Materials and Methods [ 1-‘4C!]palmitic acid and [9,10-3H]palmitic acid were purchased from the Radiochemic~ Centre, Amersham, United Kingdom. [ 9,10-3H] hexadecanol was prepared from [9,~O-3HJp~mitic acid by reduction with lithium aluminium hydride. CoA, ATP, NADPH, rat-glycerol S-phosphate, fatty acid-free bovine serum albumin and phospholipase D were purchased from Sigma ChemAddress corresPondence to: Dr. M.R. Grigor. Department P.O. Box 56, Dunedin. New Zealand.
ical Company, St. Louis, MO. Dihydroxyacetone phosphate was purchased from the same source as the di-monocyclohexylamine salt of the dimethylketal and was regenerated as directed. Unlabelled lipid standards were purchased from Applied Science Laboratories, State College, Pa., except for phosphatidic acid which was prepared from phosphatidylcholine by the action of phospholipase D. Butyl-pBD [ 2-(4-t-butylphenyl)-5-(4-biphenylyl)-l,3,4-oxadiazole) was purchased from CIBA-Geigy. The mouse preputial gland tumour was maintained in mice of the C57BL strain as described previously [l] . The tumours were homogenised in ice-cold 0.25 M sucrose. Homogenates were centrifuged at 15 000 X g in a Sorval RC-5 refrigerated centrifuge and the supernatant was centrifuged at 90 000 X g in a Beckman Model L ultracentrifuge to sediment the microsomes. The pellet was resuspended in sucrose and sedimented again before being finally taken up in 0.25 M sucrose. In the experiment to investigate the subcellular location of the wax ester acyltransferase the pellets obtained at 2 000 and 15 000 X g and the 90 000 X g supernatant were also kept. All subcellular fractions were stored frozen and thawed once before use. The protein content was determined according to the method of Lowry et al. [ 3 1.
All enzyme assays were performed at 37°C in 1 ml of 0.1 M Tris - HCl buffer, pH 7.2. Wax ester synthesis was followed using three different systems. In the first the substrates were [ l-‘4C]palmitic acid and [ 9,10-3H]hexadecanol together with ATP (10 mM), CoA (10 PM) and MgCl* (4 mM) which were added as cofactors for the endogenous acyl-CoA synthetase (acid:CoA ligase (AMP-foging), EC 188.8.131.52). The second was a direct assay for the acyl-CoA:fatty alcohol acyltransferase using unlabelled palmitoyl-CoA and [9,10-3H]hexadecanol as substrates. The third system esterified hexadecanol formed endogenously by reduction of [l-‘4C]palmitic acid by the microsomal acyl-CoA:NADPH reductase . The incubations contained NADPH (500 FM) and CoA, ATP, MgCl, in the concentrations given above. The lipid substrates were added either bound to albumin  or as 10 mM solutions in acetone. All reactions were run for 10 min. The acyl-CoA:NADPH reductase was assayed using the third system described above. Here the lipid products were transesterified by heating in a sealed tube with 1 ml 6% (w/v) NC1 in methanol for 20 min to give the total alcohol formed as the free alcohol and the fatty acyl chains as methyl esters. The synthesis of phosphatidic acid from either sn-glycerol 3-phosphate or dihydroxyacetone phosphate was assayed in similar incubations using tumour microsomes (0.2-0.6 mg protein), [9,10-3H]palmitic acid (167 PM) bound to albumin, CoA, ATP, MgC12 as above and NaF (20 mM) to inhibit the endogenous phosphatases. rat-Glycerol 3-phosphate (40 mM) of dihydroxyacetone phosphate (500 PM) were added to provide the glycerol carbons. In the latter case NADPH (500 PM) was added to reduce the acyl-dihydroxyacetone phosphate formed to sn-1-monoacylglycerol 3-phosphate [ 61. These reactions were run for 10 min. The synthesis of alkyl-dihydroxyacetone phosphate was also assayed using
tumour microsomes (0.4-1.3 mg protein), [ 9,10-3H]hexadecanol (8 PM) and dihydroxyacetone phosphate (500 PM). CoA, ATP MgClz and NaF were added as above and these reactions were run for 60 min. The reactions were stopped by adding 2 ml of chloroform:methanol (1 : 1 by vol.) and, after centrifuging, removing the lipids in the chloroform layer. This was concentrated under a stream of dry nitrogen and the lipid products were separated using thin-layer chromatography. For the neutral lipids a solvent system of hexane/diethyl ether/acetic acid (80 : 20 : 1 by volume) was used with layers of silica gel G, while the phospholipids were separated using a solvent system of chloroform/methanol/ammonia (60 : 30 : 5 by volume) and layers of silica gel H. The developed layers were stained with iodine vapour and areas corresponding to standards were scraped into scintillation vials. After adding scintillant (0.6 w/v) butyl-PBD in toluene : Triton X-100 (2 : 1 by volume), the radioactivity was determined in a Packard model 3300 spectrometer. No lipid standard was available for the alkyl-dihydroxyacetone phosphate. In this case the area of the layer below phosphatidic acid was taken to contain the alkyldihydroxyacetone phosphate. Only traces of radioactivity were recovered in this zone from chromatograms of the products of incubations carried out with no dihydroxyacetone phosphate. The identity of the products as alkyldihydroxyacetone phosphate was confirmed in an experiment where NADPH (500 PM) was added to the standard assay mixture after 60 min and the reaction was allowed to proceed for a further 30 min. The radioactive project now chromatographed with phosphatidic acid as would be expected for sn-alkylacylglycerol 3-phosphate. After reduction with lithium aluminium hydride the same proportion of radioactivity was found to chromatograph with selachyl alcohol when hexane/diethyl ether/methanol/acetic acid (50 : 50 : 10 : 1 by vol.) was used as solvent. Results Properties of the wax ester acyltransferase When palmitic acid and hexadecanol were used as substrates, ATP, CoA and MgCl, were all necessary cofactors for wax ester synthesis. They could however, be replaced by palmitoyl-CoA as in the second assay system. The apparent K, for palmitoyl-CoA was 120 PM, while concentrations of hexadecanol over 200 ,uM were required to saturate the enzyme. The kinetic properties of the enzyme did not appear to be affected by the age of the tumour. The saturating concentrations of hexadecanol were many times higher than the optimum fatty alcohol concentration observed for alkyl-dihydroxyacetone phosphate synthesis. Here concentrations of hexadecanol greater than 8-10 PM were found to be partially inhibitory. The subcellular distribution of the acyltransferase was investigated using the palmitoyl-CoA assay system. The highest specific activity (10 X that of the homogenate) and 50% of the total activity was found in the 90 000 X g pellet (microsomes). Fig. 1 shows the effect on the acyltransferase of altering the concentrations of both hexadecanol and palmitic acid. In this experiment the first assay system was used with both substrates labelled. This enabled the stoichiometry of the
Fig. 1. Synthesis of wax esters from [9,10-sHlhexadecanol, (e) and [l-“Clpalmitic acid, (0). Incubations contained CoA, ATP, MgCl2 and were performed as described in text. (A) contained pahnitic acid (50 PM). albumin (70 PM), and microsomal protein (0.7 mg ‘ ml-l ). (B) contained hexadecanol (140 #AM), albumin (100 #M) and microsomal protein (0.5 mg . ml-l). (C) as for B but with zero albumin.
reaction to be checked. The deviation from the expected 1 : 1 molar ratio is readily apparent at high concentrations of fatty alcohol and has been attributed to the presence of endogenous fatty acid in the tumour microsomes. These contain a phospholipase A which releases fatty acids from the membrane phospholipids (Harris, E.L. and Grigor, M.R.-unpublished observations). The increased incorporation of acid over alcohol at low alcohol concentrations could be the result of the formation of cholesterol esters. The tumour micro-
40 60 60 pM palmltlc acid
40 80 120 PM olelc acid
Fig. 2. Synthesis of wax esters from palmitic acid and endogenously formed hexadecanol. Incubations contained [I-14C]pahnitic acid. NADPH (500 MM), microsomal protein (0.7 mg . ml-l ), and ATP. CoA, MgCl, as described in text. Curves show incorporation of palmitic acid into total alcohols, (0); UneSterified alcohols,
(A) and wax esters, (a),
Fig. 3. Effect of free fatty acid on wax ester synthesis. Curves show Lhe incorporation of [9,10-3H]hexadecanol into wax esters in incubations which contained hexadecanol(140 /.tM), pahnitoyl-CoA (200 nM) and albumin (70 MM), (0); hexadecanol and palmitoyl-CoA (40 nM each) and albumin (20 PM), (0); hexadecanol and palmitoyl-CoA (40 pm each) and albumin (70 j&M). (A). All incubations contained 0.7 mg . mof-’ microsomai protein.
somes have an acyltransferase which will use endogenous cholesterol as substrate [ 81. The concentration of albumin in the incubations had a profound effect on the concentrations of fatty acid required to saturate the enzyme for wax ester synthesis. In the absence of albumin, fatty acid at 40 PM or more partially inhibits the incorporation of hexadecanol into wax esters, but this inhibition was completely relieved by 0.1 mM albumin. The inhibition of the acyltransferase by free fatty acids was investigated further in a system where fatty alcohols produced endogenously were esterified to produce wax esters (Fig. 2). In these assays palmitic acid was the sole lipid substrate. The alcohol produced by the reduction of the acyl-CoA was recovered both as the free alcohol and the wax ester. The yield of total alcohol was determined after transesterifying the wax. While palmitic acid at concentrations greater than 60-80 PM proved saturating for the dehydrogenase, concentrations greater than 40 PM depressed the acyltransferase. The effect of free fatty acid on the acyltransferase was also investigated using the palmitoyl-CoA assay (Fig. 3). Here the free acid cannot act as substrate. Using saturating concentrations of both palmitoyl-CoA and hexadecanol, oleic acid at 160 ,uM had only a minor effect. When the concentration of the substrates was reduced to l/4, addition of oleic acid depressed wax ester synthesis by about 40%. This inhibition however was relieved by increasing the albumin concentration to 0.07 mM. Effect of tumour age Fig. 4 shows the activity
of the wax ester acyltransferase
in several microso-
Fig. 4. Effect of tumour age on lipid synthesizing enzymes of the mouse preputial gland tumour. (A) Wax ester acyltransferase. Incubations contained Cl-” Clpahnitic acid (160 PM), C9,10-sHlhexadecanol (140 PM), albumin (80 PM), and CoA, ATP. M&l, as in text. (B) Fatty alcohol synthetase. Incubations contained [l-“Clpahnatic acid (100 PM), albumin (35 MM), NADPH (500 PM) and CoA. ATP and MgCl,as in text. (C) Alkyl-dihydroxyacetone phosphate synthetase and (D) and (E) phosphatidic acid synthesis from glycerol 3-phosphate and dihydroxyacetone phosphate respectively. Conditions for C, D and E as described in text.
ma1 preparations obtained from tumours at different stages of growth. Between 20 and 30 days the specific activity increased four-fold. This pattern is however not confined to the wax ester acyltransferase. Similar increases are also observed for the acyltransferases to both sn-glycerol 3-phosphate and dihydroxyacetone phosphate, the fatty acyl-CoA:NADPH reductase and, although at a much lower level of activity, the alkyl-dihydroxyacetone phosphate synthetase. The data has been presented as activity per mg microsomal protein. However, since the recovery of microsomal material per gram of tumour was similar for tumours of different ages, the same trends were apparent when the activities per gram tumour were plotted. Discussion Our experiments confirm the report of Snyder and Malone  that wax ester synthesis in the mouse preputial gland tumour is catalysed by an acyltransferase. Despite a suggestion that an esterase can synthesize wax esters in the livers of the rat and the dogfish (Squalus acanthias) [lo], it now appears that wax synthesis in a variety of marine organisms also involves acyltransferases which use fatty acyl-CoA as the acyl donor (refs. 11 and 12 and Quinton, R.G. and Grigor, M.R., unpublished observations). Rat liver has been shown to contain an acyltransferase which esterifies short chain alcohols  but no acyltransferase activity for long chain alcohols is detectable. The tumour acyltransferase appears to be sensitive to relatively moderate concentrations of free fatty acids. We have investigated this in a number of experiments since it represents a potential control for the age-related accumulation of wax esters observed in the tumour. Small tumours which do not contain waxes are growing in hosts which exhibit apparently normal metabolism. As the tumour grows to large sizes profound changes in the host metabolism occur, which may well alter the intracellular concentrations of fatty acids in the tumour. Free fatty acids have been proposed as effecters of a number of enzymes [ 14-161. The significance of this in vivo is difficult to assess since it is not possible to measure accurately the concentration of free fatty acids. Most of the neutral lipid in the cell is in membrane-enclosed droplets where it is unlikely to exert any affect. Alternatively such lipid as is in the cytoplasm is most probably bound to protein . Our observations that moderate concentrations of protein could relieve the fatty acid inhibition of the acyltransferase in vitro suggest that control of the acyltransferase in vivo by free fatty acids is unlikely to have any physiological significance. In light of these findings we then investigated the activity of the acyltransferase in preparations from tumours at different stages of growth. There was a considerable increase in activity of the wax ester acyltransferase between 20 and 30 days after transplantation. This is the period during which waxes begin to accumulate. However, all the lipid synthesising enzymes assayed showed similar profile, although the pattern observed for the alkyl-dehydroxyacetone phospphate synthetase does differ from that previously reported by Snyder et al. [ 21. One question of interest concerns the ability of young tumours in vivo to synthesize and accumulate alkyldiacylglycerols but not wax esters. Fatty alco-
hols are required for the synthesis of both these lipid compounds. A potential explanation for this observation is that in young tumours low levels of the fatty acyl-CoA:NADPH reductase restrict the concentration of fatty alcohol available for complex lipid synthesis. The wax ester acyltransferase has a low affinity for fatty alcohols while the alkyldihydroxyacetone phosphate synthetase was most active at only 8 (uM hexadecanol. Thus synthesis of the alkylglycerols proceeds despite the much lower activity of the alkyl-dihydroxyacetone phosphate synthetase than the wax ester acyltransferase. As the tumour grows the activity of both the reductase and acyltransferase increase several fold and the concentration of the fatty alcohol becomes sufficient for wax ester synthesis to occur. A similar explanation is proposed to describe observations made with preparations from dogfish liver. Microsomes from dogfish liver contain a wax ester acyltransferase several times more active than the alkyl-dihydroxyacetone phosphate synthetase (Quinton and Grigor, unpublished observations). However, the fish liver oil contains large quantities of alkyldiacylglycerols and only traces of waxes [18,19]. Here fatty alcohol concentrations of 100 I.IM were found necessary to saturate the acyltransferase, while only 16 PM saturated the alkyldihydroxyacetone phosphate synthetase. Acknowledgement This work was supported of New Zealand.
by a project
grant of the Medical Research
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Grigor, M.R. (1976) Biochim. Biophys. Acta 431.157-164 Snyder, F., Malone, B. and Blank, M.L. (1970) J. Biol. Chem. 245. 1790-1799 Lowry. 0.H. Rosebrough. N.J., Fur, A.L. and Randall. R.J. (1951) J. Biol. Chem. 193.265-275 Snyder, F. and Malone, B. (1970) Biochem. Biophys. Res. Commun. 41.1382-1387 Spector, A.A. and Hoak. J.C. (1969) Anal. Biochem. 32. 297-302 Hajra, A.K. and Agranoff, B.W. (1968) J. Biol. Chem. 243,3542-3543 Wykle. R.L. and Snyder, F. (1970) J. Biol. Chem. 245. 3047-3058 Grigor, M.R., Pratt, R.D. and Snyder, F. (1972) Arch. Biochem. Biophys. 150. 371-375 Snyder, F. and Malone, B. (1971) J. Am. Oil Chem. Sot. 48,85A Friedberg, S.J. and Greene, R.C. (1967) J. Biol. Chem. 242. 234-237 Sargent, J.R., Gatten. R.R. and McIntosh, R. (1971) Marine Biol. 10, 346-355 Sargent, J.R. and McIntosh, R. (1974) Marine Biol. 25. 271-277 G&or, M.R. and Bell, I.C. (1973) Biochim. Biophys. Acta 306.26-30 Lea, M.A. and Weber, G. (1968) J. Biol. Chem. 243.1096-1102 Carlson, C.N., Baxter. R.C.. UIm. E.H. and Pogell, B.M. (1973). J. Biol. Chem. 248, 5555-5561 Ramadoss. C.S.. Uyeda. K. and Johnston, J.M. (1976) J. Biol. Chem. 251. 98-107 Mishkin, S.. Stein, L., Gatmatian. A. and Arias, I.M. (1972) Biochem. Biophys. Res Commun. 47, 997-1003 18 Malins, D.C.. Wekell. J.C. and Houle. C.R. (1965) J. Lipid Res. 6. 100-105 19 Spener, F. and Mangold. H.K. (1971) J. Lipid Res. 12, 12-16